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Biology 447 Environmental Microbiology

September 2008


© 2008 Colin I. Mayfield

Biology 447 ­ Environmental Microbiology

Index: Page 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. Biology 447: General Overview & Introduction What are the goals of the course? What should I be able to accomplish by the end of the course? How to use study materials most effectively Course Organization Course Modules General Course Background Material List of Main Lecture Topics ­ Biology 447 How to allocate time for the various sections of the course Assignments: Introduction Audio Lectures in PowerPoint Background Information on Water Issues for Biology 447 Full Report from the United Nations University -World Water Day 1999 Module 1. Aquatic Microbiology Module 2. Marine Microbiology Module 3. Estuarine Microbiology Module 4. Pathogens in Water Module 5. Biodegradation Module 6. Deep Subsurface Microbiology Module 7. Groundwater Microbiology Module 8. Biological Treatment of Soil and Groundwater 3 3 4 4 5 5 6 6 8 10 12 14 14 20 48 56 60 82 150 152 185


Biology 447 ­ Environmental Microbiology

Biology 447: General Overview & Introduction

What are the goals of the course?

The course is not designed to be an intensive study of all aspects of water microbiology, but, instead, water microbiology in all of its variety is used as an example of the range of microbiological topics that directly affect environmental issues. There is an apparent concentration on groundwater and contaminated soil issues involving microbiology simply because they are less well presented in textbooks from a microbiological viewpoint. There is also a deliberate attempt in most parts of the course to put the microbiology in a context that involves other issues in the environment. These include the role of the regulating agencies, the role of the law and government agencies in setting and enforcing standards, and the assessment and reporting procedures commonly used in environmental assessment, monitoring and compliance. The assignments for the course are designed to help with this last process. You will also find that there is an evolution in the course material from the more theoretical in freshwater and marine microbiology through more applied materials in biodegradation and groundwater microbiology to very practical and applied materials in the bioremediation sections of the course. You should find, nevertheless, that many of the concepts and examples in earlier parts of the course can still be applied to the section on bioremediation. Main goals for this course: · · · · · To achieve a basic understanding of aquatic microbiology and microbial processes To know the major microorganisms of importance in freshwater, seawater, and groundwater To understand the major microbial processes involved in those habitats To understand the major environmental factors and influences in those habitats To understand how to apply basic microbiological and ecological principles in understanding the applied processes known as bioremediation of contaminated soils and groundwater.

Specifically: Freshwater microbiology · · · · · To be able to explain the activities of microorganisms in freshwater habitats, their importance and their interactions with the environment and other organisms. To be able to assess the role and importance of freshwater habitats in disease transmission by pathogenic microorganisms and protozoa. To assess and explain the concepts of productivity, growth, biomass, diversity in freshwater habitats (including estuarine habitats) and the concept of ecosystem control measures. To have a basic understanding of the methods used in aquatic microbiology and their advantages and deficiencies To be able to judge and explain the importance, relevance and future development of microbiological and other standards and regulations.

Marine microbiology · · · To understand the differences between the marine and freshwater habitat and their influences on microbial activities. To understand the specific roles of pressure, temperature and salinity. To understand the role of undersea volcanic vents in marine microbial productivity.


Biology 447 ­ Environmental Microbiology

Biodegradation · · To understand the role and importance of chemical structure of molecules and the effects of environmental factors on biodegradation by microorganisms. To understand the roles of these factors on the specific examples of naturally occurring compounds such as cellulose, lignin, proteins, etc., various hydrocarbons and pesticides.

Groundwater microbiology · · To understand the roles of microorganisms in groundwater ecosystems, especially in terms of their effects on the evolution of the geochemistry and biochemical properties of groundwater systems. To relate the microbiology of groundwater to the observed changes in naturally occurring compounds introduced into groundwater.

Bioremediation · · · To be familiar with the basic principles of bioremediation activities as they are used in contaminated soil and groundwater. To be able to devise appropriate generalized systems to perform bioremediation in contaminated systems (lectures and assignments). To apply information gained from this course and reference materials in the course and from other sources to critically evaluate bioremediation processes and applications.

What should I be able to accomplish by the end of the course?

The course deals with aspects of water microbiology from a general point of view and uses specific examples to illustrate the concepts. For instance, details of pathogenic bacteria in freshwater are not given, but, instead, an overview of their importance, the regulations surrounding their monitoring, the methods used to detect them, and their occurrence in some habitats is studied. The intention is that this serves as an example with reference sources supplied so that you can discover other aspects of pathogenic microorganisms in water systems. For instance, water treatment technologies are not presented in detail, but references to information on water treatment is given. This approach is used for the other areas of the course. You should have the following abilities and knowledge at the end of the course:

1. 2. 3. 4. 5.

Theoretical knowledge of aspects of water microbiology Practical applications of knowledge base to particular problems How to find, assess and integrate data and information on environmental issues How to produce a report on knowledge gained that presents the information effectively How to integrate knowledge from different parts of the course

How to use study materials most effectively

1. The complete lecture notes for each of the modules are given on the WWW site for the sake of providing more up-to-date material than could be provided in printed lecture notes. 2. The extra material on each Module is reference material and supplementary material; it is designed to clarify and/or complement the lectures and is NOT necessarily required knowledge. Many of the external links to other sites are dependent upon them remaining active and up-to-date; many may change or even vanish very quickly. 3. The assignments can be performed most effectively using searches and the WWW pages as starting points.


Biology 447 ­ Environmental Microbiology

4. There is a tremendous amount of material on the WWW site. One of the aims of the course is to organize the material in a comprehensible manner. Another is for you to learn techniques of organization of complex materials and to be able to find, extract, categorize, organize and assess such diverse information sources. You will find that some of them are even contradictory and certainly present different views of the same topic. One of the primary goals of the last assignment is related to that problem; to allow you to learn how to produce a good, organized and comprehensive report on a topic in bioremediation based on all available information. 5. You are NOT expected to know and be able to reproduce all of the material on this WWW site. A preliminary estimate I have done indicates that there is about 2.0 gigabytes of information directly referenced on the WWW site. If you go further into each WWW site referenced, that volume could exceed 5 Gigabytes. 6. Most of the information is background or material for the assignments. Use the search functions (local and the WWW search engines) as much as you can to narrow down information to that of use to you. No-one can possibly cope with 10 Gigabytes of information at once. To put it in perspective, a typical typed page is 1 kilobyte. One thousand kilobytes (pages) is a megabyte. A thousand megabytes is a Gigabyte. Five Gigabytes is 5 million pages! A little bit more than your average textbook!! 7. Try and use the lecture material as a guide to the other literature in the WWW site. Use the Key points and other summaries to make sure you have the essential elements of the lecture materials. Check the previous exams to see if you have enough material to answer the questions. 8. Note that the mid-term exam for on-campus students is a short answer or even a multiple choice type of exam. Details are not required. The final exam is oriented towards working out solutions to problems posed as questions. You will use the information from the course lecture materials to answer these questions. If you can use materials you found for the assignments you should do so in the examinations. You also will have a choice of questions in the final exam, but not in the midterm.

Course Organization

The course is a treatment of aspects of microbiology in various aquatic ecosystems. The ecosystems covered (to greater or lesser extents) are: · · · · · · Freshwater Marine Estuarine Groundwater Biodegradation is also a major section of the course but deals with all ecosystems, not just the aquatic ones. Combining this section with the section of the course on groundwater leads to the final section on Bioremediation. Bioremediation is the application of microbial methods to produce or stimulate some kind of biotransformation or biodegradation in order to clean up contaminated soils or groundwater.

Course Modules

The Modules are: 1. Aquatic Microbiology 2. Marine Microbiology 3. Estuarine Microbiology 4. Pathogens in water 5. Biodegradation Microbiology 6. Deep Subsurface Microbiology 7. Groundwater Microbiology 8. Biological Treatment of Soil and Groundwater The general organization of each of the Lecture Topics is similar:


Biology 447 ­ Environmental Microbiology

An Introduction is followed by a listing of the Key Points in each topic. These Key Points can form the basis for a study guide to each Topic. Various Subheadings then follow that describe the various main subdivisions of each Topic. These subdivisions contain the text version of all the lecture notes and all overheads used in the course. They may also contain extra materials or updated materials. Often, a Summary concludes the particular Topic section. The next major section is Background Materials. These are resources that relate to that particular Topic and can be Library Materials - books and articles in the library (some on reserve), WWW sources - links to resources available on the WWW. The various course modules are not of equal length and complexity. They do not correspond to individual lectures but are simply divisions of the course material. For instance the Marine, Deep Subsurface and Estuarine Microbiology modules are short but are separated to emphasize the differences between them and the module on Aquatic Microbiology that is mainly concerned with freshwater systems. The section on Biodegradation is much longer and gives a lot of detail on biodegradation pathways and microbiology. The module on Groundwater is longer since longer lecture notes are given and many examples are presented. In addition, the module contains sections on Kinetics and Rates of Biodegradation that apply generally to other modules and especially to the modules and assignment on Biological Treatment of Soil and Groundwater.

General Course Background Material

Read this material to make sure you have sufficient background in general microbiology. The background is generally equivalent to the introductory microbiology courses Biology 240 and 241. Any good introductory microbiology textbook will provide enough detail for this background. This section of the course gives a rapid overview and review of the materials from General Microbiology that are required to completely understand the various Topics presented. It is assumed that the student has some background in microbiology and therefore should be able to very rapidly work through this material. The different sections of this background material deal with overviews of microbial growth and general metabolism and biochemistry, microbial taxonomy (detailed knowledge of the taxonomy is NOT required for Biology 447 although some knowledge of commonly occurring bacteria found in the environment and their classification is very helpful), energy relationships (especially important in biodegradation), environmental determinants of bacterial and microbial growth, and some very general ecological principles (Odum's diagram).

List of Main Lecture Topics ­ Biology 447 Module 1 ­ Aquatic Microbiology A. Water Supply, Use and Distribution Issues a. Freshwater Aquatic Environments B. Aquatic Microbiology a. Pathogens b. Biomass Determination c. Activity Measurements C. Bacteria in Aquatic Systems D. Pollution of Aquatic Systems E. Biofilms (slides) F. Other Aspects of Water Pollution (slides) G. Lake Erie ­ Recent problems (slides) H. Remote Sensing


Biology 447 ­ Environmental Microbiology

I. Module 2. A. B. C. D. E. F. G.

Regulations, Guidelines and Laws Marine Microbiology Salinity Levels Dissolved Gases Large-scale oceanic currents Temperature Pressure Volcanic Vents Characteristics of marine bacteria

Module 3. Estuarine Microbiology A. Importance of Estuarine Systems B. Plants C. Adaptations of Estuarine Organisms D. Microbiology of Estuaries Module 4. Pathogens in Water A. Introduction B. Methods of detecting pathogenic viruses and bacteria C. Enteropathogenic E. coli D. Detection of water-borne parasites E. Summary (slides) F. Canadian Water Quality Guidelines G. Microbiological Indicators (slides) H. The Walkerton E. coli outbreak (summary and Walkerton Report) Module 5. Biodegradation A. General Breakdown of Naturally occurring compounds 1. General biodegradation of naturally-occurring compounds 2. Cellulose breakdown 3. Lignin Structure 4. Hydrocarbon biodegradation 5. Importance of hydrocarbon biodegradation 6. Mechanisms of biodegradation 7. Microbial degradation ­ aliphatic hydrocarbons 8. Biochemistry of hydrocarbon degradation 9. Aromatic hydrocarbons 10. Polyaromatic hydrocarbons B. Pesticides 1. Insecticides 2. Herbicides 3. Fungicides 4. Typical Use Patterns for Pesticides 5. Transport of Pesticides in the Environment 6. Pesticides in Food 7. Summary of Biodegradation of Pesticides 8. The "Benchmark" Concept 9. Alternatives to Increased use of Pesticides Module 6 ­ Deep Subsurface Microbiology Deep Subsurface Microbiology Module 7. Groundwater Microbiology


Biology 447 ­ Environmental Microbiology

A. Groundwater environments B. Groundwater Microbiology 1. Groundwater Microbiology a. Overview b. Bacteria in Groundwater c. Sampling Techniques d. Detailed Sampling Systems 2. Microbial Processes a. Chemical and Physical Conditions in Normal Groundwater b. Chemical Conditions in Groundwater contaminated with Organics c. Movement of Groundwater d. Movement of Contaminants 3. Rates of Biodegradation 4. Kinetics of Biodegradation C. Contaminated Groundwater Microbiology D. Summary of Mechanisms causing Variations in Groundwater Plumes E. Groundwater Modelling Module 8. Biological Treatment of Soil and Groundwater 1. Bioremediation 1A. Bioremediation Choices and Processes 1B. Decision support for Groundwater Intrinsic Bioremediation 1C Decision Support for Soil Remediation 1D Site Characterization 1E Summary and Overview 1F Bioremediation Computer Models 2. Factory Farms and Sewage Sludge 2A. Factory Farms and Manure Disposal 2B. Sewage Sludge Disposal 2C. Reference Materials ­ Factory Farms 2D. Reference Materials ­ Sewage Sludge 2E. Microorganisms

How to allocate time for the various sections of the course

The course is split into "modules" that do NOT correspond to individual lectures in the on-campus course; they are simply different subject areas such as Aquatic Microbiology, Biodegradation, bioremediation, etc. The different modules have, in fact, very different amounts of information and should take different times to complete.

I recommend you do the following:

1. Look over the entire course on the CDROM or the WWW site. Estimate the relative lengths and difficulty

(for you) of the different modules. Typically, the Aquatic Microbiology modules (including Aquatic Microbiology, Marine Microbiology [a very short one], Estuarine Microbiology [an even shorter one] and Pathogens in Water) would take about 25% of the course lectures. Biodegradation (including Pesticides) would be about 30%, and Groundwater Microbiology and Bioremediation would make up the remainder. The bulk of the Bioremediation section is concerned with the application of microbiology to applied problems of cleaning up contaminated sites using microorganisms.


Biology 447 ­ Environmental Microbiology

2. Look at the examples of final examinations to find out what kind of questions you will be expected to

answer. Most (but not all) require using examples to solve a particular scenario rather than a simple restatement of lecture materials.

3. Start with aquatic microbiology module and go through the first few sections on Introduction, Freshwater

Aquatic Environments, Methodology, Pollution in Aquatic Systems, Bacteria in Aquatic Systems, and Pathogens in Water. Leave the last two sections for the time being.

4. Try and get an overview of these sections - I recommend looking at the "Key Points" and making sure you

understand their importance. I also recommend that you summarize in a few key statements the main points in the sections you have read; Often a whole set of paragraphs can be summarized (and more easily remembered) in a single phrase. For example, the section on pollution has an important theme - "Aquatic Pollution is often a disturbance of the Photosynthesis/Respiration Balance - or at least it is instructive to look at it that way"

5. Then look at the last two sections on Remote Sensing and Regulations. They are designed to show you

how these problems in aquatic systems are being dealt with. Remote sensing technologies will be more widely used for monitoring aquatic systems in the future. The development of regulations and laws to try and maintain the health of aquatic systems is a very complex matter. Have a look at some of the regulations and methods, but do not try and learn any of the additional materials. Get (again) an overview of the types and general categories of regulations and laws and how they are applied. Wherever you see a complex Tabular format of numbers and different criteria you can assume that you don't have to know them. That material is for looking up as required, not for learning!

6. Move on to the Marine and Estuarine Modules in turn. They are very short because they basically compare

this environment with the freshwater environment. Therefore the differences are important. These sections on Aquatic Microbiology should take you in total about 25% of the time you allocate for the lecture materials in the course.

7. Repeat this process with the Biodegradation Section; you should again look at the entire section (read it all

very quickly) and then return and extract the most important points. Look at naturally occurring compounds, then hydrocarbons then pesticides. The chemistry gets more complex, but the principles remain the same!

8. Try and extract common features of biodegradation for the different chemical groups. Many naturally

occurring compounds (lignin) have complex structures and are resistant to decomposition, most naturally occurring compounds are not as resistant. What are the common features of resistant compounds? What environments promote decomposition and which seem to allow compounds to persist? Are there common features in the biochemistry? You will find all of these in the Module on Biodegradation.

9. Don't forget to go back and consider previous sections and how they apply to the new one. As one example

of this, the Photosynthesis/Respiration balance equation has applications in Biodegradation; the amounts of nutrients required for growth will be in balance or one will become limiting. In an aquatic environment, if massive quantities of hydrocarbons are spilled the C:N:H:O:P:S, etc. balance is no longer maintained. What will be lacking and by how much? It can be calculated from the equation. This in fact is used to calculate how much fertilizer to add to an oil spill to get optimum biodegradation. Looking forward, this equation also has obvious applications in Bioremediation processes.

10. I do NOT expect you to reproduce all of the chemical pathways and transformations given in the

Biodegradation section (especially the section on Pesticides). Examples are all that is required. If you know how 2,4-D degrades, it is not important or required that you know every other variant of 2,4-D. Similarly with all of the variants of the aromatic hydrocarbons. They are all slightly different in their biodegradation pathways but also show very strong similarities (ring fission methods, side chains, etc.). Look for


Biology 447 ­ Environmental Microbiology


11. The bioremediation section is really a summation of the course. All of the previous parts are applied to

particular contamination problems. The only part missing is that of soil microbiology. If soil is contaminated, some knowledge of soil microbiology is an advantage in remediation. The principles of soil microbiology are dealt with in another course, but, in general, aquatic systems show similar properties. The microorganisms grow slowly, they are adapted to low nutrient conditions, their biochemistry is amazingly diverse if you look at all of the microorganisms present, they respond to nutrient addition by rapid growth, and sections of the entire population can withstand different kinds of adverse or extreme conditions (cold, heat, light, etc.).

12. Typically, the sections on Groundwater and Bioremediation are the least familiar to students. They are

relatively recent developments in microbiology and, because of their economic importance, are developing very rapidly. Just examine the documents put out by the EPA in the US to see how much attention is being paid to bioremediation. Entire training systems and seminars are given by the EPA on Bioremediation.

13. When you have finished reading the course materials, try and put together an overview describing how the

various parts fit together. Even though it is not expressly pointed out in the lectures, many of the items in a module "build" on previous Modules. This should make answering examination questions easier for you to write and for me to read!


Biology 447 ­ Environmental Microbiology

Assignments: Introduction

The marking scheme for the assignments and some general points are available on the Website Mark Breakdown for Course Assignment/Examination Midterm Examination Assignment Final Examination Oncampus 20% 30% 50%

Assignment - involves a class study of an issue that has broad implications in environmental health and microbiology - The disposal processes for sewage sludge from sewage treatment plants and manure from intensive farming operations. Each group (or individual) will take one aspect of the set of problems and produce a report. The various topics are listed and you should choose one of them. Since a complete coverage of the topics is obviously desirable, whoever chooses a topic first gets to do that topic. This material will not be covered extensively in the lectures, but questions on this set of topics may be asked on the final exam. You will be given typical exam questions to clarify what is required. Assignment Topic List See the Website

More details on the assignments will be given in t he lectures and posted on the website once the course begins. View the "Announcements" page at frequent intervals


Biology 447 ­ Environmental Microbiology

Audio Lectures in PowerPoint

Many of the lectures are available on the Website as PowerPoint Slides with audio (see listing below)

Note that these audio and slide presentations are very large files to download over a slow modem link. If you use them from the WWW site, you may find that the audio files, in particular, take some time to download. Patience is required!

Module # and Title


1. Introduction 1a. The Aquatic Environment 1b. Microorganisms in Aquatic Systems

Number of Slides

25 26 12 29

1 - Aquatic Microbiology

1c. Pollution of Aquatic Systems 23 1d. Water Pollution and Microorganisms Updated Slides (entire set of 1, 1a, 1b, 1c, 1d)

2. Marine Microbiology 2 - Marine Microbiology Updated Slides for Modules 2 and 3 combined 3. Estuarine Microbiology 3 - Estuarine Microbiology Updated Slides for Modules 2 and 3 combined 14 17

4 - Pathogens in Water

4. Pathogens in Water


5 - Biodegradation Microbiology

5. Introduction



Biology 447 ­ Environmental Microbiology

5a. Hydrocarbons 5b. Pesticides Updated Slides for Module 5 (entire set of 5a, 5b and 5c)

29 38

6. Deep Subsurface Microbiology 6 - Deep Subsurface Microbiology Updated Slides for Modules 6 and 7 (combined) 7 - Groundwater Microbiology 7. Groundwater Introduction 7a. Groundwater Environment Updated Slides for Modules 6 and 7 (combined) 8. Overview 8 - Bioremediation and Applications


17 42


Other Slide Presentations

1. 2. 3. 4. BIOFILMS: and their importance Other Aspects of Water Pollution Lake Erie - Recent Problems Satellite Imagery Coral reefs at risk Microbiological Indicators Microorganisms in Groundwater - Overview Module 1

Module 2 Module 4 Module 7


Biology 447 ­ Environmental Microbiology

Background Information on Water Issues for Biology 447 General Introduction to Water Issues Use and Distribution Issues

1. Water covers 70% of the Earth's surface 2. Water in the oceans is over 96% of all water on Earth 3. About 30% of the 105,000 cubic kilometres of water that falls by precipitation reaches the oceans though river flow. 4. 70% is evaporated or transpired by plants 5. This leaves only 37,000 cubic kilometres of water for distribution to the Earth's population. This is about 6000 cubic metres per person per year at a population of 6 billion = 12,000 litres per day per person. 6. Unfortunately, that 37,000 cubic kilometres is NOT evenly distributed around the Earth' surface. 7. For instance Canada has over 22% of the Earth's standing supply of freshwater, but only about 0.005% of its population. 8. Two litres of water per person per day will sustain life. 9. Average consumption is about 250 litres per day per capita 10. Industrial consumption is about 1500 litres per day per capita in the developed countries 11. Agricultural consumption can exceed 3000 litres per day per capita in hot climates. 12. Many other complicating factors such as water quality, pollution with inorganic and organic chemicals, and contamination with microorganisms causing disease.

Full Report from the United Nations University -World Water Day 1999

UN World Day for Water, 1999

"Everyone Lives Downstream" Unsafe Water: 3.3 Billion Illnesses and 5.3 Million Deaths Yearly Price Tag for Safe Water -- $50 to $105 per Person Clean, safe water can be brought to the 1.4 billion people around the world without it for as little as $50 per person, which can prevent many of the 3.35 billion cases of illness and 5.3 million deaths caused each year by unsafe water, says a United Nations analysis. At any given time, an estimated one half of people in developing countries are suffering from diseases caused either directly by infection through the consumption of contaminated water or food, or indirectly by disease-carrying organisms (vectors), such as mosquitoes, that breed in water. These diseases include diarrhea, schistosomiasis, dengue fever, infection by intestinal worms, malaria, river blindness (onchocerciasis) and trachoma (which alone causes almost six million cases of blindness or severe complications annually). The UN warns that unless action is stepped up, the number of people without access to safe water will increase to 2.3 billion by 2025, with the number of those who die from unsafe water expected to jump sharply as well.


Biology 447 ­ Environmental Microbiology

Right now, 20 percent of the world's population in 30 countries face water shortages, a figure that will rise to 30 percent of the world's population, in 50 countries, by 2025, according to the UN, observing World Day for Water on March 22. The theme of World Water Day 1999 is: "Everyone lives downstream," meant to convey that problems in one part of a watershed, or even in a in a country abroad, can affect people great distances away. Driven by a rising global standard of living and increasing food production, water demand is increasing at twice the population growth rate," says Hans van Ginkel, Rector of the UN University (UNU), an international community of scholars engaged in research, training and knowledge dissemination to promote the UN's aims of peace and progress. In many countries, water shortages stem from inefficient use, degradation of the available water by pollution and the unsustainable use of underground water in aquifers, the UN says. For example, 40 to 60 per cent of water used by utilities is lost to leakage, theft and poor accounting. How bad is the water crisis? · · · · · Every 8 seconds, a child dies from a water-related disease 50 percent of people in developing countries suffer from one or more water-related diseases 80 percent of diseases in the developing world are caused by contaminated water 50 percent of people on earth lack adequate sanitation 20 percent of freshwater fish species have been pushed to the edge of extinction from contaminated water.

Not only is the toll a human tragedy, but it means these people are less able to carry on productive lives, and this undermines social and economic development," says Klaus Töpfer, Executive Director of the UN Environment Programme (UNEP). Dr. Töpfer notes that women and girls in developing countries spend more than 10 million person-years in aggregate each year fetching water from distant, often polluted sources. Ironically, most available fresh water is found in developed nations, which have only one-fifth of the world's population. Nearly all of the 3 billion global population increase expected by 2025 will be in developing countries, where water is often already scarce, or comes in monsoons, hurricanes and floods, draining off the land quickly. Getting Water to the Poor The U.N. states that the estimated capital cost to provide safe water in rural areas is $50 per person; $105 per person in cities. Providing sanitation can be done for an additional $30 or less per person in rural areas, $145 in urban areas. The UN estimates the overall price to bring low-cost safe water and sanitation to all those who need it today (and will in the next decade, given population growth) in rural and low-income urban areas at $23 to $25 billion per year over eight to 10 years. Current world investment is $8 billion per year, leaving a $15 to $17 billion shortfall -- an amount roughly equal to annual pet food purchases in Europe and the USA. Water can be provided with these funds in rural and low-income urban areas through the utilization of low-cost technologies that include handpumps, gravity-fed systems and rainwater collection, which would be built to serve entire rural villages or urban neighbourhoods, rather than bringing indoor plumbing to individual houses. The provisions would include pumps, pipes, the training of workers, and the development and strengthening of water management practices. This is the absolute minimum that the world community must provide to the world's poor without water," says Dr. van Ginkel. "It will save countless lives, and greatly lessen the burden on millions of those, mostly women and children, who must trudge miles each day to bring water to their homes." The Coming Water Crisis The consequences of the increasing global water scarcity will largely be felt in the arid and semi-arid areas, in rapidly growing coastal regions and in the megacities of the developing world. Water scientists say that many of these cities already are, or will be, unable to provide safe, clean water and adequate sanitation facilities for their citizens -- two fundamental requirements for human well being and dignity.


Biology 447 ­ Environmental Microbiology

The problem will be magnified by rapid urban growth. In 1950, there were less than 100 cities with a population in excess of 1 million; by 2025, that number is expected to rise to 650. By the year 2000, some 23 cities -- 18 of them in the developing world -- will have populations exceeding 10 million. On a global scale, half of the world's people will live in urban areas. Some of the world's largest cities, including Beijing, Buenos Aires, Dhaka, Lima and Mexico City, depend heavily on groundwater for their water supply, but it is unlikely that dependence on aquifers, which take many years to recharge, will be sustainable. Groundwater from aquifers beneath or close to Mexico City, for example, provides it with more than 3.2 billion liters per day, but water shortages already occur in many parts of the capital. The UN University says that as urban populations grow, water use will shift from agricultural to municipal and industrial uses, making decisions about allocating between different sectors difficult. Water scarcity is aggravated by four principle human failures: · · · · Reluctance to treat water as an economic as well as a public good Excessive reliance on inefficient institutions for water and wastewater services Fragmented management of water between sectors and institutions, with little regard for conflicts between social, economic and environmental objectives; and Inadequate recognition of the health and environmental concerns associated with current practices.

"Instead, we must adopt a new approach to water resources management in the new millennium so as to overcome these failures, reduce poverty and conserve the environment -- all within the framework of sustainable development," says Dr. van Ginkel. The UN also warns of emerging trends that indicate the world is approaching a 'water crisis' in several regions -most notably the Middle East and North.

Water Wars? Hydrologists have carefully plotted the water equation. The amount of fresh water on the planet is finite -- less than a million cubic kilometers. That was enough in 1700, when less than a billion people shared the planet, and in 1900, when some 2 billion people were alive. Now, there are more than 6 billion people and the freshwater supply is stretched to the limit. By 2025, the same amount of water must feed an additional 3 billion people. The populations of water-short countries, today estimated to be 550 million, are expected to increase to 1 billion by the year 2010. Water shortages will be especially adverse for agriculture, which uses 70-80 percent of all available fresh water in the world. Without stepped up effort, "common sense tells us that national tensions over water could jump perilously," says Dr. van Ginkel. "Conflicts over water, both international and civil wars, threaten to become a key part of the 21st Century landscape. Geography will also contribute to the water conflicts. Nearly 47 percent of the land area of the world, excluding Antarctica, falls within international water basins shared by two or more countries. There are 44 countries with at least 80 per cent of their total areas within international basins. The number of river and lake basins shared by two or more countries is now more than 300. In Africa alone, there are 54 drainage basins covering approximately 50 per cent of the total land area of the continent, including their water resources. In the coming decades, accelerating environmental pressures could transform the very foundations of the international political system. There are at least 25 million environmental refugees today, a total to be compared with 22 million refugees of the traditional kind. They are mainly located in sub- Saharan Africa, the Indian subcontinent, China, Mexico and Central America. The total may well double by the year 2010, as increasing numbers of impoverished people press ever harder on their already degraded environments, including their water resources.


Biology 447 ­ Environmental Microbiology

Appendix I Estimates of Morbidity and Mortality of Water-Related Diseases Disease Morbidity (episodes/yr.) (or as stated) 1,000,000,000 Mortality (deaths/yr.) 3,300,000 Relationship of Disease to Water Supply and Sanitation Strongly related to unsanitary excreta disposal, poor personal and domestic hygiene, unsafe drinking water Strongly related to unsanitary excreta disposal, poor personal and domestic hygiene Strongly related to unsanitary excreta disposal and absence of nearby sources of safe water Strongly related to unsafe drinking water Strongly related to lack of face washing, often due to absence of nearby sources of safe water Related to poor water management, water storage, operation of water points and drainage Related to poor solid wastes management, water storage, operation of water points and drainage Related to unsanitary excreta disposal, poor personal and domestic hygiene, unsafe drinking water Related to the absence of nearby sources of safe water Related to poor water management, water storage, operation of water points and drainage Related to poor water management in large-scale projects

Diarrhoeal diseases

Infection with intestinal helminths Schistosomiasis Dracunculiasis Trachoma Malaria Dengue Fever Poliomyelitis

1,500,000,000 (1) 200,000,000 (1) 100,000 150,000,000


100,000 200,000 --1,500,000 20,000 --

400,000,000 1,750,000 114,000

Trypanosomiasis Bancroftian filariasis Onchocerciasis Totals


275,000 72,800,000 (1) 17,700,000 (1,4) 3.34 billion

130,000 -40,000 (5) 5.29 million

People currently infected. Excluding Sudan Case of the active disease. Approximately 5,900,000 cases of blindness or severe complications of Trachoma occur annually Includes an estimated 270,000 blind Mortality caused by blindness





From: World Health Organization


Biology 447 ­ Environmental Microbiology

Appendix II Access to safe water/sanitation, life expectancy and under-5 mortality

Country Population without access to water % (1990-96) Chad Ethiopia Zambia Papua New Guinea Angola Congo Madagascar Sierra Leone Cambodia Haiti Malawi Central African Republic Tanzania, U. Rep. Of Dem. Rep. Of the Congo Lao, People's Dem. Rep. Guinea Uganda Gambia Niger Turkey Benin Cameroon Nigeria Sudan Cape Verde Berundi Comoros Kenya Togo Namibia Sri Lanka Bhutan Myanmar Paraguay Swaziland Guyana Nicaragua Yemen Indonesia Lesotho Bolivia Mozambique Nepal Senegal Dominican Republic Ghana Morocco Mali 76 75 73 72 68 66 66 66 64 63 63 62 62 58 56 54 54 52 52 51 50 50 50 50 49 48 47 47 45 43 43 42 40 40 40 39 39 39 38 38 37 37 37 37 35 35 35 34 79 81 36 78 84 31 59 89 86 75 94 73 14 82 82 69 43 63 83 38 80 50 43 78 76 49 77 23 59 66 37 30 57 59 30 19 69 76 49 62 42 46 82 61 22 62 42 94 47.2 48.7 42.7 56.8 47.4 51.2 57.6 34.7 52.9 54.6 41 48.4 50.6 52.4 52.2 45.5 40.5 46 47.5 68.5 54.4 55.3 51.4 52.2 65.7 44.5 56.5 53.8 50.5 55.8 72.5 52 58.9 69.1 58.8 63.5 67.5 56.7 64 58.1 60.5 46.3 55.9 50.3 70.3 57 65.7 47 149 177 202 112 292 108 164 284 170 134 217 164 144 207 128 210 141 107 320 47 140 102 191 116 73 176 122 90 125 77 19 127 150 34 97 83 57 105 71 139 102 214 116 127 56 110 74 220 Population without access to sanitation % (1990-96) Life expectancy at birth (1995) Under 5 mortality (per 1,000 live births) 1996


Biology 447 ­ Environmental Microbiology China Peru Ecuador El Salvadore Argentina Mauritania Pakistan Brazil Guatemala Algeria Burkina Faso Iraq Malaysia Venezuela Zimbabwe Mongolia India Côte d'Ivoire Oman Samoa Mexico Philippines Colombia Syrian Arab Rep. Egypt Honduras Belize St. Vincent Thailand Djobouti Iran, Islamic Rep. Of Botswana Cuba Korea, Rep. Of Panama Lebanon Saudi Arabia United Arab Emirates Costa Rica Dominica Maldives Bangladesh Libyan Arab Jamahiriya Trinidad and Tobago Jordan Mauritius Tunisia South Africa Barbados Fiji St. Kitts and Nevis 33 33 32 31 29 26 26 24 23 22 22 22 22 21 21 20 19 18 18 18 17 16 15 14 13 13 11 11 11 10 10 7 7 7 7 6 5 5 4 4 4 3 3 3 2 2 2 1 0 0 0 76 28 24 19 32 68 53 30 17 9 63 30 6 41 48 14 71 61 22 6 28 25 15 33 12 13 43 2 4 45 19 45 34 0 17 37 14 23 16 20 34 52 2 21 23 0 20 47 0 8 0 69.2 67.7 69.5 69.4 72.6 52.5 62.8 66.6 66.1 68.1 46.3 58.5 71.4 72.3 48.9 64.8 61.6 51.8 70.3 68.4 72.1 67.4 70.3 68.1 53.8 68.8 74.2 72 69.5 49.2 68 51.7 75.7 71.7 73.4 69.3 70.7 74.4 76.6 73 63.3 56.9 64.3 73.1 68.9 70.9 68.7 64.1 76 72.1 69 47 58 40 40 25 183 136 52 56 39 158 122 13 20 49 71 111 150 18 53 32 38 31 34 78 35 44 23 38 157 37 50 10 7 20 40 30 18 15 20 76 112 61 17 25 23 35 66 12 24 38


Biology 447 ­ Environmental Microbiology

Module 1: Aquatic Microbiology

The main problem in studies of aquatic microbiology is methodological. It is extremely difficult to obtain a "representative" sample of water and it is difficult to extract and examine the microorganisms. A suitable sampling regime in freshwater or marine systems would be complex and time-consuming; this is because of the nature of the environment. There are two types of characteristics of the environment to be considered in the design of any sampling procedure; the MORPHOMETRIC characters and the PHYSICOCHEMICAL characters. · · MORPHOMETRIC characters have to do with depth, dimension, geology of shores, sediment distribution, currents, inflow and outflow of water, etc. PHYSICOCHEMICAL characters are those such as temperature profiles, pH, inorganic ion content and distribution, pO2, oxygen profiles with depth, etc.

A typical sampling problem would be that of sampling a like system to detect microbial activity or microbial pollution. The lake must be sampled in all areas. There are numerous schemes to divide lake systems into various subsections based on morphometric or physicochemical properties. The LITTORAL zone is around the edge of a lake and is where light penetrates to the bottom. It is usually colonized by macrophyte vegetation. Below the LIGHT COMPENSATION POINT (where the rate of photosynthesis is lower than the rate of respiration - usually about 1% of the light intensity of sunlight), the PROFUNDAL zone starts and continues to the sediment base. The combined littoral and limnetic zones is called the EUPHOTIC zone. The sediment is often called the BENTHIC zone. Another division is based on temperature profiles in lake systems. The lake is divided into EPILIMNION, THERMOCLINE and HYPOLIMNION. The thermocline is the dividing line and is the point where there is a rapid temperature drop with depth. The hypolimnion has low temperature and low oxygen concentrations because of low light penetration. Consequently, there is low photosynthetic activity. In HOLOMITIC lakes there is a periodic (usually once a year in temperate zone lakes) mixing of the two zones; MEROMICTIC lakes do not exhibit this mixing and the layers are usually renamed to MONOLIMNION (similar to the hypolimnion) and MIXOLIMNION (similar to the epilimnion) with the dividing zone being the CHEMOLIMNION containing the CHEMOCLINE (similar to the thermocline). The chemocline is permanent. Another division of lakes into different "types" is based on nutritional status. OLIGOTROPHIC lakes have low nutrient status and relatively low primary productivity. EUTROPHIC lakes have high nutrient status and high productivity. All of these factors have to be taken into account when designing a sampling scheme. The sampling stations should be spaced to include all of the various zones. This entails sampling around the littoral region and taking depth profiles to include the limnetic and profundal zones. Separate methods have to be used to sample the sediment or benthic zone. A typical sampling scheme used for Great Lake studies is shown below.


Biology 447 ­ Environmental Microbiology


Attempts can be made to count the number of viable bacteria of various kinds in water (e.g. total coliforms, fecal coliforms, Pseudomonas species, etc.) These tests are usually performed to count and identify pathogens or to indicate the possible presence of pathogens (e.g. coliform tests). However, they can also be used to count the viable bacteria, fungi, protozoa in water or sediment samples in efforts to understand the ecology and population dynamics of the microbial populations. Other methods attempt to count the "total" (as opposed to "viable") numbers of bacteria and other microorganisms. These methods (such as the Direct Method of fluorescence staining with acridene orange stain) do not distinguish between living and dead microorganisms in most cases. Yet other methods use a "surrogate" technique to estimate biomass. They would, for example, use the amount of chlorophyll as a measure of the presence of algal biomass in a water sample. Direct methods; Membrane filtration of standard volumes onto 8 µM, 2 µM, 0.45 µM and 0.2 µM Millipore or Nucleopore filters followed by direct observation with or without staining. Ultraviolet fluorescence of chlorophyll can be used to detect and count algal cells and fluorescence staining methods (e.g. acridine orange, anilinonaphthalene sulfonic acid dyes, fluorescein isothiocyanate, etc.) can be used for bacteria and fungi. In these cases, a black filter is required. The black filters can be produced from the normal cellulose filters by dyeing the filter with a black dye such as Irgalan black or Dylon 44. A very thorough treatment of methods in biomass detection in various systems is given in, Jones, J.G. (1979) A Guide to Methods for Estimating Microbial Numbers and Biomass in Fresh Water. Freshwater Biological Association, Scientific Publications No. 19. Other methods: See Atlas and Bartha - Microbial Ecology or other standard microbiological textbooks for details of the Most Probable Number (MPN) technique, viable counting procedures, and biochemical methods for estimating biomass such as Protein levels, ATP and adenylate charge, lipopolysaccharide (LPS), muramic acid, and chlorophyll . Chlorophyll concentration is particularly important in the aquatic habitat because of the occurrence of photosynthetic algae and bacteria. The chlorophyll can be determined spectrophotometrically or fluorometrically. Chlorophyll a is usually measured but the chlorophylls b and c can be assayed selectively at different wavelengths with a spectrophotometer. ACTIVITY MEASUREMENTS


Biology 447 ­ Environmental Microbiology

In many cases it is preferable to assay the microbial activity rather than the biomass. There are innumerable assays for microbial activity, but most rely on some measure of metabolic activity such as respiration, photosynthesis or biochemical pathway or product. The main categories are:

1. 2. 3. 4.

Rate of increase in the number of colonies on media Direct increase in numbers on membrane filters Estimation of rates of multiplication in continuous culture systems Measurement of metabolic activities such as photosynthesis, respiration, substrate utilization or product accumulation

1. Rate of increase in the number of colonies on media. Has the usual disadvantages associated with all viable counting procedures. It may be useful to follow populations during and after a specific treatment, especially in systems where the population is known or a pure culture is being investigated. 2. Direct increase in numbers on membrane filters. Microorganisms are concentrated on a membrane filter and incubated in contact with a natural water at the temperature of the environment. It has been shown that these microorganisms will maintain their growth rates for a short period of time under these conditions. See the procedure outlined below.

3. Estimation of rates of multiplication in continuous culture systems.


Biology 447 ­ Environmental Microbiology

The growth rates or generation times of various microorganisms in water systems can be estimated using continuous culture systems. If a chemostat is operated at very slow growth rates, the growth rate of bacteria from natural waters can be estimated using those waters as the medium, even if the dilution rate of the chemostat is higher than their growth rates. This is done by measuring the washout rate of the bacteria and calculating the growth rate from the difference between this (the washout rate) and the dilution rate of the chemostat. Even if only very slow growth is occurring, the bacteria will wash out more slowly than the dilution rate. 4. Measurement of metabolic activities such as photosynthesis, respiration, substrate utilization or product accumulation. A few examples of using metabolic activities of aquatic populations as indicators of activity are given below. Any metabolic activity can be used as a measure of activity it can be measured. a) Measurement of l4CO2 uptake into algal or bacterial tissues has long been used as a measure of photosynthetic activity and thus of primary production. The aquatic ecosystem is exposed to 14Clabelled CO2 for given time periods in the dark and in the light. A typical experimental outline would be; i) Take samples of the water, place in glass bottles. Place duplicate samples in dark glass bottles. ii) Add the radioactive isotope 14C in the form of NaHCO3 (approximately 50 100 microcuries in total, 2 5 microcuries per 125 mL of sample). iii) Suspend the bottles at the sampling depth to expose them to the same light and temperature conditions as the original sample. Leave for 20 min to 6 hours (depending upon activity). iv) Remove bottles and filter through a 0.45 µM membrane filter. v) Place filters in liquid scintillation fluid ("cocktail") and measure radioactivity incorporated into cells with a scintillation counter. The dark bottle gives an estimate of "dark fixation'' or heterotrophic CO2 fixation (see later). The light bottle gives an estimate of photosynthetic activity plus heterotrophic CO2 fixation. Note: all photosynthetic organisms (plants and bacteria) will be incorporated into this assay for photosynthesis and primary productivity. However, all photosynthetic bacteria except for the Cyanobacteria ("bluegreen algae") are obligate anaerobes, so their photosynthetic activity will not occur in aerobically incubated samples. Heterotrophic CO2 fixation ("dark fixation") The heterotrophic microbial assimilation of CO2 in water bodies is the result of the activity of different groups of microorganisms. Group 1. Heterotrophic bacteria which decompose proteins generally get 3 5% of their total carbon from external CO2 by heterotrophlc fixation. This group is the main contributor to the "dark fixation'' observed in the surface layers of water. Group 2. Some bacteria have an intermediary metabolism between true heterotrophs and true chemoautotrophs and usually oxidize low molecular weight compounds such as methane, methanol, and formic acid produced during the anaerobic metabolism of organic matter. They can assimilate between 30 90% of their total carbon from external CO2 Group 3. The true chemoautotrophs are not involved in "dark fixation'' to the same extent as the above but, in certain environments, they can be of significance. They derive all of their cell material carbon from CO2 and utilize ++ the energy gained from the oxidation of reduced compounds such as H2, H2S, NH4+ and Fe and Fe+++ . The last two groups are most active in the boundary layer between the aerobic and anaerobic zones in the water column and in the interface between the sediment and the water.


Biology 447 ­ Environmental Microbiology

The observed differences in the amount of "dark fixation'' by the different groups of microorganisms make interpretation of the data from these dark fixation experiments very difficult. In nonanaerobic waters it is usually found that there is a close relationship between the dark uptake of CO2 and the production of microbial biomass. Thus, heterotrophic CO2 fixation is usually between 5 and 7% of the total biomass. The formula below is used as a first approximation for the calculation of the estimate of bacterial production;

Biomass = Dark fixation of CO2.100gC/L/day 6

It has also been found that there is a relationship between the dark fixation of CO2 and the respiration rate of the heterotrophic bacteria. They assimilate CO2 at a ratio of approximately 7g CO2carbon per 1 mg 02 consumed. This particular estimate is very useful in oligotrophic waters where the normal Winkler titration method for the estimation of 02 concentrations is not sensitive enough to detect the very low rates of oxygen consumption. The problem of the estimation of the production due to the chemoautotrophic bacteria remains; it is relatively easy to estimate the bacterial photosynthesis by incubating the bottles under anaerobic conditions. The rest of the calculation is based on an indirect method. The total dark fixation of C02 measured by the 14C method is measured ( = A). The value for the CO2 assimilated heterotrophically is calculated ( = H). The value for chemosynthesis (Pc) is then calculated

Pc = A - H or Pc = A - 0.006 P g C/L/day

where A is total dark fixation g C/L/day) and P is the total production of bacteria estimated by direct counting methods. The value H can also be measured at the surface of the water where the activity of chemosynthetic bacteria is negligible. The graph below summarizes the situation in a water column;


Biology 447 ­ Environmental Microbiology

1 = Fixation by heterotrophs as calculated above 2 = Total dark CO2 fixation 3 = Plate count or direct count of heterotrophic bacteria

b) Measurement of uptake of organic substrates. Parsons and Strickland (1961) suggested that the rate of uptake of 14C compounds could be used as an indicator of heterotrophic activity in aquatic systems. Often glutamic acid is used as the substrate.

A typical formula would be:

v = f(S + A) t

where: v = velocity of uptake (mg per hour) f = fraction of the available labelled substrate taken up in time t A = added concentration of substrate carbon (mg/L) S = concentration of endogenous substrate (mg/L) t = time (hours) of incubation


Biology 447 ­ Environmental Microbiology

Often an arbitrary concentration of natural substrate is chosen (usually about 5 mg/L). Then the data can be plotted as a graph of added substrate concentration against uptake. (A versus v). This could be: 1. Linear (indicating that uptake is proportional to concentration) 2. Hyperbolic (uptake velocity approaches a saturation level with increasing substrate concentration) If the hyperbolic line is seen, then the kinetics of uptake can be handled using Michaelis-Menton kinetics. The most common format to express these kinetics is: v = Vmax x S Ks + S where v = uptake velocity S = substrate concentration Vmax = theoretical maximum uptake velocity Ks = substrate concentration where v = Vmax / 2 The actual methodology is very similar to that described above for l4C02 incorporation for photosynthesis assay, except that the substrate is an organic compound such as acetate or glucose. These two are often chosen because they are widely used by aquatic bacteria. c) Measurement of CO2 respired by heterotrophs in aquatic systems. Early methods of measuring uptake (4b above) failed to take account of the respired CO2. A portion of the added organic material (glucose, acetate, etc.) will be respired and the CO2 may even be reassimilated. A very simple method is used to trap and measure the respired CO2. It is captured on a filter paper soaked in phenylethylamine in a closed system and counted by liquid scintillation counting methods. [ See Biometer flask ] d) Oxygen uptake measurements. In eutrophic waters the overall respiration rate of the plankton and bacteria can be measured simply by incubating the samples in the dark (to preclude 02 evolution by photosynthesis) and determining the 02 levels at various times. The bacteria can be separated partially from the plankton by differential filtration through 8 or 5 µM Millipore filters. A typical procedure would be; 1. A sample of water is split into two. Half is filtered to remove phyto and zooplankton. The samples are placed in sterile dark glass oxygen bottles. 2. The initial levels of 02 and initial numbers of bacteria are measured (membrane filter method for bacteria and Winkler titration for oxygen levels). 3. The bottles are incubated in the dark (often in situ) for 24 or 48 hours and the final bacterial numbers and oxygen concentrations are measured. If it is assumed that the bacteria grow exponentially during the incubation period, it is possible to calculate the oxygen uptake per bacterial cell per hour during the incubation. It is much more difficult to calculate the oxygen


Biology 447 ­ Environmental Microbiology

uptake in oligotrophic waters. As detailed previously, the heterotrophic fixation of CO2 can be used to estimate this or the water can be filtered to concentrate the bacteria into a smaller volume. There remains the difficulty of separating respiration due to different types of organisms; filtration is not an excellent technique because of retention of materials and organisms in or on the filters. Also many algae are very small (the nanoplankton or picoplankton fraction) and may pass through the filters designed to remove algae. It is difficult to assess algal respiration in the light because of the evolution of oxygen by photosynthesis. Algae are also capable of heterotrophic growth on carbon substrates so that heterotrophic respiration cannot be used as a measure solely of bacterial respiration. Many of the methods described can be modified so that they only examine parts of the overall population. Any method which uses an added substrate can be modified to use a specific substrate for particular groups of organisms. For example, Thiobacillus sp. can be examined by adding 10 mg/L of thiosulphate to the sample and finding the rate of thiosulphate oxidation by iodometric titration methods. Control bottles of sterilized water would be used as the control. Hydrogen and methane oxidizing bacteria could be examined by adding the appropriate substrate. Many of the methods described can also be modified to work in sediment systems, even though these are usually more difficult to work with. They often have higher specific activities because of the greater concentration of bacteria and other microorganisms. Many techniques designed for soil studies can also be used for sediment studies.

BACTERIA AND OTHER MICROORGANISMS IN AQUATIC SYSTEMS Microorganisms are involved in many geochemical cycles in fresh and ocean waters and sediments. These include the carbon cycle, the nitrogen cycle, the sulfur cycle and the cycling of phosphorus. We will use the cycling of phosphorus as an example because it is probably less familiar to you than the others and because phosphorus is an important element in controlling the productivity in fresh water systems. We will examine the amounts and rates of phosphorus cycled in typical lake systems and look at "sources" and "sinks" for phosphorus. This is an extremely complex cycling process with many different intermediate forms of phosphorus. Therefore, it demonstrates the role of microorganisms in the lake and river ecosystems very well. Example of cycling in the aquatic environment; The phosphorus cycle. Phosphorus is thought to be one of the key minerals determining activity in aquatic systems. It is often the limiting nutrient for algal growth and proliferation and has been recognized as the key element in eutrophication. It is not a common element in the biosphere since it is easily precipitated from solution with calcium, magnesium and ferric ions. In addition, a large quantity is sequestered in sediments and minerals and is unavailable to organisms. For instance, apatite minerals (typically 3Ca3[PO4]2.Ca[FeCl]2) are ubiquitous in igneous rocks and are also found in metamorphic and sedimentary rocks. These rocks contain extremely large quantities of phosphorus. There are many other kinds of apatite minerals but all have the general formula (M)10(XO4)6(Z)2 where M is Ca, Na, K, or Mg;(X) is P, As, Si, S, Cr, or Ge and (Z) is F, OH, Cl or Br. The calcium phosphates are the most important form of apatites, but the roles of apatite (predominantly hydroxyapatite Ca10(PO4)6(OH)2 and fluorapatite Ca10(PO4)6F2) in aquatic systems are not well understood. It has been often stated that apatite have no role in the phosphorus cycle of lakes because of their low solubility. This ignores the fact that many bacteria and algae can liberate P from apatite when growing in close proximity or on the surface of apatite minerals. Phosphorus is essential for biological activity because of its role in energy transfer (ATP, ADP, AMP, etc.) and as phosphodiester bonds in nucleic acids. In contrast to other element cycles, the cycling of phosphorus does not involve a change in valence state (except in the rare case of phosphine (PH3) production). The usual transformations are between inorganic and organic forms of phosphorous, and between insoluble and immobilized forms and soluble and mobile forms.


Biology 447 ­ Environmental Microbiology

The main transformations in the phosphorus cycle are between insoluble organic phosphate, soluble organic phosphate, insoluble inorganic phosphate and soluble inorganic phosphate. A version of the cycle for aquatic freshwater lakes is given below. It shows the main sinks and sources of P in freshwater and also shows the rates of transfer between different components of the cycle.

This is a simplified steady state model of the important steps in the transformation of phosphorus in a lake system. The numbers in the boxes are g P/L lake volume and the exchange rates (on the arrows) are g P/L lake volume/day. A simpler version of this cycle has been used to calculate the actual rates of transfer: The "turnover time" ( time required for loss from a phase of as much phosphorus as is present in that phase) has been calculated as ranging from about 5 min for the exchange between dissolved inorganic phosphate and phytoplankton to many days for water-sediment exchanges (about 15 days for abiotic processes and about 3 days for bacterially medicated processes). The exchange between inorganic phosphate and dissolved organic phosphorus seems to be about 8 hours.


Biology 447 ­ Environmental Microbiology

Transfer rates of phosphorus NOTE: 1. The very high sediment levels of P 2. The very low exchange rates in most cases 3. Different "sinks" of P in different levels of lakes 4. The sediment-water exchange rate is low 5. The very large amount of P available in the sediment even if the input is removed 6. Most of the cycling is in the water column 7. The "residence time" or "turnover time" can be very low; one would expect very low orthophosphate levels in the water column if bacteria and phytoplankton are present 8. Inputs of orthophosphate (from sewage plants, detergents, etc.) are likely to have an immediate effect on the levels of phytoplankton in the lake 9. The importance of detritus in the system; detritus is dead organic material, very often partially decomposed.

POLLUTION OF AQUATIC SYSTEMS We will examine the role of pollution of water systems by microorganisms in two main areas. 1. Effects of pollution on microbiological processes in water. This concerns the environmental toxicology of toxics, and the effects of nutrients on microorganisms and their activities in water systems. 2. Pollution of water supplies by microorganisms (you should complete this module before beginning module four). This is concerned with the role that pathogenic microorganisms play in making water unfit for consumption (human or animal) or making it unfit for crop irrigation purposes. 1. Effects of pollutants on microbiological processes in water. The main groups of pollutants in aquatic systems are:


Biology 447 ­ Environmental Microbiology

a) Organic compounds are degradable compounds that cause Biological Oxygen Demand (BOD) problems through high oxygen consumption as they are degraded. This causes oxygen deficits and consequent modification of other microbial processes and also fish kills. b) Inorganic compounds result from mineralization processes of organic compounds - rock leaching, run off, fertilizers, etc. Most common are phosphate, nitrate sulfate, ammonium ions and carbon dioxide. Acid rain is actually an input of inorganic ions such as sulfate and nitrate. The total atmospheric input into an aquatic system such as the Great Lakes can be very substantial; a major city may contribute 300,000 lbs/sq. mile of dust per year. Acid mine drainage can contribute ferric and ferrous iron and sulfate plus some trace minerals (uranium, chromium, etc.). Toxic industrial wastes include cyanide, chromium, arsenic, cadmium, lead and mercury. Radioactive materials constitute a special case and can include radioactive carbon, phosphorus, radium, uranium, daughter fission products such as strontium. c) Recalcitrant or xenobiotic compounds; such materials as herbicides, insecticides, larvicides, nematocides, molluscicides, fungicides, etc., can leach into or be directly added to aquatic systems. Other recalcitrant organics such as ABS (alkyl benzene sulfonate) detergents, fluorinated hydrocarbons, polyethylene, poly vinyl chloride (PVC), industrial solvents such as toluene, benzene, xylene, and trichloroethylene, polynuclear aromatic hydrocarbons such as naphthalene, phenanthrene, etc., may also enter aquatic systems from various point sources or from the atmosphere. d) Heat input is not often regarded as pollution, but can have significant effects near large scale heat outputs such as those from hydro generating stations. It is impossible to examine all of these potential aquatic pollutants. Some of them will be dealt with in more detail in different sections of the course. Instead of examining a wide range of pollutants, we will examine the case where a nutrient becomes a pollutant. This example is that of phosphorus in lake systems leading to algal blooms, fish kills, "green" lakes, etc. - the process of eutrophication. We will also use this situation as a model for a broader model of what pollution really is - a measurable deleterious effect on an ecosystem. Case 1. Phosphorus as a pollutant in lake systems. Phosphorus input to lakes is a common cause of eutrophication. In a small paperback book called "The Algal Bowl", Jack Vallentyne a scientist at the Canada Centre for Inland Waters in Burlington - gives an interesting popular account of the ecological sociological, legal, industrial and scientific problems of phosphorus input into the Great Lakes. Some of the scientific concepts and conclusions are now out of date, but it covers the political, legal and sociological aspects very well. Phosphorus in water: In an ideal ecosystem, a balance between photosynthesis and respiration is maintained (i.e. between the production and destruction of organic matter and therefore between the production and consumption of oxygen and carbon dioxide). This can be expressed as a stoichiometric equation (equation 1);

A. is the ionic composition of seawater


Biology 447 ­ Environmental Microbiology

B. is the empirical formula for algal cells The formula above applies to marine systems. The freshwater equation would be similar. In simplified diagram form, the balance between photosynthesis and respiration becomes;

For sea water, it is possible to examine the stoichiometric relationships between the various elements in the equation. The mole ratios between the elements are; N : P : C = 16 : 1 : 106 O2 : N = 9 O2 : P = 138 In the system described above (equation 1), the elements are used in these ratios for photosynthesis in the photic zone. The dead algae settle out and are mineralized (respiration) and release the same ratio of nutrients back into the water. Oxygen is evolved during photosynthesis and consumed during respiration in the ratios given. The equation is a very simple one, but seems to be generally true for marine ecosystems. The result is that, as a result of photosynthesis, both N and P become exhausted simultaneously. Perhaps at some point P was limiting, but by N2 fixation and denitrification processes the N level was adjusted to the present levels. (Alternatively, the presently observed ratios of elements in marine algae adjusted to the preexisting levels of nutrients?). We can also express these molar ratios in graphical form by plotting correlations between concentrations of N, P, O, etc. Figure 1.6 shows the result of this correlation for oceanic waters and Figure 1.7 gives the same plot for a typical lake. Note that the line passes through the origin in Fig 1.6, indicating that the stoichiometric relationship holds and that neither N nor P is limiting growth in the ocean they are exhausted simultaneously!


Biology 447 ­ Environmental Microbiology

Oceanic waters - balance of N and P

Lake Constance water N-P balance

In Figure 1.7, the line for the N:P ratio does not pass through the origin; there is an excess of N in the water, therefore phosphorus content limits growth in this lake. This is also indicated by the fact that the N:P ratio is higher; 90 versus 16 for the oceanic waters. By plotting these ratios from different parts of the ocean and lakes at various times of the year, a very good measure of the nutritional status of the lake and the nutrient limiting growth can be obtained. Of course local variations occur, but in general terms the oceans do not exhibit P limitations while fresh water lakes often do. This argument is based on Equation 1. This equation assumes that all nutrients are recycled. The equation does not hold where the mixing of the water is rapid compared with the settling time of the dead cells. Spatial separation of the photosynthesis and respiration zones (as in deep lakes) also changes the observed stoichiometry. Due to the flow rate of the water, rivers and estuaries separate the two processes in time . A consequence of these concepts is that it is possible and often valuable to regard any pollution as simply a mechanism that causes an imbalance between P (photosynthesis) and R (respiration). Phosphorusproduced eutrophication of lake systems is one example of this. It allows additional algal biomass to be produced by photosynthesis and so causes algal blooms and oxygen deficits as the biomass decays. In a very deep lake, the settling of the algal biomass produces the hypolimnion; a layer with low oxygen concentration. P and R have been separated, so this can be regarded as an analogy to the pollution of water by phosphorus eutrophication processes. The physical separation of P and R zones means that the oxygen produced at the surface is not available for decomposition or respiratory processes. Another example is the estuary of a river; this is a natural concentration mechanism for nutrients coming down the river system (Figure 1.8). It is one of the most productive ecosystems known because of this concentration effect and the by the fact that the flow of the water separates the P and R zones in the river estuary.


Biology 447 ­ Environmental Microbiology

High Productivity in River Estuary The reverse situation (where there is a net loss of nutrients due to current flows) can also occur. The best example is in the Mediterranean Sea (Figure 1.9). The pumping action removes nutrients from the lower levels; this effectively separates P and R zones and leads to nutrient-poor water.

Low Productivity in the Mediterranean Sea Phosphorus and its effects have been studied mainly in freshwater lakes such as the Great Lakes and many European lake systems. It has become apparent that the lake cannot be considered in isolation; the surrounding drainage basis must also be considered. When this is done, it is possible to calculate the input of phosphorus into a lake system and to try to isolate the effects of humanity's activities in generating extra phosphorus input into the lake. It is then possible to model the results of limiting these inputs and finding the most efficient way to slow down or prevent the eutrophication process. Typical sources of phosphorus input into a lake system are:


Biology 447 ­ Environmental Microbiology

Calculated input of P from; Human waste via sewage Detergents Runoff Industrial Agricultural arable meadow Forest TOTAL

P - g/m2/year 0.08 0.04 0.01 0.01 --0.05 0.05 0.01 0.25g/m2/year

This table is based on a population density of 150,000/km2 with a total excretion each of 3 g/day (representing a major city). To obtain some idea of the effect on a lake in the drainage area the input in g/m2/year is multiplied by the factor drainage area lake surface area. This gives the total phosphorus loading for the lake in g/m2year. For probable effects of added inorganic soluble phosphate on the eutrophication process, see the phosphorus loading and transformation model given previously . Control of phosphorus eutrophication 1. Legislated restriction of phosphorus input by, for example, improving sewage treatment facilities or removing phosphorus detergents from the market. Improvement of farming practices so that excess fertilizer use is limited. 2. Ecosystem control; this is a combination of methods which can apply to any "pollution problem." There are some very basic and simple ecological principles involved in ecosystem control measures. The background to the process is to consider the whole lake/drainage area ecosystem and to modify (or control) key elements in the ecosystem to limit input of phosphorus into the lake. The rationale is based on a very early concept in ecology; the relationships between gross production levels, species diversity and net productivity. These factors interact in a reasonably predictable fashion summarized in the following graph;


Biology 447 ­ Environmental Microbiology

The stability and productivity of an ecosystem can be defined on many factors; Unstable Homogeneous High metabolic waste Simple food chains High production Weak selection pressure Few energy pathways Stable Heterogeneous Metabolites recycled Complex food chains Low production Strong selection pressure Multiple energy pathways

Very often, the unstable ecosystem is in the first stages of development whereas the stable ones are more mature. This is true of microbial, plant, terrestrial and aquatic ecosystems. Phosphate (and many other types of pollution) causes the ecosystem to revert to a less stable phase with low species diversity and therefore fewer metabolic pathways; the productivity then increases. A lake system cannot be studied in isolation. It must be considered as part of the larger ecosystem in the drainage area of the lake. Activities (such as agriculture and deforestation) in these areas would lead to increased phosphorus input and a less stable lake ecosystem. In fact, agriculture is the ultimate extension of this ecosystem control process; it is a monoculture (zero species diversity) with extremely few metabolic pathways, and it is very unstable. A large amount of energy is "pumped" into the agricultural system and much of it is wasted. Deforestation changes a stable, mature ecosystem into a very unstable one with high waste and a large amount of runoff of nutrients. Measure to increase the stability of aquatic ecosystems 1. Reduce waste input, eliminate addition of nutrients 2. Restrict monoculture 3. Reduce pesticide use 4. Reforest drainage area 5. Control erosion


Biology 447 ­ Environmental Microbiology

6. Increase biological diversity; a) Establish new ecological niches b) Maintain lake stratification c) Selectively harvest species The opposite actions reduce stability and cause eutrophication; 1. Add nutrients, mix lake system, impose turbulence. 2. Increase use of pesticides to restrict species diversity. 3. Reduce biological complexity by; a). use of algicides b). remove ecological niches c). excessive harvesting. Mixing a lake to remove stratification can either promote or reduce the stability of the lake ecosystem. This is because destratification can have many effects; a) destroys ecological niches b) reduces gradient of chemicals and nutrients c) shortens food chains (higher energy per biomass, higher productivity) d) brings P and R into better balance, reducing net productivity e) reduces effective residence time of nutrients (e.g. PO4) decreasing total nutrient reserve.


Biology 447 ­ Environmental Microbiology


Biofilms are composed of populations or communities of microorganisms adhering to environmental surfaces. These microorganisms are usually encased in an extracellular polysaccharide that they themselves synthesize. Biofilms may be found on essentially any environmental surface in which sufficient moisture is present. Their development is most rapid in flowing systems where adequate nutrients are available Biofilms may form: on solid substrates in contact with moisture. on soft tissue surfaces in living organisms. at liquid air interfaces. Typical locations for biofilm production include rock and other substrate surfaces in marine or freshwater environments

Microbial Mats, on the other hand, are specialized microbial communities composed mainly of photosynthetic procaryotes. Thus the principle distinction between microbial mats and other biofilms is their dependence on photosynthetic primary productivity as their source of energy

Biofilms are also commonly associated with living organisms, both plant and animal. Tissue surfaces such as teeth and intestinal mucosa which are constantly bathed in a rich aqueous medium rapidly develop a complex aggregation of microorganisms enveloped in an extracellular polysaccharide they themselves produce.

Here, human dental plaque has been exposed to 5 % sucrose for 5 minutes, after which Gram's iodine (0.33% Iodine in 0.66% KI) was applied. The sucrose solution was applied to the left central incisor (which appears on the right) while the right central incisor served as a control.


Biology 447 ­ Environmental Microbiology

How do biofilms form?

Typically, within minutes, an organic monolayer adsorbs to the surface of the slide substrate. This changes the chemical and physical properties of the glass slide or other substrate. These organic compounds are found to be polysaccharides or glycoproteins. These adsorbed materials condition the surface of the slide and appear to increase the probability of the attachment of planktonic bacteria

Free floating or planktonic bacteria encounter the conditioned surface and form a reversible, sometimes transient attachment often within minutes. This attachment called adsorption is influenced by electrical charges carried on the bacteria, by Van der Waals forces and by electrostatic attraction although the precise nature of the interaction is still a matter of intense debate. In some instances, as for example, in the association between a pathogen and the receptor sites of cells of its host there may be a stereospecificity which though still reversible is stronger than that achieved strictly by ionic or electrostatic forces. If the association between the bacterium and its substrate persists long enough, other types of chemical and physical structures may form which transform the reversible adsorption to a permanent and essentially irreversible attachment. The final stage in the irreversible adhesion of a cell to an environmental surface is associated with the production of extracellular polymer substances or EPS. Most of the EPS of biofilms are polymers containing sugars such as glucose, galactose, mannose, fructose, rhamnose, N-acetylglucosamine and others. This layer of EPS and bacteria can now entrap particulate materials such as clay, organic materials, dead cells and precipitated minerals adding to the bulk and diversity of the biofilm


Biology 447 ­ Environmental Microbiology

habitat. This growing biofilm can now serve as the focus for the attachment and growth of other organisms increasing the biological diversity of the community.

Colonization and adsorption to a surface are followed by the matrix production and development of the water channels.

A mature biofilm in a flowing environment may lose bacteria to the surrounding water


Biology 447 ­ Environmental Microbiology

The biofilm may exhibit "streamers" where these cells are being lost together with some of the matrix materials

Scanning electron micrograph (SEM) of a Pseudomonas aeruginosa PANO67 biofilm that was grown in a square glass tubing flow cell. The flow cell was 3 x 3 mm across and 20 cm in length. The biofilm was grown under high shear, turbulent, flow with a flow velocity of 1 m/s (a corresponding flow rate of 540 ml/min). The arrows indicate the direction of flow in the flow cell. The flow cell was positioned in a recirculating loop attached to a chemostat. Nutrients (a minimal salts with glucose as the sole carbon) were delivered by peristaltic pump and the recycle flow rate was controlled with a vane head pump.


Biology 447 ­ Environmental Microbiology

Enlarged view of "A" above showing details of "streamers" By: Paul Stoodley, Center for Biofilm Engineering, Montana State University, Bozeman, Montana; Frieda Jørgensen, Food Microbiology Research Unit, Public Health Laboratory, Exeter, UK; Hilary M. Lappin-Scott, Environmental Microbiology Research Group, Exeter University, School of Biology, Exeter, UK

Biofilms can show surprising variation in environmental conditions within very short internal distances. Large oxygen variations occur within a few hundredths of a millimeter and significant diffusion gradients of nutrients can also be established if they are used by the bacteria in the biofilm. Another effect is that of protection of the bacteria deeper in the biofilm against toxic chemicals. These images on the next slide are micrographs of biofilm cross-sections composed of two bacterial species (Klebsiella pneumoniae and Pseudomonas aeruginosa) with progressive exposure to disinfectant. Untreated biofilm samples (control) and those following exposure to a low level (4 mg/L) of chloramine were stained with two fluorogenic compounds, frozen and cut into thin (5 µm) sections that were observed by fluorescence microscopy and photographed. The base of the biofilm that rests on the substratum is at the bottom of each image; the biofilm surface that is exposed to the overlying bulk fluid is the upper aspect of each picture. A combination of 4'6-diamido-2-phenylindole (DAPI) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) was used to stain the bacterial cells. This combination of stains distinguishes individual cells with active respiration (red-gold) from those that are non-respiring (green).


Biology 447 ­ Environmental Microbiology

Figure 1 shows the untreated control biofilm which is predominantly composed of respiring bacteria. .

Figure 2 shows the biofilm which is predominantly composed of respiring bacteria, after 30 min. exposure to disinfectant

Figure 3 shows the biofilm after 60 min. exposure to disinfectant. More bacteria have lost respiratory activity and the biofilm has become thinner.

Figure 4 shows the biofilm after 90 min. exposure to disinfectant Gordon McFeters, Center for Biofilm Engineering, Department of Microbiology, Montana State University, Bozeman, Mont., USA


Biology 447 ­ Environmental Microbiology

Biofilms may also play a role in the biodegradation of resistant chemicals since they can consist of stable aggregations of many different organisms. An example might be that of PCB degradation (below) carried out by a consortium of different microorganisms Groups of bacteria (consortia) grown on surfaces (biofilms) have been shown to be shock-resistant relative to cultures of a single type of bacteria. Growth on a surface is advantageous when compared to that in liquid because it increases the local density of the organisms, may facilitate the concentration of nutrients (especially important in low nutrient environments such as contaminated subsurface waters) and reduce exposure to shear stresses. In addition, consortia have diverse metabolic capabilities simply as a result of the genetic diversity present within the biofilm conferring to them a selective advantage over individual organisms within the environment.

Biofilms are present in groundwater and may play a role in microbial activities there.

Visualizing bacteria in environments dominated by non-biological particles is very difficult. When the field is stained with acridine orange, a stain that reacts with nucleic acids, and viewed through a fluorescent microscope, the bacteria are clearly visible as yellow/green rods William Ghiorse, Section of Microbiology, Cornell University, Ithaca, N.Y., USA


Biology 447 ­ Environmental Microbiology

Biochemistry and Interaction in Biofilms: Some recent observations: Bacteria such as Pseudomonas aeruginosa have genes that are turned on in about 15 mins after the attach to a surface. - one gene is algC and is needed to make alginate - one of the components of the polysaccharide matrix material. Many biofilm bacterial cells typically make dozens or hundreds of proteins not found in "free-floating" cells. The cells signal to each other as the approach the "quorum" or number required to initiate biofilm formation. It seems as if a certain number of cells are needed to produce enough of the signal molecules to "switch over" the cells to matrix production - this is the "quorum".

In Pseudomonas aeruginosa and similar cells, the signal molecule is known - they are acylated homoserine lactones. If the gene for these compounds are missing - no biofilms are formed. Some red algae produce compounds called substituted furanones - they have almost no biofilm on their fronds in sea water. It seems that they block the signal transmissions due to the acylated homoserine lactones since they bind to the receptor sites normally used for signaling.


Biology 447 ­ Environmental Microbiology

Other Issues in Water Pollution (Module 1) See the Powerpoint Slide Presentation in Module 1

Urbanization General Population Growth Increased Run-off and Pollution Non-point Pollution Sources (eg Agriculture) Pesticide Use (Module 5) Toxic Organic Chemicals Industrial Effluents Organic Loading Wood burning for Fuel (e.g. East African lakes - Lake Victoria) Deforestation Microbiological Contamination (Module 4)

Lake Erie - Progress and Regression

See the Powerpoint Slide Presentation in Module 1


Satellite and high altitude photography has provided a means to survey the large water areas of the planet for various disturbances and pollution events. This is an efficient way to monitor very large areas on a constant basis; many satellites make multiple passes per day over given areas. Some progress around the earth's surface covering even larger areas, but at less frequent intervals. The positions of thousands of these satellites is known with extraordinary precision, and their paths can be predicted years in advance. There are many different "kinds" of satellites circling the globe. Some are military, some civilian, some for communications (TV, phone systems, data transmissions, etc.), some for global positioning systems (GPS), some for photography in visible and non-visible wavelengths and some use radar wavelengths to penetrate cloud cover. The most useful for environmental scientists are those that examine multispectral wavelengths (ultra-violet to microwaves including visible light and infra-red wavelengths). Some examples of typical systems are given below. They include photographs showing crop coverages (Landsat), oil pollution, algal blooms in lakes, water content of soils (RadarSat), etc. Some are included just because they are interesting - the declassified military photographs from the United States fall into that category. Remember that the declassified ones are decades old - think what present technology can do A search of the Internet using the terms "satellite" and "image" (combined together) yields thousands of such images that are available on the WWW. Many images are for sale including very detailed SPOT imagery from France and detailed images from Russia. Vice-President AL Gore in the US wants to launch a satellite for public use where anyone can examine all of its images of the earth.


Biology 447 ­ Environmental Microbiology

Some examples of remote sensing images are given in the following list. Some are files stored on this server (local) and some are to remote links on the WWW.

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

NASA's "Instrument and Sensing Technology" Description (remote) RADARSAT images of parts of Canada (local) Description of RADARSAT technology and applications (remote) LANDSAT Thematic mapping images (local) List of LANDSAT images of Ontario Description of LANDSAT satellite technology and applications (remote) SPOT satellite images Description of SPOT satellite technology and applications (remote) Declassified satellite imagery from the US (remote) Other Satellite Images (local) a. Bangkok b. East Coast - US - Chlorophyll Levels in water 11. New High Resolution Satellites and public sales of images

See the Powerpoint Slides of Remote Sensing & Satellite Imagery

Regulations, Guidelines and Laws

Water is regulated in Canada at the provincial level except for cross-border issues with the United States. One example (of the exception) is the Great Lakes Commission, which oversees items of interest to both countries in the Great Lakes system. Other than these exceptions, the provincial governments have jurisdiction over most matters dealing with water pollution, contamination, health issues and with protecting water bodies and water quality. The situation in Canada is improving so that there will be more consistency between provinces in their guidelines and regulations for water-associated issues. There are deliberate attempts to harmonize regulations between most provinces. One of these efforts, perhaps the most significant, is the Canadian Council of Resource and Environment Ministers. This is a high level group, producing regulations that will become provincial guidelines when provincial legislatures agree to them. A summary of the Ontario regulations below is followed by an excellent survey and comparison of all of the regulations and guidelines in Canada and parts of the United States. This document was produced in the early stages of the Fraser River Basin Action Plan in British Columbia. This was a coordinated effort to deal with water quantity and quality issues in the Fraser River Valley together with residential and commercial development and land-use issues and problems. Note the wide differences in standards, guidelines and regulations Ontario Drinking Water Objectives, Revised 1994 Ontario Regulations - Updates 2000 Canadian Water Quality Guidelines: Detailed Treatment of Individual Toxicants in Water Bodies A review of Environmental Quality Criteria and Guidelines for Priority Substances in the Fraser River Basin by MacDonald, D. D. 1994. A review of environmental quality criteria and guidelines for priority substances in the Fraser River basin. Environment Canada, North Vancouver, B.C. DOE FRAP 1994-30 A compound specific set of Adobe Acrobat files on toxicants An excellent, very comprehensive survey of many different literature sources of environmental criteria, guidelines and regulations. Has a comprehensive set of supporting Tables, Documents and Information - by Haines, M. L., K. Brydges, M. J. MacDonald, S. L. Smith, and D. D. MacDonald. 1994. A review of environmental quality The Ontario regulations (objectives)


Biology 447 ­ Environmental Microbiology

criteria and guidelines for priority substances in the Fraser River basin. Supporting documentation. Environment Canada, North Vancouver. DOE FRAP 1994-31

Key Points 1. Morphometric and physicochemical characteristics of lakes and rivers determine the sampling

schemes required for adequate sampling

2. Zonation of lakes by temperature and oxygen levels: photic zone, epilimnion hypolimnion,

chemocline, thermocline, etc.

3. Sampling schemes based on all parameters and repeated at intervals 4. Types of methods available for determining microbial presence, activity and distribution 5. Biomass determinations 6. Chlorophyll for algal measurements 7. Most Probable Number method 8. Plate counts 9. ATP measurements 10. Fluorescence methods for bacteria and algal cells 11. Radioisotope tracing methods: 1. Photosynthetic activities in water samples 2. Heterotrophic (dark fixation activity) carbon dioxide uptake 3. Respiration by carbon dioxide evolution 4. Uptake rates of organic molecules (sugars, amino acids, etc) as measure of activity Key Concepts in Lectures

· · · · · · · · · · · · Variability of aquatic habitat - rapid change in characteristics and therefore microbial components. Use of activity measurements and biomass measurements to follow important microbial functions in water ecosystems. Importance of the effects of the choice of methods on the results that are obtained. Zonation phenomena and interactions between different layers Concepts of biogeochemical cycling of carbon, nitrogen and phosphorus Detail of phosphorus cycling in aquatic systems Effects of phosphorus on eutrophication progress in lakes. "Balance" between N P O H in lake systems, eutrophic systems and marine systems. Phosphorus in Lake Erie - amounts, effects and control measures Ecosystem control concept. General application of Odium's diagram to pollution from any source. Applications, problems and exceptions.


Biology 447 ­ Environmental Microbiology

Module Two - Marine Microbiology


The marine environment is by far the largest part of the biosphere, being about 97-98% of all the water on earth. Approximately 75% of the ocean is below 1000 M depth and is constantly cold (about 3C on average). The deepest part of the oceans (in the various "trenches" in the sea floor) is about 11,000 M deep and is at a pressure of about 1000 atmospheres. (1 atmosphere increase in pressure for each 10 M in depth).

These large pressure differences lead to different microorganisms being present at different depths in the ocean. Some, the barophiles, can be moderate (growing best at 400 atm but still able to grow at 1 atm) or extreme (growing only at higher pressures). Yet other bacteria are barotolerant (growing best at lower pressures, but able to tolerate up to 400 atmospheres in some cases). The very high pressures found at depth in the sea affect many different biochemical and biological processes. Although most research has been in the near-shore and estuarine marine environments, there is increasing interest in the off-shore and pelagic ocean. If we consider the fact that the true off-shore is where the ocean is plus 1000 metres, then 62% of the Earth's surface is in the pelagic and deep sea region. In terms of volume, this is about 98% of the world's oceans. Microorganisms are involved in most of the geochemical cycling in the oceans, but surprisingly little is known of the activities at this depth.


The composition of seawater is approximately 36 parts per thousand of salts, Their composition is: ELEMENT Na

++ ++

PERCENTAGE 30.4 3.7 1.2 1.1 0.04 0.07 55.2 7.7 0.19 0.35


Ca++ K+ Sr




CO3 and HCO3 (mainly HCO3)

Nitrogen and phosphorus are not major elements in oceans, but are present along with almost every other element in sufficient quantity for biological activity. The pH is between 6.5 and 8.3 with an average that is slightly above pH 7.0. pH values rarely have an effect on availability of ions and elements except for the CO3- and HCO3- system:


Biology 447 ­ Environmental Microbiology

This system is inherently self-balancing; the acid production (H+) releases the Ca++ and CO3 from the calcium carbonate deposited as a precipitate as a result of the change in pH to more alkaline conditions. There is then excess base that reacts in the borate buffer system to release the H+

Dissolved Gases

Carbon dioxide input is the most important gaseous exchange. The total carbon dioxide content of the atmosphere is about 600 billion tons. There is at least 100 times this in seawater, present as carbon dioxide and carbonate and bicarbonate ions. Both carbon dioxide and bicarbonate ions are utilized by plants for growth. The availability of the two species depends on the pH of the sea water. The equilibrium between carbon dioxide, carbonate and bicarbonate (above) is in favor of bicarbonate at pH levels near neutrality but at pH 9.4 carbonate is present in large quantities and is precipitated as carbonate by the calcium ions in sea water. Photosynthesis stops in sea water at pH 9.4 even in bright light, due to this precipitation. In fresh water, due to the low calcium levels, photosynthesis can continue at pH levels up to 10.1.

Large Scale Oceanic Currents

Controlled by such factors as the rotation of the earth, sunlight heating surface layers of the ocean, wind and land masses, very large scale currents are present in the ocean. There is a global circulation of ocean waters. In many cases, the deeper currents move in different directions from surface currents. In some areas of the world, there are "upwellings" of deeper currents that recirculate nutrients to the surface layers. This typically causes fast phytoplankton growth and consequent large fish populations. The anchovy harvest off the coast of South America is due to this upwelling of the Humboldt current.



Biology 447 ­ Environmental Microbiology

The temperature is usually in the range of 2 to 40C. The growth of marine bacteria is usually optimum at 18C. A higher temperature (30C for 30 min) often inhibits growth. Most of the ocean is at the lower temperature range . (around 2C). In the different currents at different depths, there are often sharp and clear differences in temperature


Pressure increases at the rate of 1 atmosphere for every 10 metres depth increase. In the deepest parts of the ocean, (at depths of 10,000 metres) the pressure can exceed 1000 atmospheres. Many marine bacteria in the deeper regions of oceans are adapted to these high pressures; they are barophilic and cannot tolerate lower hydrostatic pressures. These pressures are high enough to affect biochemical reactions due to size differences in the reactants and products; most non-barophilic marine bacteria show an increase in biochemical reaction rates at pressures around 100 atmospheres, but show a decrease in these rates as the pressures increase above 100 atmospheres.

Volcanic Vents

In the 1970s, a new community was discovered in the ocean. It was formed around thermal volcanic vents in the ocean floor and obtained its energy from the chemoautotrophic metabolism of the sulfide-rich water issuing from the vents. Sea water penetrates down several kilometres into the hot basalt layers in the Earth's crust around the spreading centres for the Earth's plates. Plate tectonics has shown that these plates are moving apart at rates of 215 cm per year as the continents move apart. The sea water in contact with the hot basalt may have a temperature of +360C (higher than 100C because of the pressures at these depths). It emerges on the sea floor as warm (825C) or hot (160-350C) water from vents. The sulphide content can be as high as 160 mmol L-1

Image from NOAA at:

There is an enormous quantity of life surrounding these vents, much more than could be supported by the input of organic materials from photosynthesis in the photic zones above. Large mussels and vestimentiferan tube worms


Biology 447 ­ Environmental Microbiology

(a new family Riftidae) up to 2.6 metres long and 5 cm thick are found around the vents. Giant white clams have been observed close to the vents. There is also a very specific set of invertebrate larvae, siphonophores and active fish populations associated with the vents.

A "black smoker" vent: From the "Ocean Planet" site The energy source is the aerobic and anaerobic chemosynthetic reduction of carbon dioxide to organic carbon using geothermally reduced inorganic compounds as the energy source. This chemosynthetic system is unique - it gets energy from a terrestrial source - not from sunlight either directly or indirectly.

Characteristics of Marine Bacteria

Many marine bacteria have an absolute requirement for sodium, potassium and magnesium ions. Some also require chloride ions and ferric iron. Since the organic matter in the ocean is produced in the top 100 to 300 metres, and over 80% of this material is metabolized before it sinks below the photic zone, there is little organic material reaching the bottom water layers. It is constantly metabolized as it sinks through the water column. Any remaining residual organic materials are usually metabolized in the topmost sediment layers. Because of this low organic matter concentration in the deeper levels of the ocean, most bacteria there have evolved to exist on such low levels; the heterotrophic bacteria in the pelagic and deep ocean are often oligotrophic (adapted to low organic matter levels) and are inhibited by high organic matter concentrations. Many are also psychrotrophic (grow at low temperature 0C and also above 20C) or psychrophilic (grow at 0C but not above 20C) due to the prevailing selection pressure for organisms surviving and growing at the normal low ocean temperatures


Biology 447 ­ Environmental Microbiology

AN ASSESSMENT OF POLLUTION IN THE OCEANS Extracted and Modified from: A Sea of Troubles

by: IMO/FAO/UNESCO-IOC/WMO/WHO/IAEA/UN/UNEP - Joint Group of Experts on the Scientific Aspects of Marine Environmental Protection (GESAMP)

Historically, concern about the state of the oceans has mainly been generated by pollution. Over the last decades, increasing understanding of the seriousness of other threats - such as overfishing and the destruction of habitats ­ and of the damage they cause, has tended to overshadow it. But it has enormous effects on health and the environment. Many studies show that respiratory and intestinal diseases and infections among bathers rise steadily in step with the amount of sewage pollution in the water. They demonstrate, too, that bathers are at risk even in lightly contaminated waters that meet the pollution standards laid down by the European Union and the US Environmental Protection Agency. A recent WHO report has estimated that one in every 20 bathers in "acceptable waters", will become ill after venturing just once into the sea.


Biology 447 ­ Environmental Microbiology

The GESAMP/WHO study - based on global estimates of the number of tourists who bathe, and WHO estimates of the relative risks at various levels of contamination - estimates that bathing in polluted seas causes some 250 million cases of gastroenteritis and upper respiratory disease every year. Some of these people will be disabled over the longer term. The global impact can be measured by adding up the total years of healthy life that are lost through disease, disability and death using a new measurement - the Disability Adjusted Life Year (DALY) ­ developed by WHO and the World Bank. When this is done, the world-wide burden of disease incurred by bathing in the sea, adds up to some 400,000 DALYs, comparable to the global impacts of diphtheria and leprosy. It is estimate to cost society, worldwide, about US $1.6 billion a year1 The toll from consuming contaminated shellfish is even greater. One study suggests that seafood is involved in 11 per cent of all the outbreaks of disease carried in food in the United States, 20 per cent of them in Australia, and over 70 per cent in Japan, which has a particularly strong tradition of eating raw fish and shellfish. Pathogenic bacteria can survive in the sea for days and weeks; viruses can survive in the water - or in fish and shellfish - for months. The particularly virulent infectious hepatitis virus - which has caused many outbreaks of the disease associated with eating shellfish - can remain viable in the sea for over a year. Shellfish, like oysters, mussels, clams and cockles, feed by filtering huge amounts of seawater - and can concentrate viruses and bacteria a hundredfold from the water in which they live.

A series of studies has found viruses in about a fifth of the shellfish taken from waters that meet US bacteriological standards for growing and harvesting them. There is strong evidence that fresh shellfish - on sale for food -frequently contain enough viruses to make many of those who eat them ill. They are often eaten raw, or after only a light steaming which is not enough to kill most of the viruses or bacteria. One US study suggested that one in every hundred people eating relatively lightly contaminated raw shellfish will be infected with a moderately serious intestinal virus disease; the risk rises to up to 50 in a 100 if the virus is highly infectious. Other studies in both the United States and the United Kingdom suggest that a quarter of those who are taken to hospital suffering from infectious hepatitis - a disease that can confine sufferers to bed for two to three months - have caught it from eating raw or lightly steamed shellfish. Some eight billion meals of shellfish are thought to be eaten worldwide each year. The GESAMP/WHO study estimates that eating sewage-contaminated shellfish raw causes some 2.5 million cases of infectious hepatitis each year. Some 25,000 of the victims die and another 25,000 suffer long-term


Biology 447 ­ Environmental Microbiology

disability from liver damage. The global burden on human health equals some 3.2 million DALYs a year - comparable to the worldwide impact of all upper respiratory infections and intestinal worm diseases ­ and costs world society some US$ 10 billion annually. This new evidence of the dangers of sewage pollution is just one example of a general reappraisal of the relative importance of different pollutants of the sea. Some of those once thought to be the most damaging worldwide are now believed to be much less important, either because more is known about them or because they have been brought under control. · The supposed effects of man-made radionuclides discharged into the sea still loom large in the minds of the general public and politicians. Although threats from accidental releases cannot be ruled out, radionuclides now probably worry scientists less than any other category of marine pollutants. Similarly, highly publicised and exaggerated concerns about the extent of contamination of the seas and their life by heavy metals cannot be justified; it is probably far less serious than pollution by nutrients and some persistent organic chemicals. The effects of even the most dramatic oil spills are generally localised; gross pollution from them disappears relatively rapidly, though some subtle effects may last for decades, with enormous economic costs. Some eight billion meals of shellfish are thought to be eaten worldwide each year. The GESAMP/WHO study estimates that eating sewage-contaminated shellfish raw causes some 2.5 million cases of infectious hepatitis each year. Some 25,000 of the victims die, and another 25,000 suffer long-term disability from liver damage. The global burden on human health equals some 3.2 million DALYs a year - comparable to the worldwide impact of all upper respiratory infections and intestinal worm diseases ­ and costs world society some US$ 10 billion annually.

· · ·

Until recently, most attention concentrated on pollutants which directly or indirectly poisoned sea life and those consuming it - or were suspected of doing so. Less attention was paid to the potential effects of the persistent organic chemicals, some of which may have much more subtle, but possibly even more damaging effects. These include changes in the structure and function of communities of marine life, through disrupting reproduction and altering behaviour, and effects at the molecular level, such as causing cancer or mutations or disrupting endocrine systems. Evidence that concentrations of these substances now in the marine environment are causing such effects is mostly inconclusive. Risks to human health usually only occur where concentrations are high, or where people are exposed to them in unusual ways, such as in the Arctic where fish and seafood form an extremely high percentage of the diet. It is now well-established that some chemicals can harm the endocrine systems of a wide range of wildlife species, both on land and at sea, and may give rise to strange `genderbending' effects. Tributyl tin, for example - which has been widely used in anti-fouling coatings on ships and in fish farming appears to have made female sea snails grow false penises, and to have severely affected oyster fisheries in some areas. Its use has now been restricted in most developed countries, but it is still being traded in some markets. It is possible that other environmental contaminants could `sneak up on us', causing further unexpected effects.

Key Points

1. Extent of marine environment 2. Main distinguishing environmental features:


Biology 447 ­ Environmental Microbiology

Salinity Temperature Pressure Dissolved Gases Chemistry and Ionic balance (buffer systems for pH) 3. How these features affect marine microbial growth and activity 4. Deep sea thermal vents and their importance


Biology 447 ­ Environmental Microbiology

Module Three - Estuarine Microbiology


The following general description of estuaries has been taken from an Australian Web site

General Description

An estuary has been defined by Pritchard (1967) as "a semi-enclosed coastal body of water, which has a free connection with the open sea, and within which seawater is measurably diluted with freshwater derived from land drainage". It is an intermediate habitat between the sea, the land and freshwaters, and is an extremely dynamic ecosystem. They are found where a river finally meets the sea, and the waters are usually calm. Estuaries have been claimed to be the most productive natural ecosystems in the world, and provide food for a variety of organisms. They are important in the commercial fishing industry, as many species use the estuary as a breeding and nursery area. The organisms that live in the estuary must be able to cope with the changes in salinity, temperature and light levels.

Importance of Estuaries

Waste Assimilation

Estuaries are places where fine sediments are deposited. River flow contains a large amount of fine sediments and the often abrupt change in current flow means that these sediments can settle out of the water column. Other processes encourage sediments to settle out. Chemical processes occur when fresh and salt water meet. These processes cause sediment and clay particles to flocculate and precipitate out of the water column. This is how many heavy metals are prevented from reaching the ocean. These potentially dangerous substances often remain "locked" to the sediment particle and become active again only when natural processes release them. This occurs gradually and acts to regulate the circulation of the metals in the environment. It is only when unnatural human activities disturb these sediments that problems can occur. This is because the substances are released in a large amount, compared to the gradual release that occurs naturally.

Provision of Habitat

An estuary is an interface between fresh and marine habitats. This results in a body of water that is brackish in nature. There are some organisms that have specifically adapted to these conditions, but most organisms in estuaries are from either fresh or salt water origins. The high level of nutrients in estuaries attracts many organisms. They live in the water body, on the water surface, within or above the bottom sediments, or amongst floating vegetation. The productive nature of estuaries provides a suitable habitat and a plentiful food supply for many plants and animals.

Provision of Food

Inputs of plant material from vegetation surrounding the estuary are broken down by bacteria and microorganisms. Nutrients come into the estuary with river flow and also with the tides. These nutrients are used by many planktonic and aquatic plants to photosynthesize and grow. Fringing vegetation such as grasses and mangrove species are vital to the input of nutrients into estuarine waters. Without these, the nutrient levels would be drastically reduced.


Biology 447 ­ Environmental Microbiology

Despite the large amount of plant material available for food, estuaries do not contain a large diversity of species. This is actually a result of the other conditions such as salinity and temperature, which make it difficult for many organisms to live in an estuary. However, those that do are often present in large numbers.

Spawning & Breeding Area

Many marine species enter estuaries at some time during their life cycle to breed. Because of their high nutrient level, and the relative shelter from wind and waves, it is an ideal environment for the growth of young.

Human Use of Estuaries

Estuaries, like many other marine ecosystems, have often been used by humans for many undesirable purposes. To provide more space for human settlement, they have been dredged and filled in, with the fringing vegetation cleared. Wastes have been dumped in them, particularly sewage, heavy metals, hydrocarbons and stormwater, most of which arrive via rivers.


Estuaries support a high level of primary productivity. The reason for this is that a large amount of nutrients come in with both the fresh and the salt water. There is a large amount of light in the shallow water; the sun's rays are able to penetrate and provide sufficient energy for photosynthesis. There is a large amount of vascular plant material from surrounding vegetation, which adds nutrients.

Mangrove swamp

Mangrove swamps are often found surrounding an estuary, and dead leaves and branches fall into the water, along with plant materials already partly decomposed by bacteria.

Adaptations of Estuarine Organisms

Estuarine organisms are derived from both terrestrial and marine origins, but the diversity of organisms that may survive in an estuary is limited by the harsh conditions. The temperature and salinity vary along the length of an estuary, as do the light levels. There is significant organic accumulation in an estuary and seagrass beds are common.


Salinity can affect an organism in different ways, and these effects may vary with any of the following factors: temperature, dissolved gases, density and viscosity. These factors will affect the levels at which the salinity changes become harmful. Salinity effects may also vary during an organism's life cycle. Generally it seems that newly hatched eggs and reproducing adults are more prone to salinity stress than organisms in the intermediate stages of the life cycle.


Biology 447 ­ Environmental Microbiology

Responses of estuarine organisms vary amongst species. Active swimmers like fish can escape changes fairly easily. Sedentary (attached) animals cannot escape these changes, but most are able to shut themselves away inside their shells. Benthic creatures can often escape salinity changes by retreating into their burrows and digging themselves in deeper if necessary. Organisms may become severely stressed if they are unable to cope with salinity changes. Many estuarine organisms are euryhaline, meaning that the organism can withstand a larger range of salinities without becoming too sick. Other organisms that are restricted to the freshwater end (low salinities) or the marine end (high salinities) are called stenohaline, meaning that they can only tolerate a narrow range of salinities. Rare occurrences such as floods (more freshwater, lower salinity) and drought (less freshwater, higher salinity) can have dramatic effects on organisms, altering the salinity gradient within the estuary and affecting where organisms can live.


The temperatures within an estuary can vary significantly during any twenty-four hour period. The shallowness of estuarine waters means that the water is subject to heating by the sun's rays, and cooling due to the incoming water from both rivers and the sea. At high tide, the water temperature is usually quite cool. Light cannot penetrate as far in deeper water, so the lower layers remain relatively cool, with only the uppermost layers becoming heated. As the tide goes out, the water is more easily heated all the way through. An Australian summer may heat shallow waters up to 50 degrees Celsius! When the tide comes back in again, it is often rapid and the water is very cool. Estuarine organisms must be able to deal with the sudden temperature changes, and many are eurythermal, meaning they can withstand the highly variable temperatures of estuarine life. Other organisms escape much of these changes by burying themselves in the bottom sediments, although they cannot escape them altogether.

Microbiology of Estuaries

The rapid variations in physical and chemical properties in estuaries lead to the establishment of unique microbial communities. The biomass of epiphytic (on estuarine plants) microbial communities is extremely large and may exceed the weight of the typical estuarine sea grasses. This epiphytic microbial community consists of algae, diatoms and bacteria. Because of the concentration of nutrients in the estuary (described in Aquatic Microbiology Module #1), the nutrient levels are high. The biomass is therefore also high. Because of the severe environmental fluctuations in temperature and salinity, the microbial species diversity is not as great as might be expected. It is, however, greater than the species diversity in the plants and animals in the estuary, since bacteria are capable of existing and growing over wider ranges of environmental conditions. As an example of this, consider the range in pH and Eh in an estuary; it is very large for both parameters, but many of the conditions are capable of supporting different groups of bacteria. The graph below shows the typical range in Eh and pH in an estuary with the tolerance range of commonly occurring estaurine bacterial groups superimposed. It is clear that the range of conditions present permits a wide range of different microbial groups (sulphur bacteria, iron bacteria, heterotrophs, etc.) to grow in estuaries.


Biology 447 ­ Environmental Microbiology

The sediment conditions fluctuate less than the water; sediment bacteria are less subject to the rapid changes in salinity and temperature and so are more similar to normal ocean or fresh water sediment bacteria

Key Points

· · · · · · ·

Definition of an estuary Environmental conditions Importance in ecology Adaptation of estuarine organisms Microbiology of Estuaries - why is it diverse and productive ? Range of conditions (Eh vs pH diagram) - how this impacts microbial activities. Concentration effects for nutrients in estuaries (from Aquatic Microbiology)


Biology 447 ­ Environmental Microbiology

Module Four - Pathogens in Water

INTRODUCTION Although current public attention is focused on chemical contamination of water supplies, only 10% of identified health problems (1971-1978) related to drinking water have been attributed to such contamination. Pathogenic micro-organisms have been responsible for 40%, while the cause of 50% of such problems has not been identified. Since water supplies are used by populations, the transmission of water-borne pathogens may be expressed in the form of epidemics or may be endemic in nature. Figure 1 shows one distinguishing feature of a water-borne epidemic. Sometimes the identification of drinking water as a source of infection is difficult due to long incubation times. Their propagation is generally a vicious circle, being introduced to water supplies via domestic effluent, infecting part of the population, who in turn reintroduce infected waste to water supplies. An examination of the micro-organisms in domestic waste is a good indicator of the degree and variety of infection in the population.

Figure 1. Typical developments in epidemic disease. Some strategies used to manage the spread of pathogens are control of the source, control of the vector, and immunization. The integration of these strategies has produced a relatively pathogen-free potable water supply in developed countries. The source is considered to be sewage effluent and the water supply as the vector, control of these usually being enough to control most pathogens. Immunization has been most important in controlling polio but with its success has come a complacency, a lowered rate of immunization and renewed higher incidence.


Biology 447 ­ Environmental Microbiology

HISTORY The determination of water-borne pathogens as the causative factor in an outbreak uses the classical detective work of epidemiological studies. In 1854, a cholera outbreak in London, England was shown to be linked to a pump that derived its water from a polluted section of the River Thames. People served by a pump that obtained its water upstream of London had a low incidence of cholera. In 1855-1856, Dudd and Shaw showed that there was an association between typhoid fever in a street in Bristol, England and the use of a particular pump for water. People on an alternate pump did no become infected. This was 30 years before the causative agent for typhoid was identified. The appreciation of water as a carrier of diseaseproducing organisms only came about in the mid-1880's with the establishment of the germ theory by Pasteur. At the beginning of this century, cities in the Ohio River watershed commonly had death rates due to typhoid on the order of 75 deaths per 100,000 people. Upon the installation of drinking water treatment plants, the death rate fell to about 15/100,000. One classical case of misinterpretation of data involves the cities of Yalta and Eupatoria which are along the Mediterranean coast. The city of Eupatoria installed water treatment systems in 1956 and registered a decrease in typhoid cases in the 3 year period preceding and following the installation. It was subsequently shown that a similar decrease had occurred in Yalta and that the pathogen in sewage effluent was transmitted along shore currents to Eupatoria. Hence the conclusions were that the vector was not drinking water supply but bathing in the effluent outfall of Yalta and that such outfall determined the rate of infection. The occurrence of pathogens in drinking water supplies is currently a serious problem in developing countries but is also of concern in developed countries. The number of water-borne disease outbreaks in the U.S. decreased steadily from 1920 - 1960 in direct relation to the increase of chlorination systems, but have increased since then. Whether this is a real trend, or a change in the ability to identify the disease source, is arguable.

PATHOGEN CHARACTERISTICS Most water-borne pathogens may be classified as viruses, bacteria, or protozoa (Table I). They cause primarily intestinal diseases, leaving the host in fecal material, contaminating the water supply, and then entering the recipient by ingestion. Their survival period in water varies widely and is influenced by many factors such as salinity, temperature, etc.. It may be generalized that cellular viruses last longer than bacteria while protozoa can extend their survival time by encystation. Viruses Although there are over 100 known water-borne human enteric viruses, infectious hepatitis A, poliovirus and viral gastroenteritis are of practical concern as water-borne viruses. All of them, with the exception of the infectious hepatitis agent, have been found in sewage and polluted rivers. Tests for the presence of viruses in water supplies are difficult and uncertain and so little is known of the survival time and concentration distribution of viruses in water sources. In general, enteric viruses survive less than three months in the environment but have been reported surviving up to five months in sewage. There is dispute over whether a minimum infectious dose is necessary as for bacteria, some researchers claiming that a single virus is sufficient for infection. Due to their small size and surface properties, viruses tend to be adsorbed onto surfaces. Table 1: Drinking water-borne disease outbreaks in the U.S., 1946-1980 Survival time (days)

Causal Agent Viral Hepatitis A virus* Polio virus, Norfolk agent




Infect. hepatitis Poliomyelitis

71 1 10

2,342 16 3,147

? ? ?


Biology 447 ­ Environmental Microbiology

Bacterial Salmonella typhi* Shigella sp.** Salmonella paratyphi* E.coli, Campylobacter sp Francisella tularensis Leptospira sp. Vibrio comma Protozoal Entamoeba histolytica Giardia lamblia* Unknown etiology ** Gastroenteritis 366 87,439 -Dysentery Giardiasis 5 47 75 19,883 150 Tularemia Tularemia Cholera Typhoid Shigellosis Salmonellosis Gastroenteritis 58 59 22 5 2 2 1 0*** 836 13,261 17,914 1,188 3,800 6 9 0 23 9 40 16 22 16

* Occurrence in groundwater supplies. ** Higher occurrence in groundwater supplies. *** Cholera remains a serious disease in undeveloped countries. (From Brock et al., 1984. Biology of Microorganisms). Bacteria Bacteria comprise the largest group of water-borne pathogens. A minimum infectious dose of several hundred to several thousand organisms is necessary to cause bacteriological infection. Pathogenic bacteria are usually poor competitors at low substrate levels found in natural waters and so tend to be eliminated by competition and predation. Low temperatures, sediment adsorption and anoxic conditions occasionally prolong their survival. Short descriptions of some of the most common bacteriological diseases are given below. Shigella sp., the cause of dysentery, is almost strictly a human affliction (minor in other primates). Most shigellosis and salmonellosis epidemics in developed countries are food-borne but a few are caused by drinking water. Transmission by drinking water is still the major route of infection in underdeveloped countries. Methodology for detection is not reliable. Die-off is rapid in sewage, although low salt concentrations and temperatures will extend survival times( 25 days @ 13 degrees C, 4 days @ 37 degrees C). Salmonellosis incidence (food poisoning) is low and peaks seasonally in mid to late summer due to favourable conditions for food-borne salmonella. Salmonella sp. Are carried by humans (1-4% of population), farm animals (13-17% incidence) and wild animals. Most Salmonella sp. cause gastrointestinal diseases, while one, which is strictly a human pathogen, causes typhoid. Enteropathogenic E. coli produce gastroenteritis and urinary infections. Carrier rates vary but may be 16% in mothers of newborne infants, 7% in food handlers and 3% in children. Farm animals are also carriers. Concentrations of E. coli in effluents to natural waters reduce to 5% of original levels in less than 5 days. Tularemia is passed principally by ticks, rodents and direct contact with sewage. Water contamination occurs from rodents. The disease spreads via the lymphatics and bloodstream. It grows intracellulary and causes lesions in the lungs, liver, spleen and brain. Leptospirosis, which begins as a wound infection, is an occupational disease among workers in close contact with polluted water. Pigs, dogs, rodents and humans are carriers and is excreted in urine of infected animals.


Biology 447 ­ Environmental Microbiology

Cholera is a serious, highly contagious disease causing dramatic and fatal loss of water and electrolytes. Healthy carriers may make up 1-9% or even 25% of the population.

Protozoa Protozoans entering the host body by ingestion are usually in cyst form. Two protozoans of major concern as water-borne pathogens are Giardia intestinalis and Entamoebia histolytica. Giardiasis is the most prevalent waterborne disease in the United States. One to ten cysts of the flagellated Giardia is the minimal infection dose and causes serious diarrhea. Beavers have been implicated as a source where no obvious human contamination was found. Cysts of E. histolytica, which cause amebic dysentery, survive for long periods of time at low temperatures and damp conditions in clean water, but only a few days in fecal material. Cryptosporidium has also been associated with gastrointestinal disease outbreaks in a number of North American cities (including KitchenerWaterloo). Another protozoan, Cyclospora cayetanensis, has been a recent problem associated with imported fruit that has been in contact with feces. Methods for detection are currently being refined. STANDARDS The variety of pathogenic organisms in water supplies is large but their concentrations are low. Testing for each one in order to monitor water quality is an expensive and unsure proposition. Tests for some pathogens are unreliable and may require an unacceptably long incubation period for the quick response which may be required for public safety. Thus, the concept of an indicator organism is used to indicate the possible presence of diseasecausing constituents. Such an indicator organism should behave as follows: · · · · · · be applicable to all water - be present when pathogens are have no aftergrowth in water - be absent when pathogens are have constant characteristics - persist longer than pathogens be harmless to humans - correlate quantitatively with pathogens be present in greater numbers than pathogens be easily, accurately and quickly detected.

The coliform group nearly fulfills these criteria. E. coli is considered a reliable indicator of bacterial pathogens. Protozoans and viruses, however, usually survive longer than E. coli and may also survive disinfection which is otherwise adequate for bacteria. Filtration is usually successful in extracting protozoans and viruses and should be used in conjunction with disinfection. The U.S. has consistently been the first to establish standards in water quality issues. The spread of disease via drinking water was realized in the mid 19th century and the first standards for microbiological quality of drinking water were set in 1914 by the U.S. Public Health Service. The fermentation test at 37 degrees C was used and 1 in 5 samples were allowed to test positive for "Bacillus coli" ( now E. coli ) group. The standards were revised in 1925 in recognition of statistical aspects of bacteriological distribution. Further revisions in 1943 and 1962 required the use of the fermentation test at 44.5 degrees C to identify fecal coliform separate from non-fecal coliform, sampling from the water distribution system as well as at the treatment plant, and further adjustments to the statistics of sampling and results. Until 1974, federal law applied to common carriers only (e.g.. Railroads) while some states passed their own laws. In 1974 the Safe Drinking Water Act provided national quality standards enforceable by the E.P.A.. These standards are given in table III, along with the Canadian guidelines. The first comprehensive Canadian guidelines were published in 1968 and revised in 1978. In 1986 a federalprovincial advisory committee was established to revise and update the guidelines on a continuing basis. They are unenforceable at the moment, but laws may be introduced at the provincial, territorial or federal levels. The Ontario Ministry of the Environment currently samples and analyses multiple drinking water supplies. Municipalities usually conduct parallel monitoring.


Biology 447 ­ Environmental Microbiology

Table IIa: U.S.A. maximum contaminant levels for coliform organisms Detection Technique Used Membrane filter <20 20 or more Fermentation tube 10-ml standard portions 100-ml standard <5 5 or more <20 20 or more Number of Samples per Month

Maximum # of Coliform Bacteria Arithmetic mean of 1/100 ml of all samples each month 4/100 in no more than 1 sample 4/100 in no more than 5% of all samples None in >10% of portions None in >2 portions in more than 1 sample None in >2 portions in 5% of the samples None in >60% of portions None in 5 portions in more than 1 sample None in 5 portions in more than 20% of the samples

Table IIb: Canada - Ministry of National Health and Welfare maximum acceptable concentration for total coliforms. 1. No sample should show > 10 total coliform/100 ml - none of which may be fecal coliform. 2. No consecutive sample from the same site should show the presence of coliform. 3. For community drinking water supplies: a) Not > 10% of samples should show coliform presence; and b) Not > 1 sample from a sample set taken from the community on a given day should show coliform presence. The United Kingdom had a Drinking Water Directive that was legally enforceable but defined the required water quality simply by the word "wholesome". In 1974, the U.K. entered the European Community which has published a set of guidelines. Enforcement of the guidelines is left to member countries.

SEWAGE TREATMENT The first human concern with water quality derived from the realization that raw sewage was the source of destructive epidemics of cholera, typhoid, dysentery and other diseases. Sewage treatment is now also performed for aesthetic and environmental reasons and variably attempts to control pathogenicity, toxicity, turbidity, pH, and BOD, among other things. The standards for effluent quality are not influenced by drinking quality standards since purification for distribution systems is the quality bottleneck. Rather, standards of water quality for recreational use is most important with respect to pathogenicity of sewage discharge. Treatment of waste is usually identified as primary, secondary or tertiary, although distinctions are somewhat arbitrary since modern plants use different processes in single operations. Primary treatment involves removing suspended solids with screens and the use of gravity settling ponds. Due to their size, most protozoal cysts settle out in the ponds after 11 days . Secondary treatment uses biological treatment to decompose organic matter to cell material and byproducts, and the subsequent removal of cell matter, usually by gravity settling. Aerobic processes are preferred for their higher rates of decomposition. Attached growth or suspended growth methods are used and may include mechanical aeration and agitation to quicken activity. Activated sludge process returns 20 - 50% volume of digested sludge containing a developed culture of micro-organisms to freshly introduced sewage to maintain an active culture. Intestinal pathogens are greatly reduced by predation (by ciliates, rotifers and Bdellovibrio ), competition, adsorption, and settling. Furthermore, pathogenic microbes tend to grow poorly or not at all under aerobic


Biology 447 ­ Environmental Microbiology

conditions. Secondary treatment typically reduces pathogen concentration by 90 - 99%. Anaerobic processes such as septic tanks do not reliably destroy intestinal pathogens. Tertiary treatment is any practice beyond secondary. Treatment processes may include filtration for clarification, activated carbon to remove xenobiotic compounds, coagulants and lime for phosphorus removal, high pH for ammonia-stripping, desalinization, and disinfection. All of these processes are very effective in destroying pathogens.

DRINKING WATER SUPPLIES The supply of potable water consists of a source, a distribution system, and a delivery point at which the water quality is sufficient for consumption. Although many parameters such as salinity, aesthetics and toxins are important, only pathogens are discussed here. Location of the intake must consider the position of sewage outlets although this can be mitigated through proper treatment as discussed in the previous section. Sources of pathogens other than sewage outlets are wildlife watersheds ( e.g.. Giardia ), farm lots ( e.g.. Salmonella ), garbage dumps, and septic tank systems. Groundwater has often been considered a safe source since it is usually clear and the connection to pathogen-bearing sources is not obvious. Table II shows a more objective perspective. Groundwater systems generally contain fewer pathogens due to their filtering action. Most pathogenic groundwater problems derive from lack of well head protection. Table III: Waterborne disease outbreaks in the U.S., 1971-1979 Community water systems Surface water, no treatment Ground water, no treatment Deficiencies in treatment Deficiencies in distribution Miscellaneous Non-community water systems Surface water, no treatment Ground water, no treatment Deficiencies in treatment Deficiencies in distribution Miscellaneous 8 44 36 7 5 100 2 41 39 17 1 100 Outbreaks (percent) Illnesses 13 11 36 34 6 100 23 2 50 24 1 100

From Bitton and Gerba, 1984. Groundwater pollution microbiology. More than 50% of pathogens in water die within 2 days, and 90% within one week. Therefore storage reservoirs are reasonably effective in controlling them. Community water supplies can be disinfected by chlorination and ozonation. A benefit of chlorination is that a chlorine residue remains in the water during distribution which protects against recontamination. An undesirable side effect is that chlorination forms trihalomethanes, some of which are suspected carcinogens. Ozone is an excellent disinfectant that is more effective against viruses and protozoa than chlorine but is expensive and does not impart a residual antimicrobial activity as it is an unstable gas. In Europe, chlorination and ozonation are commonly used together.


Biology 447 ­ Environmental Microbiology

Figure 2. Number of water plants chlorinating water vs. Disease Other germicides, not amenable to community systems but with application to individual systems, are UV irradiation, silver, and ionizing irradiation. Protozoan cysts often elude disinfection by chlorine although ozone is more effective. Filtration is effective in removing them. All processes used in preparing water for drinking ( filtration, coagulation, settling, etc. ) help in reducing pathogens. Proper design of a delivery system can avoid problems. Breaks in the sewage system by natural (earthquakes) or man-made (construction) causes may allow for cross-contamination between the sewage system and the potable water distribution system or sources. The crossing of sewage lines over potable water lines should be avoided, and proximity of each to the other considered. Bibliography Atlas R.M. and Bartha R., 1987. Microbial Ecology: Fundamentals and Applications. Benjamin/Cummings, Menlo Park, Ca., 533 pp. Bitton G. and Gerba C.P., 1984. Groundwater Pollution Microbiology. Wiley & Sons, Toronto, 377 pp. Brock T.D., Smith D.W. and Madigan M.T., 1984. Biology of microorganisms. Prentice-Hall, Engelwood Cliffs, N.J., 847 pp. Craun G.F., 1985. A summary of waterborne illness transmitted through contaminated groundwater. J. Env. Health, 48:3, p 122-127. Crewe W. and Owen R.R., 1981. The occurrence of sewage-borne pathogens in the U.K.. Wat. Pollut. Control, p. 632-637. Dept. of National Health and Welfare, 1989. Guidelines for Canadian Drinking Water Quality. Can. Gov. Publ. Centre, Cat. #H48-10/1989E, 20 pp. Geldreich E.E., 1986. Potable Water: New directions in microbial Regulations. ASM News, 52:10, p 530-534. Kidson R.J., 1984. Maintenance of drinking water quality: The essential decision. Wat. Pollut. Control, p. 23-30. Linsley R.K. and Franzini J.B., 1979. Water Resources Engineering. McGraw-Hill, Montreal, 716 pp. Mance G., 1986. Water quality standards in relation to the European Community. Wat. Pollut. Control, p. 25-33. Ongerth J.E., 1990. Evaluation of treatment for removing Giardia cysts. Jour. AWWA, 82:1, p. 85-96. Stanier R.Y., Adelberg E.A. and Ingraham J.L., 1976. The Microbial World. Prentice-Hall, Toronto, 871 pp. Williams R.B. and Culp G.L., 1986. Handbook of Public Water Systmes. Macmillan, Agincourt, Ont. 1113 pp.


1. Introduction One of the problems involved with the reliable detection of waterborne pathogens is that there is no one method suitable for all pathogens. The pathogens in the Enterobacteriaceae occur only sporadically in water and the environment is not conducive to growth and survival of these pathogens. This, together with many other technical reasons, makes the coliform test of dubious value in assessing the actual risk of ingesting water. In addition, detection of the actual pathogens themselves is technically difficult and often time consuming.


Biology 447 ­ Environmental Microbiology

For instance, since the pathogens are usually present in very low concentrations in most water samples, the degree of concentration required to obtain usable numbers of the pathogenic organisms is often very high. This leads to other problems in water where other kinds of contaminants such as detritus, organic materials or other organisms are present. As examples of the problem, to detect Giardia cysts, over 400 litres of water is usually filtered. For Cryptosporidium this value may be +1200 litres; for viruses it may be as high as 2000 to 5000 litres. Other reasons for the difficulty are that recovery of organisms from the samples (even after concentration) is difficult due to possible damage to the organisms by the harsher environment in water compared to their normal habitat. This can, in the extreme cases, lead to organisms in water that cannot be cultured on normal media, but still retain their pathogenicity. These organisms are called "viable, but non-culturable".

Methods for detecting pathogenic viruses and bacteria from water samples

Viruses Concentration of viruses is an important facet of their detection and identification. The most common method involves passing very large volumes of water through filters that are electronegative or electropositive in nature. The virus particles are adsorbed to the surface of the filter by electrostatic charges and are then eluted by passing smaller volumes of a protein-containing liquid (e.g. beef extract).

Detection is often achieved through a tissue culture of the viruses in human or primate tissue samples. More recently, immunofluorescence (antibodies linked with fluorescent dyes such as FITC - see Biology 446, Chapter on Methodology) , enzyme-linked immunosorbent assays (ELISA both direct and indirect), nucleic acid probes, the Polymerase Chain Reaction (PCR) and radioimmunofocus assays (RIFA) have become more common. Bacteria The most common indicator of potential pathogen contamination is still the coliform test in its various guises. The Total coliform test has many forms; the most common so far has been the membrane filter method in which a known volume of water is filtered through a 0.45 M or 0.22 M filter and the filter incubated on M-endo or LESEndo agar. Red colonies with a metallic sheen are considered coliforms. A slower method, the Most Probable Number method is also used and involves serial decimal dilution of the water sample followed by adding 5 aliquots of the dilutions to 5 tubes of nutrient media. If the dilutions are chosen correctly, some of the serial decimal dilutions will have some tubes with growth and some without. A statistical procedure based on these sets gives the number


Biology 447 ­ Environmental Microbiology

of bacteria in the original sample. This test is sometimes referred to as "dilution to extinction" - an unfortunate phrase ! More recently, with changes in many countries drinking water regulations with regard to coliforms, no coliforms at all are allowed in 100 mL of sample. This permits a simple presence/absence test to be used where 100 mL of sample is placed in nutrient medium at suitable concentrations and incubated; any growth means the water has coliforms and so fails the regulations. A remaining problem is the time taken to assay for coliform number or presence/absence; up to 3 days is required for some of the tests. The quickest tests (P/A and membrane filter) take 24 hours. Very often, quicker results reporting is essential or desirable. Rapid detection tests have been developed for these situations. They vary in their acceptance by the regulatory bodies, but at least one (Defined Substrate Systems) has achieved broad use and regulatory standing. In this test (various commercial versions are available), the water sample is filtered through 0.45 membrane filters, removed and incubated at 35C on a medium containing 4-methylumbelliferonebeta-D-galactoside. If coliforms are present, they break the bond between the methylumbelliferone and the galactoside, releasing the fluorescent umbelliferone derivative. The degree of fluorescence is measured with a fluorimeter and shows the degree of contamination by coliforms.

A variant of this test (the Colilert method) uses ortho-nitrophenyl-beta-D-galactosidase (ONPG) and 4-methyl umbelliferyl-beta-D-glucuronide (MUG) for detecting total coliforms and Escherichia coli in a single solution. The coliforms break down ONPG with their beta-galactosidase enzymes releasing the yellow coloured indicator portion of the molecule. If E. coli is also present, the enzyme glucuronidase hydrolyzes the MUG to glucuronide and the indicator portion 4-methyl-umbelliferone that fluoresces under ultraviolet light. This permits separate and independent estimates of total coliform and E. coli counts in the same sample. Levels as low as 1 CFU/100 mL have been reliably detected with this method. This test can also be used with the MPN or P/A tests to improve resolution and sensitivity.

For a complete assessment of the coliform bacteria and methodologies to determine their numbers, etc. see the Canadian Water Quality Guidelines article.

Gene probe and PCR technology (Polymerase Chain Reaction References General Molecular Biology Methods Donis-Keller Lab.:Laboratory Manual) could also be used to produce more sensitive and specific test for coliforms or E. coli

Bibliography on new Methodologies for Microbial Water Quality 68

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Bernhard, A.E., and Field, K.G., 2000a, Identification of nonpoint sources of fecal pollution in coastal waters by using host-specific 16S ribosomal DNA genetic markers from fecal anaerobes: Applied and Environmental Microbiology, v. 66, no. 4, p. 1587-1594. Bernhard, A.E., and Field, K.G., 2000b, A PCR assay to discriminate human and ruminate feces on the basis of host differences in Bacteroides-Prevotella genes encoding 16S rRNA: Applied and Environmental Microbiology, v. 66, no. 10, p. 4571-4574. Carson, C.A., Shear, B.L., Ellersieck, M.R., and Asfaw, A., 2001, Identification of fecal Escherichia coli from humans and animals by ribotyping: Applied and Environmental Microbiology, v. 67, no. 4, p. 1503-1507. Dombek, P.E., Johnson, L.K., Zimmerley, S.T., and Sadowsky, M.J., 2000, Use of repetitive DNA sequences and the PCR to differentiate E. coli isolates from human and animal sources: Applied and Environmental Microbiology, v. 66, no. 6, p. 2572-2577. Dunbar, J., Ticknor, L.O., and Kuske, C.R., 2001, Phylogenetic specificity and reproducibility and new method for analysis of terminal restriction fragment profiles of 16S rRNA genes from bacterial communities: Applied and Environmental Microbiology, v. 67, no. 1, p. 190-197. Farag, A.M., Goldstein, J.N., Woodward, D.F., and Samadpour, M., 2001, Water quality in three creeks in the backcountry of Grand Teton National Park, USA: Journal of Freshwater Ecology, v. 16, no. 1, p. 135-143. Farnleitner, A.H., Kreuzinger, N., Kavka, G.G., Grillenberger, S., Rath, J., and Mach, R.L., 2000, Simultaneous detection and differentation of Escherichia coli populations from environmental freshwaters by means of sequence variations in a fragment of the beta-D-glucuronidase gene: Applied and Environmental Microbiology, v. 66, no. 4, p. 1340-1346. Hagedorn, C., Robinson, S.L., Filtz, J.R., Grubbs, S.M., Angier, T.A., and Reneau, R.B., Jr., 1999, Determining sources of fecal pollution in a rural Virginia watershed with antibiotic resistance patterns in fecal streptococci: Applied and Environmental Microbiology, v. 65, no. 12, p. 5522-5531. Hager, M.C., 2002, Detecting Bacteria in Coastal Waters (Part 1 of Feature article): Stormwater, accessed June 3, 2002, at Hager, M.C., 2002, Detecting Bacteria in Coastal Waters (Part 2 of Feature article): Stormwater, accessed June 3, 2002, at Harwood, V.J., Whitlock, J., and Withington, V., 2000, Classification of antibiotic resistance patterns of indicator bacteria by discriminant analysis--Use in predicting the source of fecal contamination in subtropical waters: Applied and Environmental Microbiology, v. 66, no. 9, p. 3698-3704. Kariuki, S., Gilks, C., Kimari, J., Obanda, A., Muyodi, J., Waiyaki, P., and Hard, C.A., 1999, Genotype analysis of Escherichia coli strains isolated from children and chickens living in close contact: Applied and Environmental Microbiology, v. 65, no. 2, p. 472-476. Kreader, C.A., 1995, Design and evaluation of Bacteroides DNA probes for the specific detection of human fecal pollution: Applied and Environmental Microbiology, v. 61, no. 4, p. 1171-1179. Landry, Natalie, New Hampshire Department of Environmental Services, Microbial Source Tracking Survey, accessed May 31, 2002, at Long, Barry, National Park Service, Tracking Microbial Sources Using Genetic Analysis of Ribosome Patterns, accessed May 31, 2002, at Luows, F.J., Rademaker, J.L.W., and de Bruijn, F.J., 1999, The three Ds of PCR-based genomic analysis of phytobacteria--diversity, detection, and disease diagnosis: Annual Reviews of Phytopathology, v. 37, p. 81-125.


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Malakoff, D., 2002, Microbiologists on the trail of polluting bacteria: Science, v. 295, p. 2352-2353. Olive, D.M., and Bean, P., 1999, Principles and applications of methods for DNA-based typing of microbial organisms: Journal of Clinical Microbiology, v. 37, no. 6, p. 1661-1669. Parveen, S., Murphree, R.L., Edmiston, L., Kaspar, C.W., Portier, K.M., and Tamplin, M.L., 1997, Association of multiple-antibiotic-resistance profiles with point and nonpoint sources of Escherichia coli in Apalachicola Bay: Applied and Environmental Microbiology, v. 63, no. 7, p. 2607-2612. Parveen, S., Portier, K.M., Robinson, K., Edmiston, L., and Tamplin, M.R., 1999, Discriminant analysis of ribotype profiles of Escherichia coli for differentiating human and nonhuman sources of fecal pollution: Applied and Environmental Microbiology, v. 65, no. 7, p. 3142-3147. Parveen, S., Hodge, N.C., Stall, R.E., Farrah, S.R., and Tamplin, M.L., 2001, Phenotypic and genotypic characterization of hman and nonhuman Escherichia coli: Water Research, v. 35, no. 2, p. 379-386. Rademaker, J.L.W., Luows, F.J., and de Bruijn, F.J., 1998, Characterization of the diversity of ecologically important microbes by rep-PCR genomic fingerprinting: Molecular Microbial Ecology Manual, v. 3.4.3, p. 1-27. Samadpour, Mansour, University of Washington, Identification and Tracking of the Sources of Microbial Pollution in Watersheds and Distribution Systems, accessed May 31, 2002, at Stewart, Jill, NOAA, Microbial Source Tracking in South Carolina Surface Waters, accessed May 31, 2002 at Toivanen, P., Vaahtovuo, J., and Eerola, E., 2001, Influence of major histocompatibility complex on bacterial composition of fecal flora: Infection and Immunity, v. 69, no. 4, p. 2372-2377. Wiggins, B.A., Andrews, R.W., Conway, R.A., Corr, C.L., Dobratz, E.J., Dougherty, D.P., Eppard, J.R., Knupp, S.R., Limjoco, M.C., Mettenburt, J.M., Rinehart, J.M., Sonsino, J., Torrijos, R.L., and Zimmerman, M.E., 1999, Use of antibiotic resistance analysis to identify nonpoint sources of fecal pollution: Applied and Environmental Microbiology, v. 65, no. 8, p. 3483-3486. Wiggins, F.A., 1996, Discriminant analysis of antibiotic resistance patterns in fecal streptococci, a method to differentiate human and animal sources of fecal pollution in natural waters: Applied and Environmental Microbiology, v. 62, no. 11, p. 3997-4002.


Biology 447 ­ Environmental Microbiology

Enteropathogenic Escherichia coli in Walkerton, Ontario.

Escherichia coli O157:H7 - Information Sources · · · ·

General Information on Escherichia coli O157:H7 from the Center for Disease Control, Atlanta, USA Technical Information from CDC Additional Information from CDC Summary of Cases in the US in 1999 - (Adobe Acrobat file - pdf)

Summary of the situation in Walkerton Well 5 was contaminated during a heavy rainfall in May 2000. Bacteria from a farm manure pile (probably) entered the well through overland flow and/or through transport in groundwater after percolating down through the soil. The overburden (the depth of soil above the aquifer) was very shallow at Well 5 . The soil around Wells 5, 6 and 7 was also very permeable (see below) so bacterial contamination could easily percolate down to the aquifer.


Biology 447 ­ Environmental Microbiology

Well 5 was an extremely shallow well and, when the pump was operating, water was drawn down from the surface water around the well. When the pump was not operating, natural springs occurred in the depression (standing water) around the well. In other words there was a direct connection between the surface water and the well as it pumped from the shallow aquifer. (see description below)

More detailed study of Well 5 showed that the aquifer was also very shallow and open to contamination (below)


Biology 447 ­ Environmental Microbiology

Other problems emerged with other wells. Well 7 has an overflow device (a pipe connected to the well head) that allowed excess water to run out when the well was not operating. This was required since Well 7 is another artesian well (like Well 5) where the water flows even with the pump turned off. The "overflow" water entered a depressed land area (with some standing water). During a period of high rainfall, the area could have been flooded. If the well was turned on during that time, only a small plastic flap valve (below) prevented water "backing up" the pipe in to the well.


Biology 447 ­ Environmental Microbiology

Coupled with these technical issues was the issue of management and reporting of contamination events. Reports from private laboratories that showed the presence of coliforms or Escherichia coli in the drinking water that were supplied to the Public Utilities Commission did not also have to be reported to the Ministry of the Environment or the Health Unit. The PUC was supposed to do that. This has been changed since the Walkerton contamination (see new Ministry reporting guidelines). The Toronto Star called this the "broken loop" - (see below)

Then the chlorination plant either broke down and did not inject chlorine at the required level or it had been faulty for some time (this will emerge during the inquiry). Levels of chlorine in the drinking water are supposed to be at or above 0.5 ppm. Documented cases are in the log books for the wells where these criteria were not met. In addition,


Biology 447 ­ Environmental Microbiology

many reading over a long period of time were stated to be at either "0.5" or "0.7" ppm - an unlikely coincidence for all levels to be essentially identical over a long period of sampling.

Detection of Waterborne parasites

Water can contain many different protozoal parasites. The most significant are Entamoeba histolytica, Cryptosporidium spp. and Giardia spp.. Giardia outbreaks have become more common and in fact is the most prominent cause of waterborne illness in the U.S., accounting for 20% of all waterborne disease cases. Cryptosporidium is less common but there have been numerous outbreaks around the world. The largest outbreaks occurred in Milwaukee, Wisconsin affecting 400,000 individuals . A lake used as a water supply became contaminated and the oocytes of Cryptosporidium survived through water treatments of coagulation, flocculation, rapid sand filtration, sedimentation and chlorination. A local outbreak occurred in the Region of Waterloo after Grand River water was added to the normal groundwater supply (see the details of the Mannheim water treatment system to see the present Region of Waterloo water treatment scheme). Giardia is a flagellated protozoan with a life cycle consisting of two stages; the first is a cyst that produces two flagellated trophozoites that, when ingested, attach to the lining of the intestines. These cysts can survive for long periods (up to 2 months at 8C and are much more resistant to chlorine than most bacteria. The only practical identification method is microscopic examination of water after concentration by filtration. No culture method for Giardia is available. Cryptosporidium is a protozoan parasite that is coccoidal in form and develops within the gastric or intestinal mucosal epithelial layer in mammals. It has a complex life cycle consisting of sexual and asexual stages and produces a very stable and resistant oocyte structure which "germinates" after ingestion to form 4 sporozites that infect the lining of the intestine cells. Many stages follow including trophozoites that undergo asexual multiplication to form type I meronts and then merozoites that can infect new cells. The oocysts are formed from gametocytes from Type II meronts that form merozoites that go on to form microgameteocytes and macrogametocytes. The oocytes are then excreted and enter the water supply. The oocytes are even more resistant to disinfection than Giardia. A major source of Cryptosporidium is from cattle and other animals. Most likely, manure pits and storage areas overflowing into local rivers and streams of the Grand River or cattle grazing on river or stream bans were the cause of the regional infection by Cryptosporidium. No culture method is available for Cryptosporidium.


Biology 447 ­ Environmental Microbiology

Entamoeba histolytica Entamoeba histolytica has three stages in the life cycle. The trophozoite and precyst stages are not as resistant as the cyst stage to disinfection. Even the cyst stage is not able to tolerate temperatures above 50 ºC, sunlight or extended disinfection periods. The only source of the organism is from humans and a few primates


Biology 447 ­ Environmental Microbiology

Summary of Testing for Bacteriological Quality

Historically, water has played a significant role in the transmission of human disease. Typhoid fever, cholera, infectious hepatitis, bacillary and amoebic dysenteries and many varieties of gastrointestinal disease can all be transmitted by water. The introduction of water treatment with disinfection and the implementation of bacteriological surveillance programs to ensure the delivery of safe water have resulted in a dramatic decrease in the occurrence of water-related illness. The occasional occurrence of waterborne disease outbreaks, however, points out the continuing importance of strict supervision and control over the quality of public and private water supplies. Contamination by sewage or human excrement presents the greatest danger to public health associated with drinking water, and bacteriological testing continues to provide the most sensitive means for the detection of such pollution. Although modern microbiological techniques have made possible the detection of pathogenic bacteria, viruses and protozoa in sewage and sewage effluents, it is not practical to attempt to isolate them as a routine procedure from samples of drinking water. Pathogens present in water are usually greatly outnumbered by normal intestinal bacteria, which are easier to isolate and identify. The presence of such organisms indicates that pathogens could be present; if they are absent, diseaseproducing organisms are probably also absent. It should be emphasized that no bacteriological analysis of water can take the place of a complete knowledge of the conditions at the sources of supply and throughout a system. Contamination is often intermittent and may not be revealed by the examination of a single sample. The most a bacteriological report can prove is that, at the time of examination, bacteria indicating faecal pollution did or did not grow under laboratory conditions from a sample of water. Therefore, if a sanitary inspection shows that a well is subject to contamination or that water is inadequately treated or subject to contamination during storage or distribution, then the water should be considered unsafe irrespective of the results of bacteriological examination The Coliform Group The coliform group merits consideration as an indicator of pollution because these bacteria are always present in the intestinal tracts of humans and other warm-blooded animals and are excreted in large numbers in faecal wastes. Although the sanitary significance of some coliform strains is questionable, all members of the group may be of faecal origin, and it should be assumed that they are of faecal origin unless it can be proven otherwise. Finally, water is not a natural medium for coliform organisms, and their presence must at least be regarded as indicative of pollution in its widest sense. Definitions The coliform group has been defined in the 16th edition of Standard Methods for the Examination of Water and Wastewater as follows: · · all aerobic and facultative anaerobic, Gram negative, non-spore-forming, rod-shaped bacteria that ferment lactose with gas formation within 48 hours at 35°C; or all organisms that produce a colony with a golden-green metallic sheen within 24 hours on an Endo-type medium containing lactose.

These definitions are not to be regarded as identical but, rather, refer to two groups that are roughly equivalent in sanitary significance. Both groups contain various species of the genera Escherichia, Klebsiella, Enterobacter and Citrobacter. Two characteristics-- b-galactosidase positive and cytochrome oxidase negative--should be added to provide common traits to link the definitions. The b-galactosidase test would provide a definitive test for lactose fermentation, whereas the cytochrome oxidase test would serve to exclude members of the genus Aeromonas, which are frequently responsible for false-positive coliform reactions The faecal coliform group includes that portion of the total coliform group that is capable of forming gas within 24 hours in EC medium at 44.5°C or that produces a blue colony on m-FC broth within 24 hours at 44.5°C. This group comprises the genera Escherichia and, to a lesser extent, Klebsiella and Enterobacter. The organism most commonly thought of as an indicator of faecal pollution is Escherichia coli. Complete identification of E. coli in terms of modern taxonomy would require an extensive series of tests that would be impractical for routine water bacteriology.


Biology 447 ­ Environmental Microbiology The detection and identification of the faecal coliform group in accordance with the simpler operational definitions given above are currently preferred. A membrane filter method has been developed for the direct enumeration of E. coli.

Differentiation of Organisms It was recognized at an early date that some strains included in the total coliform group were not common in faecal material. Organisms of the Klebsiella, Enterobacter and Citrobacter genera (intermediate­aerogenes­cloacae [IAC] subgroups) have been found in soils and on vegetation; in faeces, however, they are present in much smaller numbers than E. coli, which is characteristically the predominant coliform in warm-blooded animal intestines. Attempts have therefore been made to differentiate members of the coliform group and to relate their physical and biochemical characteristics to their natural sources and habitats. MacConkey defined the aerogenes group on the basis of · fermentative reactions with five sugars and the · ability to produce acetylmethylcarbinol in the Voges-Proskauer (VP) reaction. Coliforms can also be differentiated by the ratio of carbon dioxide to hydrogen produced. Coliforms derived from non-faecal sources produced two or more times as much carbon dioxide as hydrogen; in faeces-derived strains, the ratio was 1:1. Low-ratio cultures also produced indole from tryptophan.

Clark and Lubs were able to correlate the gas ratio data with the much easier to perform methyl red (MR) test. Low-ratio cultures--faecal coliforms--turned the methyl red indicator a brilliant red. Koser18,19 found the MR and VP tests inadequate for faecal coliform characterization and suggested a citrate utilization procedure to differentiate coliforms in polluted water (citrate-) from those in unpolluted water (citrate+). Based on an analysis of the literature on coliform differentiation, Parr chose the indole, MR, VP and citrate tests as the combination of four procedures that would yield the best classification and introduced the mnemonic IMViC to facilitate the expression of results. IMViC types ++­­, +­­­ and -+--, or the Escherichia group, were to be considered of faecal origin. A second approach to coliform differentiation is the elevated temperature test originally proposed by Eijkman in 1904. Geldreich, Clark and their co-workers carried out an extensive evaluation of these procedures and reached the following conclusions: 44.5°C was the best temperature for separation of the faecal coliform group; false negatives and false positives tend to cancel each other out; and the EC broth described by Perry and Hajna was the best medium for the test. When these findings were applied to studies on coliforms isolated from faeces,2 soils and vegetation, a greater positive correlation was found with the elevated temperature procedure (96.6%) than with the IMViC series of biochemical reactions (87.2%).

A new method to differentiate coliforms is based on the selective ability of E. coli to metabolize 4methylumbelliferyl-b-D-glucuronide (MUG). When MUG is used both as the sole source of energy and the indicator in a medium, it is hydrolysed by E. coli to form 4-methylumbelliferone, which fluoresces under longwave ultraviolet light. Survival and Aftergrowth Although all the coliform genera (Escherichia, Klebsiella, Citrobacter and Enterobacter) are present in fresh faeces and in fresh pollution from faecal sources, they may not all persist in water for the same length of time. Escherichia coli, for example, is generally most sensitive to environmental stresses and least likely to grow in the environment. Klebsiella, Citrobacter and Enterobacter, on the other hand, are more likely to persist and to grow on organic-rich materials or in organic-rich waters. They may also form a biofilm within the distribution system that is resistant to chlorination and other eradication measures. Regrowth of coliforms in the distribution systems presents a serious problem to water purveyors: the sporadic positive coliform results make it difficult to assess the true hygienic status of the water. Although identification to species of positive coliform tests should be performed, the presence of organisms apparently as a result of "aftergrowth" should not be ignored. Applications to Water Studies


Biology 447 ­ Environmental Microbiology

The faecal coliform test has been shown to be an indicator of the potential presence of enteric pathogens in water. A relationship between the faecal coliform density and the frequency of Salmonella detection has been demonstrated. At faecal coliform densities of 1 to 200 CFU (colony-forming units) per 100 mL, Salmonella was detected in 28% of the water samples examined; this frequency rose to 98% in waters with a faecal coliform count above 2000 CFU per 100 mL. Studies on survival in river water, well water and septic tank water have shown that faecal coliforms persist longer than Salmonella organisms. Because it is relatively specific for faecal contamination, the faecal coliform measurement is preferred for monitoring raw water quality and for indicating the potential presence of pathogens at source. It is also of value in testing untreated drinking water supplies. Any untreated supply that contains faecal coliforms should receive disinfection. The total coliform test, on the other hand, is less reliable as an indicator of faecal pollution. However, because of its superior survival characteristics, the total coliform group is preferred as an indicator of treatment adequacy in drinking water supply systems. The presence of any type of coliform organism in treated water suggests either inadequate treatment or contamination and therefore should not be tolerated. Heterotrophic Plate Count Although attainment of a coliform level of less than 10 CFU per 100 mL in a given sample is considered to satisfy the bacteriological requirements for potable water, there are many micro-organisms commonly present in drinking water whose numbers far exceed those of the coliform group and that can interfere with the development of coliforms.

The heterotrophic plate count (HPC, formerly known as standard plate count) provides an index of the level of this general bacterial population. No single medium, temperature or incubation time will ensure the recovery of all organisms present in water. The 16th edition of Standard Methods for the Examination of Water and Wastewater does, however, specify requirements that will permit a meaningful standard count of selected members of the bacterial population. These counts can be used for quality control in water treatment plants and as a measure of quality deterioration in wells, distribution lines and reservoirs.

In some jurisdictions, the background colony counts on total coliform membrane filters are used as a convenient and inexpensive index of the HPC.

Elevated background counts not only are an indication of increased concentrations of the general bacteria population but can also suppress the development of any coliform bacteria that may also be present. The general population of bacteria in potable water may include some genera that could, under special circumstances, constitute a health risk.

Some species of Pseudomonas can become serious secondary pathogenic invaders in post-operative infections, burn cases and the very young. Flavobacterium has been reported as a primary pathogen for some surgical patients. The HPC is not a true indicator of potential pathogens of this type, as no constant relationship appears to exist between the HPC and the number of pathogens that might be present. It does seem reasonable to assume, however, that chance occurrences are proportionately greater as the general bacterial population increases:

Muller cited evidence from three typhoid fever outbreaks in Germany that supports the use of the HPC as an indicator. A sudden rise of a colony count that had been low for several years signaled the beginning of the 1926 outbreak in Hanover. Four thousand cases of disease were attributed to these unspecified organisms. The high colony counts were detected before the water in the distribution system yielded E. coli or coliforms and two weeks before the first cases of typhoid fever were reported. In addition, disease outbreaks have been attributed to un-chlorinated water supplies in which coliforms were not detectable by conventional methods.


Biology 447 ­ Environmental Microbiology

Canadian Water Quality Guidelines (See website for details)

General Topics 1. 2. 3. 4. 5. 6. 7. Derivation of Guidelines Summary of Guidelines for Canadian Drinking Water Quality Guide - Recreational Uses 1 Guide - Recreational Uses 2 Boil Water Advisories Drinking Water Treatment Units 9th National Conference on Drinking Water May 16, 17 & 18 2000

37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. Dinoseb Diquat Diuron Dichloroethylene (1,1) Fluoride Gasoline Glyphosate Hardness Iron Lead Magnesium Malathion Manganese Mercury Methoxychlor Metolachlor Metribuzin Microbiological Monochlorobenzene Nitrate NTA (nitriloacetic acid) Odour Paraquat Parathion pH Phorate Picloram Protozoa Radionuclides Radon Selenium Silver Simazine Sodium Sulphate Sulphide Taste Total Dissolved Solids Temperature Terbufos Tetrachloroethylene Toluene Trichloroethylene Trifluaralin Trihalomethanes Turbidity Uranium Vinyl Chloride Zinc

Non-Chemical factors 1. 2. 3. 4. 5. 6. 7. 8. 9. Bacteria Protozoa Microbiological Quality Taste Colour Turbidity pH Temperature Hardness

All Individual Compounds & Issues 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. Aldicarb Aldrin Aluminum Ammonia Arsenic Asbestos Atrazine Azinphos Bacteria Barium Bendiocarb Benzo-a-pyrene Boron Bromate Bromoxynil Cadmium Calcium Carbaryl Carbofuran Carbon tetrachloride Chloramines Chloride Chlorophenols Chlorpyriphos Chromium Colour Copper Cyanazine Cyanide Diazinon Dicamba Dichloroethane (1,2) Dichlorobenzene Dichloromethane Diclofop-methyl Dimethoate


Biology 447 ­ Environmental Microbiology

Key Points

· · · · · · · ·

General properties of epidemiology of water-borne diseases Main causal agents of water-borne disease and their survival times in water How these factors (main causal agents and survival times) affect the relevance and importance of setting and using standards. The characteristics of "good" standards How present standards such as the coliform test match or do not match these characteristics Importance of sewage treatment and water treatment on water-borne disease Main detection methods for viruses, bacteria and protozoa. Example of each. Problems associated with such detection methods. Relationship of detection methods to standards.


Biology 447 ­ Environmental Microbiology

Module Five - Biodegradation Microbiology

Overview of Factors Affecting Biodegradation

Biodegradation is the partial or complete conversion of the compound of interest to its elements. It usually mediated by microorganisms but many macro-organisms can also carry out biodegradative processes. The term "biodegradation" is usually applied to compounds that are xenobiotic - compounds manufactured or used by humans in the course of their activities and thereby introduced as a "foreign" substance (xeno = foreign) into an environment. It is also often applied to the study of the "biodegradation" of naturally occuring compounds such as lignin or cellulose - typically, in that case the compounds studied are those that are more resistant to decomposition. You will see the terms "biotransformation", "partial biodegradation" and "complete biodegradation" used in some literature. These terms are used to distinguish between the complete decomposition of a compound to its elemental form (complete biodegradation) and an intermediate stage of "partial biodegradation" to less complex molecules. This is somewhat of an artificial distinction. To restrict the use of the term "biodegradation" to mean "complete biodegradation" is too limiting. "Biotransformation" has also come to mean the changing of a compound to another reasonably stable molecule (often one that is useful or one that is less or more toxic than the original) Biodegradation of a given compound is a complex result of the interacting factors listed in this diagram. It is a function of the chemical structure of the compound, the environmental conditions, the organisms present and their quantities, the adsorption, release and solubility of the compound, the general bioavailability of the compound, interactions with other compounds present in the environment, kinetics of growth and metabolism, threshold effects, co-metabolic processes, acclimation effects, and others.

The diagram shows the many interactions that should be considered when examining biodegradation processes. Predicting the outcome is difficult if not impossible in a "new" situation. Field trials are an essential part of discovering what will happen to new chemicals introduced into the environment. On the basis of past results from many other chemicals under many different environmental conditions, it is, however, sometimes possible to infer which types of compounds might be more or less biodegradable under specified conditions. The actual rates (or kinetics) of biodegradation are always difficult to predict.


Biology 447 ­ Environmental Microbiology


Biology 447 ­ Environmental Microbiology

Biological Factors



The role of organisms, both micro- and macro-organisms, in biodegradation is complex. It is a function of the organism's presence in an environment, their ecology, their biomass, their metabolism (enzyme complement and efficiencies), growth rate and kinetics (of both growth and metabolism). When a compound subject to biodegradation enters an environment, its immediate and eventual rate of biodegradation will be a result of the interactions of these biological factors with the chemical structure of the compound, the environmental factors, factors affecting its bioavailability and the other factors listed below in the diagram. Some biological factors that might not be immediately obvious are the rates of predation of microorganisms by protozoa in the different environments, the effects of the rhizosphere (the soil zone around plant roots affected by exudates from the roots) on microbial growth, the lytic effects of viruses (bacteriophages) and Bdellovibrio and organisms that excrete enzymes that can destroy the cell walls of other microorganisms. Therefore, predicting the outcome is difficult if not impossible in a "new" situation. Field trials are an essential part of discovering what will happen to new chemicals introduced into the environment. On the basis of past results from many other chemicals under many different environmental conditions, it is, however, sometimes possible to infer which types of compounds might be more or less biodegradable under specified conditions. The actual rates (or kinetics) of biodegradation are always difficult to predict.




Environmental factors (physical, chemical and biological) can have effects on the biological activities of microorganisms (growth rates, kinetics of biodegradation, etc.), sorption of the compound to materials in the environment, toxicity, bioavailability, and the observed recalcitrance of the compounds

Chemical Factors



There are over 100,000 organic chemicals produced commercially, yet probably only a few hundred of these have been well studied for their environmental effects or their impact on ecosystem or human/animal health. The main industries involved are:

Petrochemical Industry - largest producer in terms of volume. It includes the oil and gas industry, refineries and production of basic "building block" chemicals such as ethylene, benzene and vinyl chloride.

Saturated aliphatics (mainly gasoline and fuel oils) make up the bulk of the chemicals (about 70%) in gasoline but the remaining 30% includes many aromatic hydrocarbons such as xylenes (about 10%), toluene (about 5%), 1,2,4trimethylbenzene (about 3%), 1-methyl-3-ethylbenzene (about 3%), benzene (about 2%), etylbenzene (about 1.5%), C11-alkylbenzenes (about 1.5%), 1,3,5-trimethylbenzene (about 1%), and methyl-tert-butyl ether (?%). There are many more compounds in smaller quantities including some metals such as manganese, lead, (less so in North America) and others. The next group from the petrochemical industry is the "bulk" chemicals that are used for further organic syntheses. These include ethylene, ethylene glycol, propylene, propylene glycol, isopropyl alcohol, acetic acid, formaldehyde, benzene, acrylonitrile, toluene, xylenes, and styrene. In lesser quantities are the chlorobenzenes, anilines, nitrobenzenes, phthalates, naphthalene and many others.


Biology 447 ­ Environmental Microbiology

Some compounds produced from ethylene glycol are themselves significant in quantity. They include many plasticizers, copolymers. de-icing chemicals, surfactants, detergents, textile finishing chemicals, and industrial solvents. Degreasing solvents are widespread and include trichloroethylene, tetrachloroethylene, trichloroethane, methanol, and chlorfluorohydrocarbons (CFCs). Replacements for these compounds are now being introduced and include isopropyl alcohol, terpenes, hydrochlorofluorocarbons, and pyrrolidones. Typical disinfectants include phenol, cresol , phenylphenol, hexachlorophene and sulfonic acid derivatives like chloramine-T. Many disinfectants have been "de-listed" in the past (i.e. removed from approved lists of the EPA and similar agencies) because of concerns about their safety. PCBs and chlorinated dioxins are well known chemicals used as heat transfer fluids, hydraulic fluids, transformer insulation and solvent extenders. They have been phased out for many uses but large quantities have already entered the environment. Dioxins are typically products of combustion or occur as impurities produced during the synthesis of chlorinated organic chemicals.

Plastics Industry - closely related to the petrochemical industry and uses very large quantities of some products such as styrene,

vinyl chloride, aniline, methyl acrylate, terephthalic acid, toluene, benzene and methanol. They also modify the properties of plastics using a very wide range of specialized organic chemicals

Pesticide Industry - with the plastics industry, the pesticide industry is an important consumer of petrochemical feedstocks. There are many different chemical classes of compounds manufactured and used as pesticides. The more common ones are examined in this course (Module 5) Paint Industry - solvents are the major ingredient in paint formulations although "water-borne" paints are becoming more

common. The common solvents are toluene, xylene, methyl ethyl ketone (MEK), and methyl isobutyl ketone. The replacement "water borne" formulations include alkyl acetates, polyglycol ethers and ketones.

Wood Preservation Industry - the compounds used to impregnate or treat wood to prevent decay include chlorinated benzenes, chlorinated phenols (pentachlorophenol especially), creosote and fungicides such as tributyl tin and 8-hydroxyquinoline. Large quantities of metals such as arsenic, copper, tin and some chromium are used - often in an organometallic formulation (naphthenates, etc.) in the so-called "pressure treated wood".

Other industries using organic chemicals in significant quantities include the pharmaceutical industry, the metal finishing industry (degreasing), and the explosives industry.

Organisms - Overview

The most striking feature of a survey of the microorganisms involved in biodegradation processes is their large numbers, ubiquitous presence and varied capabilities. Rather than list the organisms involved (including the bacteria, fungi, actinomycetes, protozoa, etc.) a very brief treatment of the reasons for this versatility is given.


Biology 447 ­ Environmental Microbiology

Microorganisms as a group show a very wide tolerance range for environmental factors - very low to very high pH levels, 0C to +80C temperature, low to high redox potentials (Eh), low to high nutrient levels, resistance to toxic compounds and elements, etc. Microorganisms as a group also show enormous biochemical versatility. One can find examples of microorganisms growing on almost every conceivable substrate, using almost every energy source, carbon source, sources of nitrogen, phosphorus and sulfur, using simple to complex molecules. Obviously, not all substrates and energy sources are used in all environments, but the ecological range of the microorganisms is very wide. Microorganisms tend to change their phenotypic composition more readily than higher organisms. The bacteria are haploid and so immediately express any mutation in the single set of genes - there are no recessive/dominant effects to prevent this immediate expression. Microorganisms grow very fast - at some of their growth rates, with an unlimited source of nutrients, a culture derived from a single cell could surpass the weight of the Earth in just over 24 hours. You can calculate this in an Excel

Spreadsheet using doubling times for the bacteria of 20 mins and then 10 mins.

They are present in very large numbers in most environments - soil contains between 10000 and 100 million microorganisms per gram - most are inactive or even dormant, but are available if suitable nutrients are delivered to the environment.

Effects on Growth



Environmental factors have a large influence on the growth rates (kinetics) and extent of growth (biomass production) of microorganisms. Their effects are usually expressed as a tolerance range with an optimum point. This is really a laboratory phenomenon, most microorganisms are growing in low nutrient conditions, well below their maximum possible growth rate, in environmental conditions that are not at their optimum value. Any given nutrient can be a limiting nutrient for growth and any given environmental parameter (pH, temperature, osmotic pressure, moisture content, etc.) is also probably at suboptimal levels for any given microorganism. Statistically - they have to be at suboptimal levels since there is only one optimum and many (an infinite number !) of suboptimal levels. Environmental factors (physical, chemical and biological) can have effects on the biological activities of microorganisms (kinetics of biodegradation, etc.), sorption of the compound to materials in the environment, toxicity, bioavailability, and the observed recalcitrance of the compounds. All of these can affect the growth rates.




There are only a few generalizations that are possible regarding the effects of chemical structures and groups on the biodegradation of compounds. This is because of the enormous complexity of the factors affecting biodegradation (as you have seen). The research studies performed have often been targeted more towards specific groups of chemicals (pesticides, solvents, etc.) than towards finding general principles of structure-function effects. Also some structure-function effects change the bioavailability of compounds and those changes may be different in different environments. In addition, acclimation effects due to structure-function effects may (and probably are) different in different environments. Some generalizations that are possible include the following: Xenobiotics Molecules that are "new" to the environment will tend to be more resistant than molecules that have been in existence for millions of years. Simply due to the mutation and evolution of microorganisms capable of their breakdown, these "older" molecules will have recognized biodegradation pathways. Note that we are using human time scales here - if the compound disappears in a conveniently short time according to our time scale then it is


Biology 447 ­ Environmental Microbiology

"biodegradable". In fact, some naturally occurring compounds persist for long times in certain environments - Peat has been found to persist for +25,000 years in some Russian peat bogs, for instance. We are introducing newer xenobiotic chemicals to the environment at an increasing rate. It is not surprising, therefore, that in the early stages of this introduction in the 1940s to the 1960s, no account was taken of their persistence in the environment The results were DDT, PCBs, and chlorinated hydrocarbons in general, and other very persistent molecules. Xenophore effects: It seems clear that the addition of certain groupings or elements at given positions on molecules leads to persistence of that molecule. Very often the new molecule is also a xenobiotic (did not occur in nature until we synthesized and released it). Examples of this xenophore effect: 1. Addition of halogens, sulfonic acid, methoxy and NO2 groups (xenophores) increases persistence (but not always) 2. Addition of more xenophores tends to make the molecule more resistant 3. The position of the xenophore has a significant effect - a xenophore at one position can make a molecule more persistent in one environment but make it less persistent in another. 4. Typical xenophore groups (at least in aerobic environments) are: o Halogens (chlorine, bromine, iodine) o Nitrite o sulfonic acid groups o methoxy groups o branched methyl groups o branched alkanes versus unbranched alkanes o ......... and many other examples

Physical Factors - Overview

The physical factors that can affect biodegradation include: · · · · · · pH temperature osmotic pressure pressure (at depth in marine environments or in deep groundwater, for instance) salinity soil moisture level

Microorganisms have a range of tolerance values for these factors and, if the conditions are not within that range, there will be no biodegradation activity.




The literature on microbial metabolism is vast ! Rather than look at that literature this section examines how chemicals enter microbial cells, especially those chemicals that are only slightly soluble or are effectively insoluble. The question then is really - How do microorganisms biodegrade compound like PCBs, dioxins, and heavy fuel oils that are essentially insoluble ?


Biology 447 ­ Environmental Microbiology

It is easy to understand the process involved if hydrolytic enzymes are excreted by the microorganisms and they break down the larger insoluble molecules to smaller more soluble sub-units or fragments. This process certainly occurs with molecules such as cellulose and lignin - both naturally occurring molecules from plant debris. This is not the case for smaller molecules (lower molecular weight) that are effectively insoluble - How do microorganisms degrade those ? The key feature is the mechanism by which the molecules are transported into the cell where they can be metabolized. There seem to be 3 main mechanisms: · · Only the small amount of the chemical that IS soluble in water enters the cell - further amounts of the chemical are then only available through further partitioning of the chemical into the water phase. The microorganisms secrete compounds (surfactants or emulsifiers) that convert the molecule into very small droplets (maybe much less than 1 M) and these are assimilated by the organism in some fashion. Note that the increased surface area due to this emulsification process may be enough in many cases to explain the increased activity simply due to faster partitioning of the compound into the water phase. Direct passage through the cell membrane by close contact with the compound (solubilization into the lipid phase of the membranes)


Once the compound is inside the cell, then normal metabolic processes can take place and transform the molecules.

For details of these biodegradation metabolic processes, see the: The University of Minnesota Biocatalysis/Biodegradation Database

Chemical Factors



There are many chemical factors that can influence the activities and biodegradation of compounds introduced into an environment. They include: · · · · · · · · Nutrient status Oxygen concentrations * The Eh or Pe - the "reducing potential" of the environment. Presence of toxic compounds Inorganic chemicals in the environment acting as sorbants, buffers, surfaces, etc. Acid/base composition (effects on pH) Carbon dioxide levels Other gases

This Eh or Pe is a function of the amount of oxygen present in the environment, and if it is absent, the Eh is usually controlled by the presence of specific ionic forms of chemicals. See the Groundwater Module (Module 7) for further details


Acclimation - Overview

It is often observed that a chemical, especially pesticides used in fairly large quantities, exhibit a phenomenon known as "Acclimation" ; the second and subsequent applications are degraded more quickly than earlier ones. This has been seen with 2,4-D, MCPA, TCA, dalapon, chlorpropham, monuron (all herbicides), methyl parathion, azinphosmethyl (insecticides), naphthalene, anthracene (polyaromatic hydrocarbons), phenol, 4-chlorophenol, 4nitrophenol, 1,2- and 1,4-dichlorobenzene, pentachlorophenol and 3,5-dichlorobenzoic acid. The length of the acclimation period varies considerably and may range from hours to months. It varies with concentration of the compound, organisms present in the system, and the environmental conditions.


Biology 447 ­ Environmental Microbiology

It is thought to occur for the following reasons: · · Growth of initial small populations of microorganisms capable of metabolizing the compound Presence of toxic compounds (including the chemical being examined) - the toxic compound may have to be removed before significant degradation can occur. If the compound itself is toxic, then it may then inhibit the faster-growing microorganisms capable of degradation, giving a selective advantage to slowergrowing organisms that also degrade it - but initially more slowly until they increase in biomass. Appearance of new genotypic variations of microorganisms are needed. Mutation and selection may occur. Diauxic growth - the microorganisms grow on an easily available substrate before using a less available one. This might be a factor in systems where only a few different types of bacteria are present. Enzyme induction and the "lag phase"

· · ·





EPA Summary Paper

Basic Concepts of Contaminant Sorption Introduction The Robert S. Kerr Environmental Research Laboratory (RSKERL) has developed a number of Issue Papers and Briefing Documents which are designed to exchange up-to-date information related to the remediation of contaminated soil and ground water at hazardous waste sites. In an attempt to make the content of these documents available to a wider audience, RSKERL is developing a series of Summary Papers which are condensed versions of the original documents. Understanding the processes which dictate transport and fate characteristics of contaminants in soil and ground water is of paramount importance in designing and implementing remediation systems at hazardous waste sites. Sorption is often the most significant of these processes. This summary paper addresses the basic concepts of sorption in soil and ground water with an emphasis on organic contaminants having the characteristics of those often found at existing hazardous waste sites. The Concept of Sorption Sorption can be defined as the interaction of a contaminant with a solid. More specifically, the term can be further divided into adsorption and absorption. The former refers to an excess contaminant concentration at the surface of a solid while the latter implies a more or less uniform penetration of the solid by a contaminant. In most environmental settings, this distinction serves little purpose as there is seldom information concerning the specific nature of the interaction. The term sorption is used in a generic way, to encompass both phenomena.


Biology 447 ­ Environmental Microbiology

There are a number of factors which control the interaction of a contaminant with soil or aquifer materials. These include chemical and physical characteristics of the contaminant, composition of the surface of the solid, and the fluid media encompassing both. By gaining an understanding of these factors, logical conclusions can often be drawn about the impact of sorption on the movement and distribution of contaminants in the subsurface. The failure to take sorption into account can result in a significant underestimation of the amount of a contaminant at a site as well as (underestimation of) the time required for it to move from one point to another. Soil and aquifer materials are primarily composed of sand, silt clay, water, and a highly variable amount of natural organic carbon, the presence of which profoundly complicates a soil's sorptive properties. The combination of these characteristics describe the surfaces offered as sorptive sites to contaminants in water passing through the subsurface matrix. It can be deduced that sandy materials offer little in the way of sorptive surfaces to passing contaminants while silts and clays, particularly those having substantial amounts of organic matter, provide a rich sorptive environment for many types of contaminants. Even the most porous and highly productive aquifers, composed of sands and gravels, usually have some fine grained material, small amounts of which can result in a substantial increase in the sorptive behavior of the aquifer material. Most organic contaminants of environmental concern have a limited solubility in water and therefore tend to be associated with the solid rather than the water phase. But even with limited solubilities, many hazardous chemicals can be detected at measurable, and often toxic concentrations in water. One of several ways used to describe the distribution of contaminants between the solid aquifer matrix and water is the sorption coefficient Kp:

The value of Kp is dependent upon the characteristics of the contaminant, the type of aquifer material, and the amount of soil organic carbon. In order to better understand the utility of this concept assume Kp = 1.0 which dictates that the contaminant is equally distributed between the liquid and solid phases. Since soil is about 2.5 times more dense than water, 2 liters of aquifer would contain 1 liter of water and 2.5 kg of soil. Therefore, 1.0 mg/l of the contaminant would be associated with the water and 2.5 mg (70 percent) would be sorbed to the solid phase. As can be seen from this example, sorption tends to complicate remediation techniques that require pumping water to the surface for treatment. The slow desorption of contaminants from the solid to the liquid phase can significantly reduce the effectiveness of a pump-and-treat system by progressively lowering contaminant concentrations in water pumped to the surface. It is not uncommon to pump a system until contaminant concentrations in the pumped water meet a mandated restoration level, while the aquifer's solid phase still contains a substantial contaminant mass. Therefore, when the pumps are turned off, concentrations in the ground water soon return to their previous level. The quantity and distribution of contaminants in the subsurface is of paramount importance in designing extraction or in-situ remediation systems. In this regard, it is necessary to obtain the best information possible on contaminant sorption. Therefore, tests to determine a sorption coefficient (Kp) should be made with the contaminants of concern, as well as soils and aquifer material from the specific site.


Biology 447 ­ Environmental Microbiology

Bioavailability - Overview

There can be many reasons why a particular compound, although biodegradable in testing, is not biodegraded: · · · · · · Required nutrients are missing Environmental conditions are unsuitable Toxic substance concentration is too high Compound may be at too low a concentration Compound may not be bioavailable

Required nutrients are missing

One or more nutrients required for growth are missing. The "limiting" nutrient is the one that is exhausted first in the growth cycle. This assumes that growth is required for biodegradation; this may not be true if sufficient biomass is already present, or if the compound is degraded by enzymes already present. · Environmental conditions are unsuitable

There are many environmental conditions that can inhibit growth or metabolism; high or low pH levels, high or low Eh (redox potentials), high or low temperatures, etc. A good example is in some peat bogs where organic materials may be over 20,000 years old. · Toxic substance concentration is too high

Some conditions (high hydrogen sulfide levels, high acid concentrations, etc. may inhibit growth or metabolism. · Compound may be at too low a concentration

If a compound is degraded through the growth of microorganisms on that compound, there will be a minimum concentration below which growth will not occur. There will be an even lower concentration that will not even provide maintenance energy levels sufficient for the organism. · Compound may not be bioavailable

Bioavailability can be affected by: · · · · A. Sorption o o Adsorption is the retention by solid surfaces of solutes from a solution. Absorption is the uptake and retention of solutes within the mass of a solid. A. Sorption to some solid materials in the environment B. Presence in a NonAqueous Phase Liquid (NAPL) C. Confinement or entrapment in physical soil or aquifer matrix D. Complexation

The term Sorption is used to cover both Adsorption and Absorption Chemistry of Sorption: Organic compounds can be sorbet by a wide variety of constituents of soils and groundwater systems. The main sobbing constituents are clays and organic matter. The clays are of two types. The first, where the Si and Al layers in the crystalline lattices are in an approximate 1:1 ratio are


Biology 447 ­ Environmental Microbiology

the kaolinite type or "non-expanding lattice type. The second type has an approximate 2:1 ratio of Si and Al and is the montmorillonite or expanding lattice type. The second expanding lattice type swells when in contact with water and has an enormously greater water-holding and sorptive capacity. This is because materials can enter into the lattice as it swells and attach (sorb) to the vastly increased surface area thus produced. The clays have a net negative charge on their surfaces. Materials can be sorbed by hydrogen bonding (usually the larger molecules do this), van der Waals forces, ion exchange or chemisorption. The organic fraction is often responsible for sorbing many compounds, especially those that are hydrophobic in nature. The extent of this retention is correlated wit the octanol-water partition coefficent (see below) since the humic organic materials behave as hydrophobic molecules. The hydrophobic molecules may also diffuse into the complex polyaromatic structure of the humic acids and humins. Sorbed Substrate Availability In many instances, the sorbed molecules are less available for metabolism by microorganisms. Sometimes they are so tightly sorbed (the herbicide paraquat on clay or organic matter, for example) that they are effectively completely unavailable and persist for very long periods. This occurs to a lesser extent with other pesticides and organic molecules that are sorbed to organic matter and clays. The effect can become complex because the surfaces that sorb the compounds also sorb microbial nutrients and can thus stimulate growth. This is especially true when the microorganisms themselves are attracted to or sorbed on these same surfaces. Movement in Soil and Groundwater The sorption of organic molecules onto organic matter is of particular relevance in groundwater systems. In a normal aquifer, the organic matter content on the inorganic matrix is usually quite low. The presence of this organic matter causes the effect of "retarding" the passage of the organic molecules through the aquifer as the groundwater flows through it. (see Module 8 - Movement of Groundwater and Contamination of Groundwater) B. NAPLs NAPLs - NonAqueous Phase Liquids In some cases, the compounds are not in an aqueous phase, but exist in liquids that are immiscible with water. These NAPLS are typically petroleum products spilled from refineries, tankers, or organic solvents leaking into the environment. If their density id less than water, they will separate into a floating phase on the surface (most oil and gasolines, for example). The organic compounds partitioned into NAPLS are usually less available than in the aqueous phase. The surface area of the NAPL in contact with the aqueous phase becomes important since the partitioning between the phases is one mechanism whereby the compounds become amenable to aqueous phase decomposition by the microorganisms. There are other mechanisms that can stimulate decomposition of compounds in NAPLS: --- extracellular hydrolytic enzyme activity, activity of excreted surfactants and emulsifying agents and direct microorganism contact with the NAPL coupled with direct uptake into the microbial lipid materials.

Solubility of various organic compounds in water


Biology 447 ­ Environmental Microbiology

Group Compound Solubility (mg/L) or ppm Aliphatic Hydrocarbons Heptane (6Carbons) Octane (8C) 2.9 0.66

Nonane (9C)


Decane (10C)


Hexadecane (16C)


Eicosane (20C


Aromatic Hydrocarbons

Naphthalene Biphenyl

31 7.2














Palmitic acid 4-chlorobiphenyl

0.0035 0.96



Octanol Water Partition Coefficient This measure is the partition coefficient derived when a compound is mixed with water and octanol and allowed to equilibrate. It is supposed to represent the degree of bioconcentration that compound would undergo in animal tissues because octanol is a surrogate for the lipids in animal tissues. A compound that is very slightly soluble in water (hydrophobic) is usually soluble in lipids (or octanol). Compounds soluble in water (hydrophilic) are not soluble to the same extent in lipids. Compound Logarithm of the octanol-water partition coefficient. Log Kow


Biology 447 ­ Environmental Microbiology

Dioxane Acetone Pyridine Benzene Toluene Xylene Diphenyl phthalate Diphenyl ether Decane Tetradecane Dioctyl phthallate -1.1 0.23 0.71 2.0 2.4 3.1 3.3 4.4 5.6 7.6 8.8


DNAPLs - Dense NonAqueous Phase Liquids

These solvents are heavier than water and sink to the bottom of aquifers, lakes or streams. Similar principles apply to microbial bioavailability and utilization of compounds dissolved in these DNAPLS, but with the added complication that they are even less available because of the DNAPL "sinking" behaviour. C. Entrapment or Sequestration Compounds can became "entrapped" inside soil or sediment structures (like soil aggregates) and may become less bioavailable. This is a physical effect - the compound is simply sheltered from microbial attack since the microorganisms cannot penetrate the physical structures. This often because the very small pore sizes in such structures prevent physical access. D. Complexation

Thresholds - Overview

Since many organic pollutants are present only in very low concentrations in the environments, the concept that there might be a minimum concentration below which they would not be biodegraded is and important and interesting one. Even though the concentrations of these compounds in the field are low, they still can have important effects. · · · · Very low levels of a compound, when applied to an entire population, may cause significant risk to some of that population. Compounds can be bioconcentrated to higher levels in target organisms and cause effects. Even at these low concentrations, some compounds are toxic, especially to aquatic organisms Some standards for human and ecosystem health are set very low because the risk analysis procedures tell us that the risk is only acceptable at these low acceptable concentrations.

The concept of a threshold concentration that is below that needed for microorganisms to take in, assimilate and biodegrade becomes very important in these cases. Some examples ?


Biology 447 ­ Environmental Microbiology

Concentrations where biodegradation ceased or was much slower than predicted: Chemical 2,4-D herbicide Aniline Phenol Sevin insecticide 2,4-dichlorophenol Carbofuran 2,4,5-T herbicide 1,2-, 1,3- and 1,4dichlorobenzenes Source Freshwater Freshwater Freshwater Freshwater Freshwater Soil Soil Biofilms on glass beads in a treatment column Concentration mg L-1 or mg kg-1 soil 2.2 0.1 0.0015 3.0 2.0 10 100 0.2 to 7.1

The threshold is the lowest concentration of compound that sustains growth .Above the threshold level of a compound, the organisms are able to grow and bring about detectable chemical changes. Organisms all require a minimum concentration of an energy source (and everything else they require) in order to survive - even though they are not able to proliferate (or even increase in cell size). This maintenance energy level is almost certainly related to the observed threshold levels for compounds supplying that energy. Note: this concept of a threshold assumes that growth is required for biodegradation - but see co-metabolism and secondary substrate metabolism. In both cases compounds can be metabolized when they are present in concentrations below that required to provide only for maintenance energy yields. In essence, the organisms are growing on another energy source and the compound in question is being "accidentally" metabolized even at the very low concentrations.




Co-metabolism can be defined as: "The transformation of an organic compound by a microorganism incapable of using the substrate as a source of energy or of one of its constituent elements"

(Alexander, 1967. Agriculture and the Quality of Our Environment (N.C. Brady, Ed.) pp 331-342. Am. Soc. Adv. Sci. Washington D.C.)

What this means is that the organism in question derives no benefit from the co-metabolism of the compound. No energy is available, nor are any of the (C, N, P, S, etc) elements in the compound used as a significant source of material for biosynthetic activity. The term "secondary substrate metabolism" is also in the literature - this means that the organism is actually growing on a second substrate and is transforming another substrate at the same without gaining benefit. This undoubtedly occurs, but is very difficult to detect and prove in natural environments, even though it can be demonstrated in pure cultures. For this reason, co-metabolism is taken to be either case: 1. The organism in question is NOT growing on another substrate - it may not even be proliferating at all.


Biology 447 ­ Environmental Microbiology

2. The organism IS growing on another substrate (known or unknown) but is also co-metabolizing the compound in question. Mechanisms of co-metabolism 1. Initial enzyme or enzymes change the substrate to a product that is not further transformed by the other enzymes in the organism to anything that can be further metabolized. Do enzymes have many different substrates ? 2. The original substrate is transformed to a product that inhibits later enzymes in a metabolic sequence or .inhibits the growth of the organism. 3. The organism needs a second substrate to achieve some later reaction - that substrate is missing. The first explanation is the most likely in most cases. It implies that co-metabolism is "accidental" or "fortuitous". An enzyme can react with a compound, produce no energy for the cell, nor incorporate any of the elements of the compound into biosynthetic processes, but still achieve partial transformation of the compound. It may be that many of the compounds co-metabolized are similar to normal substrates of the cells, differing sufficiently so that their immediate products cannot be further metabolized. For example: Enzyme A ----------> Enzyme B -------------> Enzyme C

Substrate A ----------> Product B ------------> Product C Substrate Ax-----------> Product Bx [not metabolized by enzyme C] Substrate Ax is "sufficiently similar" to Substrate A that Enzyme A can transform it to Bx, but Bx is "sufficiently different" to B so as to prevent further metabolism by Enzyme C.

Recalcitrance - Overview

Recalcitrance (resistance to biodegradation) may be due to biological, environmental or chemical reasons. There are attempts to predict this recalcitrance using various measures short of a full biodegradability investigation. A. Biological and Environmental Factors

There are innumerable interactions between the biology, chemistry of the compounds and the environment that can cause recalcitrance of given molecules. Rather than repeat them, we will examine below the situation where a particular compound, although biodegradable in testing, is not biodegraded: · · · · · · · Required nutrients are missing Environmental conditions are unsuitable Toxic substance concentration is too high Compound may be at too low a concentration Compound may not be bioavailable Microbial population does not produce correct enzymes

Required nutrients are missing


Biology 447 ­ Environmental Microbiology

One or more nutrients required for growth are missing. The "limiting" nutrient is the one that is exhausted first in the growth cycle. This assumes that growth is required for biodegradation; this may not be true if sufficent biomass is already present, or if the compound is degraded by enzymes already present. · Environmental conditions are unsuitable

There are many environmental conditions that can inhibit growth or metabolism; high or low pH levels, high or low Eh (redox potentials), high or low temperatures, etc. A good example is in some peat bogs where organic materials may be over 20,000 years old. · Toxic substance concentration is too high

Some conditions (high hydrogen sulfide levels, high acid concentrations, etc. may inhibit growth or metabolism. · Compound may be at too low a concentration

If a compound is degraded through the growth of microorganisms on that compound, there will be a minimum concentration below which growth will not occur. There will be an even lower concentration that will not even provide maintenance energy levels sufficient for the organism to survive in an active state (some may sporulate). · · Compound may not be bioavailable (see details) Microbial population does not produce correct enzymes

As a result of many possible factors, the microorganisms present in a given environment might not be able to produce enzymes capable of degrading a particular compound. This would probably be a result of the environment not supporting those microorganisms. However, it is conceivable that the organisms are simply "missing" from that habitat, even though the environment would support their growth

B. Chemical - Structure-function relationships

In addition to these biological and environmental factors, some chemical structures and molecules are resistant to the range of enzymes produced by living organisms including microorganisms. See: Chemical Structures leading to recalcitrance

Predicting recalcitrance and "Biodegradability" of a new compound.

Approximately 1 to 2% of the new compounds submitted to the Environmental Protection Agency in the US each year have been tested for biodegradability. In some cases, this is obviously not required, but in many cases the information would be useful. There is an obvious need to be able to predict which compounds need further examination and confirmatory testing on their biodegradability or their recalcitrance. There have been a number of approaches to this problem: · · Assessing the similarity of the new molecule to known examples (the "benchmark" process) Assessing the physical and chemical properties of the compound rather than the biochemical properties. These properties have included solubility, boiling point, melting point, molecular weight,


Biology 447 ­ Environmental Microbiology



molar refractivity, octanol-water partition coefficient, measures of the hydrophobic nature of the compound and others. Analysis of functional groupings in the molecule (e.g. the strength of the C-halogen bond in aromatic halogenated compounds as a measure of its biodegradability in anaerobic environments this is the bond that is "cleaved" first during the initial dehalogenation - removal of the halogen step) Predictions based on molecular topology - or the shape and size of the molecule.

Toxicity - Overview

Toxicity is too large a field to cover in this course. Briefly, toxicological analyses attempt to measure the effects of chemicals on different types of organisms. The dose-response of chemicals applied to these organisms is used to calculate a toxicity concentration that is then applied to regulate the allowable concentration of that chemical in the field. Toxicology tends to be divided into four main areas - Hazard identification, exposure assessment, dose-response measurements and quantitative risk characterization by estimation. The final step - Risk Assessment - is calculating by integrating all of the available data from different sources, the risk that an individual will develop symptoms by a given time for a specified set of exposure conditions. The risk is the probability (from 0 to 1) of this happening to an individual. Toxicologists generally believe there is a threshold dose below which there are no toxic effects in any individual in a population. This is thought to be true for most biological effects except carcinogenicity where a single molecule could in theory cause a deleterious mutation. The basis for this belief is that are doses of toxic chemicals where it is impossible to find differences between treated and untreated organisms. Unfortunately, every test has a statistical "resolving power" or limit of detection based on the number in the sample population exposed and the statistical analysis of the results. It also ignores the effect of a very small dose on pre-existing toxicity from other chemicals. Even that small addition may then cause a small increase in effects. The complicating factors in determining a toxic level of a compound can be numerous. They include the following: · · · · · · · · · Different organisms respond differently to the same dose (or dosage - the received "dosage" versus the applied dose) Organisms at different stages in the life cycle respond differently Organisms at different ages, nutritional states or health states respond differently Compounds can be accumulated, metabolized, immobilized or excreted to varying extents by different organisms or at different stages in the life cycle, age, or nutritional state of the same organisms. It is not always clear which organisms to use as the "indicator" - the most common, the most sensitive, the most valuable, all of the above ? Bioavailability of compounds may play a large role in the actual toxicity Compounds can show antagonistic, neutral or synergistic effects when applied in combinations. Unknown toxic effects can occur - e.g. the teratogenic effects (birth defects) of the drug Thalidomide were not detected during testing because the test animals did not exhibit these effects. Regulation of toxic chemical levels inherently involves the "analysis of the risk" involved at different exposure levels. Risk analysis is difficult and an inexact science. Communication of that risk to the general public is even more difficult.


Biology 447 ­ Environmental Microbiology

What level of risk will people accept ? Most decide on 1 in a million when asked.......until it applies to them or their close relatives.

Integrated Risk Assessment System from the EPA List of Chemicals on IRIS "Top 20" hazardous chemicals (1999) from the EPA Downloadable Database from IRIS - Chemical substance data Toxicological Reviews of Chemicals from the EPA Ecotox software download Ecotox Thresholds (ETs), are defined as media-specific contaminant concentrations above which there is sufficient concern regarding adverse ecological effects to warrant further site investigation.


Microbial degradation of chemicals in the environment is an important route for removal of these compounds. The types of compounds range from plastics through organic chemicals (both industrial chemicals used in large quantities and trace chemicals such as pesticides) to organometallics such as methylmercury. The biodegradation of these compounds is often a complex series of biochemical reactions and is often different when different microorganisms are involved. The particular details of each biodegradation scheme are not particularly important, but the general series of reactions and enzyme types involved are relatively straightforward. The biodegradation schemes and pathways for biodegradation of many chemicals can best be understood by considering the same pathways for natural chemicals such as hydrocarbons, lignins, cellulose and hemicellulose. Many of the pathways, particularly the later stages of metabolism, are similar. The following treatment will deal with lignin and hydrocarbons in some detail, cellulose and hemicellulose in lesser detail and will then consider groups of more toxic chemicals; · · Naturally occurring compounds Hydrocarbons


Biology 447 ­ Environmental Microbiology

· ·

Polyaromatic hydrocarbons (PAH's) Pesticides including organophosphates, chlorinated hydrocarbons, dithioates, carbamates, phenylureas, phenoxvacetates, dipyridinium herbicides, etc.


Biology 447 ­ Environmental Microbiology

General biodegradation of naturally-occurring compounds

There are some compounds which are naturally-occurring and are also recalcitrant; they are resistant to biodegradation in the sense that they are not rapidly degraded in nature. One of the most important of this group is lignin. Lignin is a complex polymeric compound produced from the breakdown of plant materials in soil or water. The general breakdown pattern of plant materials is given below;

Generalized plant material biodegradation Microorganisms respond to the presence of organic materials by growing rapidly and using the easily available parts of the organic material. Different populations of microorganisms then use more resistant parts until a recalcitrant or resistant fraction remains. The order of use is often; · · · · carbohydrates proteins cellulose and hemicellulose lignins and associated compounds.


Biology 447 ­ Environmental Microbiology

There are distinct populations of microorganisms able to degrade each of these categories of organic materials. The environmental conditions play a large role in determining the speed at which the materials are used. The physical form of the materials (finely divided versus granular, crystalline cellulose versus amorphous cellulose, etc.) also plays a large part in determining the rate of reaction. The most important environmental factors are temperature, pH, water content, and oxygen content. There are therefore two main determinants of the fate of organic materials added to a soil or water system: · · the environmental conditions the chemical structure of the compound. Each of these factors works by affecting the microbial colonization and breakdown of the materials. Two examples will be used; cellulose breakdown and lignin breakdown.

Cellulose breakdown

Cellulose is a carbohydrate consisting of 1-4 linked glucose units in a linear chain. It is degraded by a very wide range of bacteria and fungi and is degraded under a variety of different environmental conditions. Typically bacteria of the genera Streptomyces, Cytophaga, Cellulomonas, Nocardia, and Vibrio are involved in cellulose breakdown while Clostridium is an important anaerobic cellulose degrading organism. Some bacteria can degrade it at very high temperatures (C. thermocellum). Anaerobic metabolism results in low molecular weight fatty acids and carbon dioxide while carbon dioxide is the main metabolite under aerobic conditions. Aerobic decomposition is thus more efficient than anaerobic; more cellulose-carbon is transformed to CO2 and biomass. Many fungi are also extremely active in cellulose decomposition; Trichoderma, Chaetomium, Aspergillus, Fusarium, and Phoma are particularly active in soils. The enzymatic processes are similar in most microorganisms; extracellular cellulase enzymes are produced. There are several different compounds in the cellulase enzyme system. The normal cellulase enzyme complex contains three types of enzymes; a Cl enzyme which acts primarily on native cellulose and not on partially degraded cellulose, a Cx enzyme which acts upon partially degraded cellulose molecules in two ways; the endo 1-4 glucanases break the cellulose internally at random while the exo 1-4 glucanases attack the end of the chains resulting in cellobiose molecules. The degradation of the cellobiose and other small fragments is by the enzyme bglucosidase that forms glucose from these fragments.

Cellulose biodegradation enzymes

Lignin Structure

Lignin is a complex polymer found in plants, especially in woody plants. It is an amorphous, three-dimensional aromatic polymer composed of oxyphenylpropane units. It is formed by polymerization in the plant of cinnamyl alcohols; p-coumaryl alcohoI, coniferyl alcohol and sinapyl alcohol


Biology 447 ­ Environmental Microbiology

Lignin alcohols There are three main types of lignin, depending on the type of plant; 1. guaiacyl lignin from conifers, lycopods, horsetails and ferns. Mainly coniferyl alcohol units with small amounts of coumaryl and sinapyl alcohol units. 2. guaiacyl-syringyl lignin from dicot angiosperms and some gymnosperms. Equal amounts of coniferyl and sinapyl units with minor amounts of coumaryl units. 3. guaiacyl-syringyl-p-hydroxyphenyl lignin in the highly evolved grades and woody tissues of conifers. Equal amounts of all three types of units.

Model of lignin structure


Biology 447 ­ Environmental Microbiology

Main bond types linking phenyl-C-C-C (phenylpropane units) are;

Main bonds linking phenyl propane units O - (ether links) are very common Phenyl-C-C-C structure is common Highly aromatic nature of material Large number of methoxy groups Large number of - O - cross links between subunits Lignin biodegradation The lignin molecules are degraded by the "white-rot fungi" and some bacteria. There are several hundred species of white rot fungi including members of the Agaricaceae, Hydnaceae, Polyporaceae, and the Thelephoraceae. The white rot basidiomycetes are probably the most efficient microorganisms at degrading lignin. The soft rot fungi include Chaetomium spp., Paecilomyces and Allescheria spp. Bacteria such as Pseudomonas, Xanthomonas, Acinetobacter, Bacillus, Arthrobacter, Micrococcus, Aeromonas, Chromobacterium, and Flavobacterium, have all been implicated in lignin breakdown. Good evidence has been given for Streptomyces, Bacillus, Nocardia, and Pseudomonas spp. in that they have released 14CO2 from radiolabelled lignin molecules.


Biology 447 ­ Environmental Microbiology

Hydrocarbon biodegradation

For a good introduction to nomenclature and models of organic molecules, see the site by Dave Woodcock from Okaganan College.

Main types of hydrocarbons Natural sources of hydrocarbons tend to be complex mixtures of the different types above. The particular composition varies so greatly that it is often possible to analyze a particular hydrocarbon mixture with such discrimination that the oil well and certainly the oil field, where it originated can be identified. This is usually done by gas chromatographic techniques, yielding "fingerprints" of the particular oil;


Biology 447 ­ Environmental Microbiology

Gas chromatographic analysis of crude oil Analysis by gas chromatographic techniques are essentially fractional distillation methods; higher boiling point hydrocarbons are delayed in the GC column and so elute later in the process. In other words, different fractions of the oil "boil off" at different temperatures. This is the basis of oil refining, where the crude hydrocarbon is heated and the different components are collected at different temperatures. After refining, the gasoline, diesel fuel, kerosene, etc., have a much less complex series of different chain lengths of hydrocarbons. Octane is a component of gasoline and it is the standard by which the gasoline is rated - the "octane rating". Diesel fuel is less volatile (higher chain lengths). The crude hydrocarbon also contains other components such as metals (vanadium, nickel) and sulfur as well as many different aromatic compounds.

Importance of hydrocarbon microbiology

a) Oil spill problems (marine and freshwater) b) Degradation of stored oil supplies c) Use of hydrocarbons as carbon source for microbial production of food (?) d) Oil prospecting using microbial distributions in soils and air e) Use of bacteria to release oil from oil sands f) Waste disposal - methane production, etc.


Biology 447 ­ Environmental Microbiology

g) Soil gases and groundwater evolution

Mechanisms of degradation

Hydrocarbons are only degraded in water. Bacteria and fungi do not grow in hydrocarbons, they grow on water in or surrounding the hydrocarbons. All hydrocarbons are soluble to some extent. This is why the process of degradation is able to start and then continue. Without the presence of water, oil is not degraded. 2. Emulsion formation is an important part of hydrocarbon degradation. There are two kinds of emulsion; a). Oil-in-water emulsion; are common and occur when small quantities of oil are present in large quantities of water. Droplets of oil are suspended in water. b). Water-in-oil emulsion; are less common and occur when large quantities of oil are present in water. There are droplets of water suspended in a matrix of oil. The damage caused by oil spills in marine or freshwater systems is usually caused by the water-in-oil emulsion. A thick layer of crude oil on the surface of water will take up about 50% water by weight and remain a free-flowing, oily liquid. It can spread over the water surface to form an oily film and wilI eventually disperse and be degraded. When the water content reaches about 80% by weight, the consistency of the emulsion changes - it becomes a thick semi-solid mass with a grease-like consistency. It is often called "chocolate mousse" at this stage because of both its consistency and its brown colour. This process does not readily occur with fuel oils such as diesel oil and kerosene, but occurs easily with light and heavy crude oils - the usual cargoes of oil tankers.

Microbial degradation of aliphatic hydrocarbons

Many types of microorganisms can degrade hydrocarbons. Bacteria, yeasts, and filamentous fungi all have taxa that degrade some types of hydrocarbon molecules. Bacteria - The autotrophic bacteria Thiobacillus and Desulfovibrio can both metabolize the sulfur component of crude oil. Thiobacillus can metabolize S to H2SO4 (sulfuric acid) and Desulfovibrio metabolizes this under anaerobic conditions to sulfide. Both sulfide and sulfuric acid can damage metal containers and fuel systems. This is not true utilization of hydrocarbons as carbon sources, but is important in practical terms; any water present in oil storage tanks may lead to the growth of these organisms and subsequent damage. The heterotrophic bacteria utilize carbon from the hydrocarbons as a source of carbon and energy for biomass production. There are many genera of bacteria which carry out these reactions on different hydrocarbons. They include Bacillus, Pseudomonas, Mycobacteria, and actinomycetes (especially Nocardia spp.) Fungi - Many yeasts are active in soils and water systems in hydrocarbon degradation, as are some very common genera of mycelial fungi such as Trichoderma, Aspergillus and Cladosponum. Note: None of these microorganisms degrade all of the possible hydrocarbon molecules at the same rate if at all. There is a definite preferential use of certain chain lengths of aliphatic hydrocarbons and each organism may have a different spectrum of activity. In general terms, the utilization of n-alkanes (non-branched alkane hydrocarbons) is reasonably well established; Solids Gases --> Liquids C19 C20 C21 ..-> Solid at room

Chain Length ---> C1 C2 C3 C4 C5 --> C10 C11 C12 C13 Not susceptible Attacked by many


Biology 447 ­ Environmental Microbiology

to microbial action


temp. Not susceptible

Methane is attacked only by certain bacteria. This means that a crude oil subjected to microbial action will lose some of the hydrocarbon chain lengths and retain others; it will be "enriched" in those hydrocarbons not susceptible to microbial decomposition.

Biochemistry of hydrocarbon degradation

There are many different reactions involved with the degradation of hydrocarbons by microorganisms. We will deal only with the more common reaction types. A) Hydroxylation at C1. B) Hydro-peroxidation C) Dehydrogenation reactions D) Subterminal reactions


Biology 447 ­ Environmental Microbiology


Biology 447 ­ Environmental Microbiology

Note that the first three processes deal with a terminal -CH3 group while subterminal oxidation process "splits" the alkane at a subterminal site. There are some generalizations that can be made concerning the biodegradation of aliphatic hydrocarbons (alkanes, alkenes and alkines [ alkynes] ). 1. The chain length has a significant effect on biodegradability; it is almost the same for alkanes, alkenes and alkines [alkynes]. Not a simple relationship to number of carbon atoms. 2. Aliphatic compounds are more easily degraded with decreasing saturation and increasing reactivity; Thus;

3. The degree and type of branching in a structure has a marked effect on the biodegradability of aliphatic compounds. In general terms, branching decreases biodegradability. In aliphatic compounds with very large numbers of carbons, the presence of branching may bring the branch chain length down to the number of carbon atoms which can be degraded; this would promote biodegradability compared to the compound with the same number of carbon atoms but in a non-branched linear chain. Many microorganisms, including several Pseudomonads, are able to use linear alkanes as their sole source of carbon and energy . The OCT-plasmid of Pseudomonas oleovorans contains two operons, alkBFGHJKL and alkST, which encode all proteins necessary for the degradation of n-octane and other five- to twelve-carbon linear alkanes . Branched isomers, such as isooctane, are less susceptible to biodegradation than n-octane. The conversion of n-octane to 1-octanol is catalyzed by a group of proteins collectively referred to as the "alkane hydroxylase system." It has three main components: alkane 1-monooxygenase, and the two soluble proteins rubredoxin, and rubredoxin reductase. Rubredoxin reductase transfers electrons from NADH to rubredoxin. This protein then passes electrons to alkane 1-monoxygenase, an enzyme localized in the cytoplasmic membrane. The final product of this pathway, octanoyl-CoA, enters the beta-oxidation cycle and is used as both a carbon and energy source . The alkyl hydroperoxide reductase enzyme system of Salmonella choleraesuis (formerly S. typhimurium) is composed of two enzymes (AhpC and AhpF) which reduce organic hydroperoxides and hydrogen peroxide Homologs of these enzymes are found in a variety of Gram-positive and Gram-negative species. .


Biology 447 ­ Environmental Microbiology

Graphical Map of n-octane biodegradation:

modified from

Aromatic hydrocarbons

For a good introduction to nomenclature and models of organic molecules, see the site by Dave Woodcock from Okaganan College. (web)

The aromatic ring structure and the convention for naming various substituent groups is given below. If a phenol is considered, then other compounds with groups at the different carbon position can be named either by the ortho, meta, para convention, or by the 1,2- 1,3- and 1,4-, etc. convention. For instance, a chlorophenol with a chlorine atom at position number 3 would be 3-chlorophenol or meta-chlorophenol. Benzene itself is quite susceptible to decomposition in some ecosystems.


Biology 447 ­ Environmental Microbiology

. Adding another substituent group has a significant effect on the rate of degradation of the new compound. For example, adding different substituent groups to benzene (producing mono substituted benzenes) produces the following result; Group added to benzene Persistence (half-life) Days -COOH -OH -NO2 -NH2 -OCH3 (methoxy) -SO3H (sulfoxy) 1 1 +64 4 8 16

If PHENOL phenol is modified by the addition of halogens to produce CHLOROPHENOL or BROMOPHENOL, the following results are obtained; Phenol Substituent Decomposition period half-life (Days)

None 2-chloro 3-chloro 4-chloro 2-bromo 3-bromo 4-bromo 2,4-dichloro 2,5-dichloro 2,4,5-trichloro 2,4,6-trichlorophenol 2,3,4,5-tetrachloro Pentachlorophenol

2 14 +72 9 14 +72 16 9 +72 +72 6 +72 +72

The effects of these substituent groups may be summarized; 1. Single substituent groups on benzene affect the degradation in the order (COOH or OH), NH2, OCH3, SO3H, NO2 (increasing persistence).


Biology 447 ­ Environmental Microbiology

2. Meta substitution for halogens on phenol causes the greatest persistence. 3. Ortho- and para-substitution of halogens has less effect. . 4. Increasing amounts of chlorination or bromination in a molecule increase persistence

Metabolic pathways for aromatic hydrocarbon decomposition There are many pathways of breakdown for aromatic compounds. Many of them have catechol as a central intermediate. The next stage is ring fission of catechol, followed by incorporation into the normal biochemical pathways of the cell. This can be by two distinct mechanisms; ORTHO or META cleavage 1. ORTHO cleavage. The aromatic ring is fissioned between the two hydroxyl groups on catechol with the production of muconic acid. 2. META cleavage. This occurs adjacent to, rather than between, the two hydroxyl groups of catechol and yields a semi-aldehyde.

Molecular Models

Toluene Benzyl alcohol Benzaldehyde Benzoic acid Anthracene 3-hydroxy-2naphthoic acid Salicylic acid Catechol


Biology 447 ­ Environmental Microbiology

Generalized aromatic metabolism One important mechanism in the breakdown of many compounds is the removal of the halogen atoms, which confer much of the resistance to decomposition of such diverse compounds as the insecticide DDT , polychlorinated biphenyl (PCB), pentachlorophenol (PCP), halogenated benzenes (e.g. hexachlorobenzene), etc. This is often a process of dehalogenation involving several mechanisms, by which halogen atoms are removed from the molecules. These processes can occur under both anaerobic (reductive dehalogenation) and aerobic conditions.

Ortho and meta cleavage patterns Some compounds (such as the chlorinated benzenes) appear to be only dehalogenated under aerobic conditions, whereas other compounds can be dehalogenated under either anaerobic or aerobic conditions. Yet other compounds (some pesticides and halogenated 1 and 2 carbon compounds) undergo only reductive


Biology 447 ­ Environmental Microbiology

dehalogenation. Another important concept in aromatic biodegradation (and with other compounds) is that of cometabolism. In this process, a compound is transformed by a microorganism even though the organism is unable to gain energy by the transformation. It occurs "by accident", in that an enzyme produced by an organism is able to metabolize a compound but the product of the reaction is not metabolized. It is obviously important in mixed cultures or natural situations more than is indicated by pure culture work since different microorganisms may be able to metabolize these different products of previous transformations

Biological Transformations of aromatic hydrocarbons

Utilization of benzene Surprisingly little information was available before 1968. Early observations showed that bacteria grown on benzene were able to metabolize catechol (see above). Experiments with Pseudomonas putida by Gibson and his colleagues showed two routes to catechol from benzene


Metabolism from benzene to catechol

Generalized metabolic routes from benzene to catechol. The enzymes involved have been extracted from a number of bacteria including Pseudomonas, Moraxella and Arthrobacter. A three component enzyme system was found to be operative:


Biology 447 ­ Environmental Microbiology

Three-component enzyme system The conversion from cis-benzenediol to catechol was by a dehydrogenase enzyme - cisbenzenediol dehydrogenase - coupled with NAD. Metabolic routes of monoalkyl benzene utilization Alkyl benzenes such as toluene, ethylbenzene, propyl-, and butylbenzenes, phenyl alkanes (long chain), biphenyl, biphenylmethane and secondary alkylbenzenes are metabolized according to the generalized scheme given below;

Metabolism of monoalkyl benzenes In addition, there is metabolism of the side chains in the alkyl benzenes containing long side chains. Beta oxidation of the side chains occurs. All possible combinations have been discovered; some bacteria metabolize odd-length side chains, some use even length side chains and some use both. See the details of toluene metabolism (aerobic and anaerobic) below. Biphenyl metabolism has become important because of the presence of these molecules as part of the environmental pollutants polychlorinated biphenyls and DDT and its breakdown products. Some bacteria are able to break the biphenyl ring structure. They usually follow the meta cleavage pattern described above. Utilization of dialkyl benzenes. The dialkyl benzenes such as the xylenes and the 4-alkyltoluenes undergo a variety of microbial biodegradation processes. 1. The methyl groups are metabolized to carboxyl groups; thus m-xylene becomes m-toluate, p-xylene becomes ptoluate, toluene becomes benzoate, and 1,2,4-trimethylbenzene becomes 3,4-dimethyl benzoate. 2. The general metabolic route for utilization of the dialkyl benzenes is given above. 3. The ability of Pseudomonas spp. to utilize the toluates is linked with the presence of a transmissible plasmid (TOL) in the organism. The same applies to xylene metabolism with the XYL plasmid. The functions of these plasmids are fairly general; for instance the TOL plasmid in P. arvida allows the organism to grow on 1,2,4-


Biology 447 ­ Environmental Microbiology

trimethylbenzene, 3-ethyltoluene, 3,4-dimethylbenzoate, 3- ethylbenzoate, 4-ethyl-3-methyltoluene and 4-ethyl-3methylbenzoate as well as some of the alcohol and aldehyde intermediates m the process. The TOL plasmids thus seem to possess the coding for many of the enzymes in the whole metabolic sequence.

Examples of aromatic compound biodegradation pathways (toluene and xylene)

Toluene 1. Anaerobic pathway: Toluene is one of the most water soluble aromatic hydrocarbons. Anaerobic toluene mineralization under denitrifying conditions was demonstrated for pure bacterial cultures. This page shows only its anaerobic pathway. The aerobic degradation of toluene is documented elsewhere in the UM-BBD. Anaerobic toluene mineralization is assumed to proceed via benzoate (benzoyl-CoA) as intermediate. The following tentative pathway for anaerobic toluene oxidation to benzoate (benzoyl-CoA) was compiled from Beller and Spormann 1997, J. Bacteriol. 179 (3): 670-676 and Biegert et al. 1996, . In vitro studies were conducted using Azoarcus sp. strain T and Thauera aromatica, respectively. In these studies, benzylsuccinate, E-phenylitaconate, and benzoyl-CoA (benzoate) were identified as intermediates in anaerobic toluene metabolism. Oxidation of benzylsuccinate to E-phenylitaconate is


Biology 447 ­ Environmental Microbiology

dependent on CoA (succinyl-CoA), suggesting that the true metabolic intermediates may be benzylsuccinyl-CoA and E-phenylitaconyl-CoA. Compounds in brackets were not identified yet but were postulated based on other experiments. 2. Aerobic pathway The biodegradation of toluene has been well-studied at the molecular level and it, thus, serves as one of the principal models for understanding the mechanisms of bacterial benzene ring metabolism. The diagram below shows only its aerobic pathway; the anaerobic pathway is diagrammed above. The enzyme that starts one branch of this pathway, toluene 1,2-dioxygenase, has many other catalytic abilities


Biology 447 ­ Environmental Microbiology


Biology 447 ­ Environmental Microbiology


Biology 447 ­ Environmental Microbiology


The metabolic enzymes in this pathway have been shown to have similar specificity for toluene, para-xylene, and meta-xylene. Davey and Gibson (1974) report that para-xylene is degraded to 4-methylcatechol by the oxidation of a methyl substituent to an alcohol group and the addition of another alcohol group. Murray and Duggleby (1972) report that 4-methylcatechol is degraded by the meta (or alpha-ketoacid) pathway, in which the aromatic ring of 4methylcatechol is cleaved in the meta-position. Gunther and Schlosser (1994) report that 4-methylcatechol is also degraded by ortho-cleavage to 4-methylmuconolactone, a dead end-product


Biology 447 ­ Environmental Microbiology


Biology 447 ­ Environmental Microbiology

Polyaromatic hydrocarbons

Details and molecular models of 660 polyaromatic hydrocarbons from the National Institute of Standards and Technology

These compounds are fused ring aromatic hydrocarbons - common in coal products and coal byproducts. They are ubiquitous pollutants in the atmosphere and are relatively resistant to biodegradation; they can therefore accumulate to substantial levels in the environment. Since some of the larger species are carcinogenic, they can pose a significant health hazard. There are more than 70 compounds classed as PAHs and they have from 2 to 7 rings. They have been detected in a wide range of soils and sediments, including some ancient sediments. Significant quantities are present in both industrial and domestic effluents and sometimes cause problems in waste water treatment. Another source is from hydrocarbon spills, the more biodegradable aliphatic and aromatic fractions are removed leaving the more resistant fractions; these often include PAH's which can sink to the bottom of water bodies and become permanent pollutants of oceans, lakes and rivers. They are produced in large quantities from coking operations and gasification processes using coal and from other sources of incomplete combustion such as automobile exhausts, power generation plants, refuse burning and industrial emissions. They can also have a natural origin in coal deposits, from natural aromatics such as terpenes, sterols and quinones from plants which volatilize and can become condensed to PAHs. Plant lignins also may become progressively decomposed to humic substances which can become larger during maturation of the pest or coal deposits and can eventually produce PAH's. Several species of bacteria have produced PAHs in agar devoid of hydrocarbons but containing glycerol. Algae can also synthesize PAHs. In summary, PAHs are mainly of anthropogenic origin, but also have geological and natural biological origins. 3D Molecular models

naphthalene anthracene phenanthrene pyrene 2,3-benzanthracene 1,2-benzanthracene chrysene triphenylene 3,4-benzopyrene 1,2-benzopyrene perylene 1,2,5,6-dibenzoanthracene anthanthrene indene fluorene coronene


Biology 447 ­ Environmental Microbiology

Biodegradation of PAH's Factors affecting biodegradability include; a). The number of fused rings b). Number and position of substituents of the rings. c). Degree of ring saturation. The following set of results are taken from a study of oxygen utilization by resting cells of Pseudomonas putida and Flavobactenum sp. by McKenna and Heath, 1976. The results are similar to those obtained by other workers.

Oxidation rate

P. putida

Naphthalene = 100% Compound

Flavobactenum sp.

Phenanthrene = 100%

1. a. Effect of number of fused rings on oxidation rate Naphthalene Anthracene Phenanthrene 1,2 benzanthracene 2,3 benzanthracene chrysene pyrene triphenylene 100 9.8 67 0 2.7 0 1.1 0 79.8 36 100 10.1 1.1 27.9 10.1 0

1. b. Effect of alkyl and phenyl substituents on naphthalene ring oxidation 1-methylnaphthalene 1-ethylnaphthalene 1-phenylnaphthalene 2-methylnaphthalene 2-ethylnaphthalene 2-vinylnaphthalene 41.7 39.0 0.0 81.0 70.3 78.8 60.0 36.3 0.0 85.3 45.0 40.0


Biology 447 ­ Environmental Microbiology 2-phenylnaphthalene 1,3-dimethylnaphthalene 1,4-dimethylnaphthalene 1,5-dimethylnaphthalene 1,6-dimethylnaphthalene 2,3-dimethylnaphthalene 2,6-dimethylnaphthalene 2,6-di-tert-butylnaphthalene 2,7-di-tert-butylnaphthalene 2,3,5-trimethylnaphthalene 2,3,6-trimethylnaphthalene 1,2,3,4-tetramethylnaphthalene 0.0 1.4 39.1 8.8 8.8 1.4 88.0 15.5 0.0 0.0 9.0 7.0 0.0 8.9 0.0 19.9 0.0 4.4 84.2 62.8 0.0 0.0 17.5 9.6 0.0

1. c. Effect of position of methyl substituent on oxidation rate

1. d. Effect of saturation on oxidation rate Naphthalene 1,2-dihydronaphthalene Tetralin cis-Decalin trans-Decalin Phenanthrene 9,10-dihydrophenanthrene 1,2,3,4,5,6,7,8-Octo-phenanthrene Perhydrophenanthrene indene indane Hexahydroindane fluorene perhydrofluorene 0. 3. 100 32.2 15.1 4.4 4.4 67.0 12.0 9.3 1.2 32.4 24.6 6.8 32.4 0.0 79.8 59.8 15.2 0 0 0 23.3 25.5 0.7 14.9 13.0 1.0 36.7 3.0

Notes and Summary: 1. There is no significant oxidation of compounds with more than three rings. Four-membered ring systems are oxidized at negligible rates. Five-membered ring systems underwent no significant oxidation. (Table la)


Biology 447 ­ Environmental Microbiology

2. Naphthalene is oxidized readily by both bacteria, phenanthrene is oxidized more slowly by Pseudomonas than by Flavobactenum. Anthracene metabolized slowly by Pseudomonas, slightly more quickly by Flavobacteriunt (Table la) 3. Naphthalene nuclei with a small alkyl group (methyl, vinyl or ethyl) were oxidized rapidly (Table lb) while those with phenyl were extremely resistant. 4. A substituent at position 2 allowed faster oxidation than at position 1 (less steric hindrance) (Table lb). 5. Other methyl-substituted ring systems (Table 1c) were oxidized slowly. 6. Substituted naphthalene with more than 1 methyl group (Table lb) on one ring had at least a small amount of oxidation. 7. Methyl substitution of methyl groups on both rings (Table lb) led to decreased rates of oxidation. 8. Increased ring saturation (Table ld) led to decreased oxidation. 9. As long as one aromatic ring was available, (Table ld) some measurable oxidation rate was achieved. 10. The perhydro compounds (cis- and trans-decaline, perhydrophenanthrene, hexahydroindane and perhydrofluorene) were resistant to decomposition. In comparison to these PAH oxidations, the oxidation of single ring aromatics showed that benzene and cyclohexane were not oxidized to any significant extent, whereas toluene and the xylenes were oxidized at an initial moderate rate. This rapid rate for the xylenes may be because they "mimic" the naphthalene in that they can attach to the same active site of the oxygenase enzymes involved in the oxidation process. The table below shows the results of experiments with natural populations of microorganisms on various substrates. The non-growth substrate was monitored, but the organisms were actually growing on either phenanthrene or naphthalene. The non-growth substrates were oxidized by a process called "co-oxidation" or "cometabolism". Neither the Flavobacterium nor the Pseudomonas putida used in the previous series of experiments were able to carry out this process of co-metabolism with these substrates, but the natural populations allowed significant degradation to occur. In summary, for the PAHs, the number of aromatic rings, the size, position and number of attached ring substituents and the degree of saturation all influenced the breakdown rates. In general terms, increased number of fused rings, size and number of hydrocarbon substituents, and degree of ring saturation all lead to decreased oxidation rates

Examples: phenanthrene and naphthalene biodegradation pathways

Phenanthrene: Phenanthrene is degraded by some soil bacteria through one of two different routes. In one route,1-hydroxy-2naphthoic acid is oxidized to 1,2-dihydroxynaphthalene, which is further degraded via the Naphthalene Pathway to salicylate which can be further metabolized. In the other pathway, the ring of 1-hydroxy-2-naphthoic acid is cleaved and further metabolized via the Phthalate Pathway. It has been demonstrated that naphthalene and phenanthrene share a common upper metabolic pathway . In addition, the metabolism of phenanthrene by Streptomyces flavovirens and the marine cyanobacterium Agmenellum quadruplicatum PR-6 is more similar to that reported in mammalian and fungal enzyme systems than those catalyzed by bacteria. Both oxidize phenanthrene to phenanthrene trans-9,10-dihydrodiol via a monooxygenase-epoxide hydrolase-catalyzed reaction rather than by a dioxygenase. The metabolic formation of 1methoxyphenanthrene from phenanthrene was first reported in Synechococcus sp. PR-6. This organism may


Biology 447 ­ Environmental Microbiology

detoxify other 1-phenanthrols. The metabolic fate of 1-methoxyphenanthrene in Synechococcus sp. PR-6 remains to be elucidated. (Kiyohara H, et al. 1994) From:

See Website for Details

Naphthalene: Naphthalene dioxygenase, the enzyme which initiates bacterial naphthalene metabolism, is used in a biotechnological process to synthesize the blue jean dye indigo. This versatile enzyme has many other catalytic abilities From:


Biology 447 ­ Environmental Microbiology


Biology 447 ­ Environmental Microbiology

Biodegradation of Pesticides

Insecticides Chlorinated hydrocarbons

I) DDT and analogues The outline below gives a general picture of the many interconversions that DDT can undergo in the environment. Many of the analogues of DDT are breakdown products of the DDT itself. The main reaction route in microorganisms is through TDE (DDD) and TDEE. It is a direct reductive dechlorination process and is carried out by bacteria in the soil and water. Studies on the metabolic fate of the other products of DDT metabolism are limited.

The importance of DDT is emphasized when examining the distribution of the material in the environment.


Biology 447 ­ Environmental Microbiology

ii) BHC (lindane) The gamma isomer of BHC was extensively used as an insecticide; studies on its biodegradation are limited. It does not accumulate to the same extent as DDT in animal tissues. iii) Chlorinated cyclodiene insecticides These compounds represent one of the more persistent groups of pesticides. Examples are aldrin, dieldrin, heptachlor, isodrin and endrin. Aldrin and Dieldrin have been banned in many jurisdictions and are only used for particular purposes in many other jurisdictions.


Biology 447 ­ Environmental Microbiology

In soil and water, degradation is probably microbial but very often leads to the formation of an epoxide ring structure in the molecules. This is formed from the CH=CH group of the least chlorinated ring. This process of epoxidation converts aldrin to dieldrin, isodrin to endrin, and heptachlor to heptachlor epoxide. All the epoxides are more stable than the parent form.

Oxidation processes of cyclodiene compounds to yield more stable toxic intermediates. Microbial activity thus results in the formation of "stable toxic intermediates". The rates of disappearance of the various cyclodiene insecticides are as follows: Time for 95% disappearance (Years) 1-6 5-25 3-5 Averages (Years) 3 8 3.5

Pesticide Aldrin Dieldrin sti Heptachlor Heptachlor epoxide sti

sti = The so called "stable toxic intermediates" They are not completely inert (as can be seen by their eventual breakdown by microorganisms).


Biology 447 ­ Environmental Microbiology

Organophosphorous compounds

The group contains many well known insecticides such as malathion, parathion, methyl parathion, mevinphos, sevin, and diazinon. Their persistence is of a lower order of magnitude than the organochlorine insecticides above. They are not particularly persistent pesticides, with half lives in soils measured in weeks or months. They are derivatives of the nerve gases sarin and tabun from World War II (although they were never used). Both groups of chemicals work by inhibiting the acetyl-choline-esterase enzyme enzyme They are metabolised by many different microorganisms, particularly members of the Pseudomonas, Arthrobacter, Streptomyces, and Thiobacillus genera and by the fungi in the Trichoderma genus. The formula of the organophosphorus insecticides is relatively simple;


Parathion There are many sites of attack for microorganisms in these molecules. There are therefore many different possible breakdown patterns. For example, in malathion, the sites of attack are marked A,B,C, and D and can be attacked by phosphatase, mixed function oxidase, and carboxyl esterase enzymes: A = phosphatase and mixed function oxidases B = mixed function oxidase C = phosphatase D = carboxyl esterase Many different products can be formed. It has been shown that autoclaving soil destroys about 90% of its ability to degrade malathion. However, a treatment with sterilizing levels of gamma irradiation does not affect the soil's ability to degrade malathion. It was possible to extract a fraction of the soil with 0.2N NaOH that activity degraded malathion. It is therefore possible that some organic compound in soils is able to chemically degrade some organophosphorus insecticides.



Biology 447 ­ Environmental Microbiology

Carbaryl is an example of the carbamate insecticides. Its formula is;

Other examples are 3-keto carbofuran, carbanolate, propoxur, carbofuran and landrin. They are moderately persistent insecticides. Typical mechanisms of biodegradation are by; 1) Ring hydroxylation 2) N-methyl hydroxylation


Insecticides range in persistence from organophosphates (1 week to 16 months), through carbamates (2 months to 14 months), to organochlorines (18 months to 25 years). In most cases, the persistence can be explained by the chemical structure and by the degree of water solubility. "Stable toxic intermediates" can be produced, but these are eventually degraded in most cases. Bioaccumulation can occur with the more lipid soluble and persistent insecticides such as DDT. In certain cases, chemical breakdown of the organophosphorus insecticides is possible.


Phenoxyalkanoic acids and derivatives The three main groups are the phenoxyacetic, phenoxypropionic, and 4-phenoxybutyric derivatives. Many of them are the acids of these groups, but more recently, forms such as esters and amines are becoming more common. The basic structure is;


Biology 447 ­ Environmental Microbiology

1). Phenoxyacetic group 1 CI 2 CI = 2,4-dichlorophenoxyacetic (2,4-D) 3H 1 CI 2 CI =2,4,5-trichlorophenoxyacetic acid (92,4,5-T) 3 CI 1 CH 2 CI =MCPA 3H

2). Phenoxypropionic group 1 CH3 2 CI =(mecoprop) 3H

1 CI 2 CI =[2(2,4,5-trichlorphenoxy) propionic acid] - silvex 3 CI


Biology 447 ­ Environmental Microbiology

3. Phenoxybutyric group 1 CH3 2 CI =MCPB ([4-(2-methyl-4-chloro-phenoxy) butyric acid] 3H

1 CI 2 CI =2,4-DB[4-(2,4-dichlorophenoxy)butyric acid]

1 CI 2 CI =2,4,5-DB[4-(2,4,5,trichlorophenoxy)butyric acid] 3 CI

A closely related compound to the phenoxy group is; Sodium 2,4-dichlorophenoxy ethyl sulfate (Sesone herbicide)


Biology 447 ­ Environmental Microbiology

Degradation of Phenoxyalkanoic Herbicides Plants are able to degrade these herbicides in their tissues, but the rates of degradation are very low. The rates of degradation vary considerably in soils. Most of the available results are from the phenoxyacetic particularly 2,4-D itself. The patterns of microbial breakdown may be summarised as follows; 1). 2,4-D is rapidly degraded in soil. MCPA is more persistent and 2,4,5-T is even more resistant to decomposition. 2). Microorganisms in soils can become "adapted" in two separate ways a). by enzyme induction b). by selection for 2,4-D-degrading organisms 3). Such adaptation often leads to the rapid degradation of other members of the group of phenoxy herbicides. 4). Many microorganisms can degrade more than one of the groups. 5). There appear to be two main pathways of biodegradation; a). Via hydroxyphenoxyacetic acid b). Via phenol A typical metabolic scheme is;

s-triazine group

The most commonly used a-triazines are the herbicides atrazine , prometryne, simazine, and ametryne. They are


Biology 447 ­ Environmental Microbiology

used in very large quantities (especially atrazine and simazine) in weed control in field crops.

1. Atrazine

2. Simazine

The triazines are moderately persistent in soils (atrazine about 12 months, simazine about 12-14 months). The mechanisms of microbial decomposition are classified according to the "site of attack".

General formula for triazine herbicides

Biodegradation of Triazine Herbicides 1. Attack can occur at C2 by hydroxylation;

e.g 2. Attack can occur at C4 or C6 by oxidative removal;

e.g 3. Attack by cleavage of triazine ring to produce straight chain compound. Reaction is slow in soil.

Substituted urea There are many different substituted urea herbicides being used. Typical examples include monuron, linuron, diuron, neburon, chlorbromuron, etc. Most are phenyl substituted ureas;


Biology 447 ­ Environmental Microbiology


The phenyl substituted urea herbicides resist chemical breakdown in soils and are degraded by microorganisms. Typical persistence is of the order of 4-18 months in most soils. Typical schemes of biodegradation include dealkylation;

Bipyridylium group Diquat and paraquat are the most commonly used bipyridylium herbicides.


Paraquat They are extremely resistant to microbial attack. The resistance is such that they are probably not biodegraded to any significant extent in soils. Their half-life has been quoted as "plus 1000 years" in terms of biodegradation. They are sensitive to ultra violet light decomposition (photolysis) and disappear quickly if in sunlight. Many microorganisms which degrade the compounds in pure culture have been found, but they are so tightly adsorbed to soil particles in the field that they are simply not available for microbial decomposition Thiolcarbamates Typical examples are CDEC, pebulate and diallate. Vapourisation and biodegradation are the most significant factors in the losses from soils. Very little information is available on the metabolic pathways involved, or on the products formed. Chlorinated aliphatic acids The most important are TCA (trichloracetic acid) and dalapon (2,2-dichloropropionic acid). The persistence is of the order of 3 - 6 months. Microbial decomposition is the most important factor. Hydrolytic processes are the most common biodegradative pathways


Biology 447 ­ Environmental Microbiology

Miscellaneous The other chemicals used as herbicides include the trifluaralin group, the chloracetamides, amitrole, phenyl, carbamates, and pentachlorophenol.

Fungicides Aliphatic compounds

These compounds (carbon disulfide, ethylene, dibromide, chloropicrin, formaldehyde, allyl alcohol, etc.) are often used as soil fumigants to kill fungi and nematodes. They are degraded at various rates by the microorganisms in soil. Chloropicrin and allyl alcohol are probably the most persistent. They are often degraded by soil fungi, even though they are fungicidal!

Quinones (dichlone)

Chloranil and dichlone are effective fungicides. They are SH enzyme inhibitors and are usually degraded by hydrolysis.

Organometallics (semesan)

Many different organomercurials have been used as fungicides as seed dressings, crop protectants and general fungicides. There has been very little work done on the biodegradation of these compounds, but the microorganisms involved seem to have a high specificity for particular compounds such as 2-chloro-4hydroxymercuriphenol or cyano-(methylmercuri)guanidine. Most are no longer in use because of their toxic effects. They have largely been replaced by the organo-tin compounds such as

Triphenyltin hydroxide


Biology 447 ­ Environmental Microbiology


These compounds are very common fungicides (thiram, zineb, ferbam and vapam) and are broken down in most soils within 2 days to 2 weeks. A typical formula is that of thiram; tetramethyl thiuram disulfide




The phenols used as fungicides are usually halogen and nitro derivatives of an aromatic hydrocarbon. Pentachloronitrobenzene (PCNB) is an example and is used as a stable soil fungicide. It is active against a narrow range of soil fungal plant pathogens. It is not readily degraded and probably has a half life of 2 to 3 years in soils. Major decomposition products of PCNB are PCA, PCTA and PCP;


Certain antibiotics (such as griseofulvin) have been used as treatments for individual plants to protect them from plant pathogens. It is very rapidly degraded in soils.


Biology 447 ­ Environmental Microbiology

Miscellaneous Compounds

There are many other compounds used as fungicides. Copper salts (copper naphthenate, dexon [a sulfonate compound]) and the captan group are examples. Copper salts are very slowly degraded as are the captan, while dexon is rapidly degraded. Other fungicides in common use are the substituted phthalimides such as captan, folpet and captafol;

and the benzimidazoles such as Benomyl and thiabendazole;

Typical use patterns for pesticides

The insecticides are typically used in lower concentrations and total volumes than are the herbicides and fungicides. This is because of their higher efficacy at low dose rates against their target organisms. A typical "spray regime" for a horticultural crop is usually specified in some detail by, for example, the Ontario Ministry of Agriculture. Very often, different types of pesticides are recommended for different soil types and the total number of different sprayings and pesticides applied during a growing season is also specified


Biology 447 ­ Environmental Microbiology

Transport of pesticides in the environment.

The questions that should be asked regarding transport of pesticides are; I). What are the transport mechanisms? ii). What proportion of the materials is transported? iii) What is the fate and behaviour of the material during transport? iv) What are the biological consequences? The answer to these questions involve complex treatments of meteorology, aerosol behaviour, volatility and solubility of the pesticides, leaching behaviour, absorption and absorption properties, photodecomposition, biodegradation, chemical degradation, biomagnification, bioaccumulation, detoxification, effects on nontarget organisms, toxicology, etc., etc., These usually involve complex ecological modelling processes. The field is now called the "environmental dynamics" of pesticides and is an amalgam of the areas above. A general treatment of the problem addresses the different "sinks" for pesticides in the environment and the transport rates and biodegradation rates, etc. between those sinks and compartment. The diagram below shows some of the compartments which take part in the process.


Biology 447 ­ Environmental Microbiology

To take only one example of a microbial process, the disappearance and fate of a pesticide in the soil is dependent upon many factors. These may be summarised as; a). Type of soil; composition (clay, silt, sand), structure (bulk density, surface area) and prior treatment (chemical and agricultural). b). Type of chemical; physical properties such as solubility, vapour pressure, stability, sensitivity to light, etc. and chemical properties (such as those which affect the adsorption and absorption to organic and inorganic compound). c). Climatic conditions; rainfall, temperature, sunlight, humidity, etc. d). Biological populations; type, nutrient requirements, adaptations, lifecycles, etc. e). Method of application; granular, solution, suspension, powder, in organic solvents, as wettable powders, etc. The pesticides can enter the soil directly by surface application, by injection, as a powder, by incorporation of decomposed organic matter, or indirectly by surface runoff, foliage runoff or inaccurate spraying practices. Once in the soil the pesticide can be removed by; a). Leaching b). Microbially mediated decomposition c). Volatilisation d). Plant uptake e). Nonbiological decomposition Nonbiological decomposition may involve such processes as photodecomposition and chemical modification on surfaces by catalysis with clay minerals, metal oxides, organic materials, etc. All have been demonstrated. Microbially mediated decomposition is the major, and sometime the only, mechanism of permanent removal or modification of pesticides in soils. An extensive literature exists on insecticide decomposition processes, less exists on herbicides and even less exists for fungicides. One of the main mechanisms is cometabolism. Cometabolism Most of the available evidence now indicates that the microbial activity is the main route by which chemicals are degraded in the environment. Although plants and animals degrade a number of compounds, their activities are usually small in comparison with those of the microorganisms, especially the heterotrophic bacteria and fungi in the same habitats. The mineralisation of organics ( or complete degradation of an organic molecule to inorganic molecules) is almost always a consequence of microbial activity. Mineralisation is usually a process that results in increased biomass of microorganisms since some of the carbon in the organic materials is used for growth. Energy is released and the populations or communities increase in numbers and biomass. With many chemical compounds, a process quite different from the normal mineralisation process occurs. It became apparent that some chemicals were metabolised in soils and in water, even though no microorganisms were found that were capable of using them as nutrients or sources of energy. There compounds are metabolised in non-sterile soils but are not in soils that have been sterilized. These compounds include DDT, 2,4,5-T, aldrin, heptachlor and many other chlorinated and non-chlorinated molecules. No microorganisms able to use these as sources of energy or nutrients have ever been isolated, yet they undergo transformations in chemical structure in


Biology 447 ­ Environmental Microbiology

soils. The process is usually due to the phenomenon of cometabolism (or co-oxidation). The microorganisms are growing on another substrate but are capable of performing the transformation known as cometabolism. The populations responsible for these transformations do not increase in number and the compounds are often metabolised very slowly. The rate does not increase with time as it would if the responsible microbial population increased over time. A typical graphical result of the two types of transformations (normal and cometabolism) is:

Cometabolic Processes will be examined in more detail in the Module on Groundwater

Summary of Biodegradation Mechanisms of Pesticides

There are many mechanisms involved on the biodegradation of pesticides and other contaminants. These may be summarised as follows: Type reactions (with examples) of microbial transformations of chemicals. CATEGORY/REACTION

Dehalogenation RCH2C1 Õ RCH2OH ArC1 Õ ArCH Propachlor (C,S) Nitrofen (S)



Biology 447 ­ Environmental Microbiology ArF Õ ArCH ARC1 Õ ArH Ar2CHCH2C1 Õ Ar2C=CH2 ArCHCHC12 Õ Ar2C=CHC1 Ar2CHCC13 Õ Ar2CHCHC12 Ar2CHCC13 Õ Ar2C=CC12 RCC13 Õ RCOOH HetC1 Õ HetOH Deamination ArNH2 Õ ArOH Decarboxylation ArCOOH Õ Ar4 ArCHCOOH Õ Ar2CH RCH(CH3)COOH Õ RCH2CH2 ArN(R)COOH Õ ArN(R)H Methyl oxidation RCH3 Õ RCH2OH Õ RCHO Õ RCOOH Hydroxylation and ketone formation ArH Õ ArOH R(R')CHR" Õ R(R')CHOH(R") R(R')(R")CCH3 Õ R(R')(R")CCH2OH B-oxidation ArO(CH2)nCH2CH2COOH Õ ArO(CH2)nCOOH Epoxide formation O Heptachlor (S,C) RHC=CHR'Õ RCH-CHR' Nitrogen oxidation R(R')NR" Õ R(R')N(O)R" Sulfur Oxidation RSR' Õ RS(0)R' Õ RS(O2)R' =S to =0 (AlkO)2P(S)R Õ (AlkO)2P(O)R Sulfoxide reduction RS(O)R' Õ RSR' Triple bond reduction RC=CH Õ RCH=CH2 Double bond reduction Ar2C=CH2 Õ Ar2CHCH3 Hydration of double bond DDT (C) Buturon (S,C) Phorate (S) Aldicarb (S,C) Parathion (S,C) Trideomorph (S) Dichlorphenoxyacetic acids (S,C) Benthiocarb (S) Bux insecticide (S) Denmert (S) Bromacil (S) Biofenox (S) DDT (C) Dichlorfop-methyl (S) DDD(S) Fluchloralin (S) Flam-prop methyl (S,C) Pentachlorophenol (S,C) DDT (C) DDT (C,S,W) DDT (C,S) DDT (S,C) N-Serve (S), DDT (C) Cyanazine (S)


Biology 447 ­ Environmental Microbiology Ar2C=CH2 Õ Ar2CHCH2OH Nitro Metabolism RNO2 Õ ROH Oxime metabolism RCH=NOH Õ RC=N Nitrile/amide metabolism RCº N Õ RC(O)NH2 Õ RCOOH Bromoxynil (S) Aldicarb (S,C) Nitrofen (S) DDT (C)

Note: S = reaction observed in soils, C = reaction observed in cultures.

There are also mechanisms which are not strictly degradative; they form conjugates between the pesticide and other chemicals or chemical groups and so modify the chemical. These may be more or less harmful than the original compounds. These reactions include methylation, ether formation, N-acylation (ArNH2 Õ ArNHC(O)H), nitration, N-nitrosation [(Alk)2NH + NO2 --- (Alk)2NNO], dimerization, and nitrogen heterocycle formation;

The "benchmark concept"

Because of the difficulty of dealing with the large number of pesticides on the market, the concept of using "benchmark" chemicals to assess the potential damage of new chemicals to the environment has been developed. The process basically goes as follows: 1). Define the properties of pesticides which control their activities in the environment. 2). Develop lab methods to measure these properties. 3). Measure these properties for chemicals currently in use. 4). Determine the relationship between these properties and the field behaviour of pesticides. Any new pesticide chemical can then be compared to the presently existing standards or benchmarks and the behaviour in the environment can be predicted.


Biology 447 ­ Environmental Microbiology

Alternatives to increased pesticides use

Finding alternatives to increased use of pesticides is necessary because of high costs and resistance phenomena ,as well as because of concern about environmental contamination. There are a number of approaches to the problem. The most promising is called "integrated control measures". It consists of using all available methods, including efficient and controlled pesticide use, to control pests. It can include development of resistant varieties, application of pesticides at critical and carefully monitored points in the life cycle of the pests, monitoring programs to assess efficacy of pesticide applications, use of biological control measures, use of high efficiency sprays, etc. These programs require a high level of technical expertise to design and operate, together with careful monitoring and sophisticated advisory services for the farmer.

Key Points

· · · · · · · · ·

Range of properties and consequent biodegradation of naturally occurring materials - simple sugars, carbohydrates, amino acids, proteins, cellulose, hemicellulose, and lignin. Factors in lignin chemical structure and physical nature that slow down biodegradation. Environmental conditions that affect biodegradation rates - aeration, temperature, moisture content, etc. Main types of hydrocarbon molecules and importance of hydrocarbon biodegradation processes. Effect of the physical state of hydrocarbon emulsions that affect degradation (oil in water and water in oil) How hydrocarbons are degraded by microorganisms - which general categories of microorganisms are involved and the importance of the presence of water and (almost always) molecular oxygen. Importance of chain length of hydrocarbon in biodegradation (straight and branched chain aliphatics and side chains on aromatics) Main 4 types of aliphatic hydrocarbon biodegradation biochemistry - how they differ and importance of molecular oxygen Importance of chemical structure of aromatic hydrocarbons on biodegradation: o Side groups - position effect o Side groups - different molecular structures Halogen atoms Different side groups (amine, carboxyl, sulfoxyl, etc) Main two metabolic pathways- ortho and meta - (details of biochemistry are required) The significance of catechol and catechol-like structures in aromatic biodegradation Some examples of more complex aromatic hydrocarbon breakdown patterns - their similarity to the simple benzene example in terms of catechol-like intermediates and overall similarity of patterns. (e.g. monoalkyl benzenes and dialkyl benzenes) The role of: o The number of rings o Number and position of substituents o Degree of ring saturation on PAH biodegradation. Details of the actual rates and structures (except for general overall ring structures of main PAHs) are NOT required.

· · · ·

· ·



Pesticides - Major Groups (herbicides, insecticides and fungicides) and major subdivisions within these groups (e.g. insecticides - organochlorines, organophosphates, carbamates). The main structural formulae for each of these subdivisions but NOT the complete details of the formulae for each member of each group (e.g. the major structural features of organochlorines, but NOT the details of every breakdown product of DDT; the general structure of phenoxyalkanoic herbicides but NOT the detailed structure of every member of that group). An overview of the breakdown patterns for each of the groups. Compare these details to those given in hydrocarbon breakdown pathways - you should see many similarities and even be able to predict the degree of resistance of different member of groups based on their chemical structure. An overall knowledge of why certain pesticides are persistent is important. The concept of "stable toxic intermediates" as demonstrated by aldrin-dieldrin type breakdown patterns.


Biology 447 ­ Environmental Microbiology

· · · · · · · ·

The importance of the role of environmental conditions (temperature, pH, light, adsorption, etc.) on the breakdown of pesticides The concepts of biodegradation of (for example 2,4-D herbicides) by induction of microbial enzymes versus selection of populations that break down the compounds. Mechanisms of recalcitrance - what features of molecular chemistry and environmental factors lead to persistence ? The importance of resistance to breakdown on the transport and environmental fate of pesticides in the environment. How are they transported ? What factors affect their fate and transport ? How are they removed (not only by biodegradation but by leaching, chemical reactions, plant uptake, and volatilization) The process and importance of co-metabolism (in pesticides and other compounds) The final Table "Summary of Biodegradation of Pesticides" is for reference only - you are NOT expected to know all of the possible reactions. Look at the various types of reaction (dehalogenation, etc..) and see if you have seen this reaction in the hydrocarbons or pesticides biodegradation sections. General overview only of fungicides - not much information available on actual breakdown patterns in some cases. Know what the "Benchmark concept" is and what "alternatives to increased pesticide" use means.


Biology 447 ­ Environmental Microbiology

Module Six - Deep Subsurface Microbiology

Deep aquifers (hundreds or thousands of metres below surface) have only recently been investigated other than by petroleum or sulfur companies seeking deposits or concerned with the impact of microorganisms on their drilling and mining activities. The first major project to investigate the deep subsurface was started in 1986 at the U.S. Department of Energy site at Savannah River. The drilling, for the first time, was carried out with microbial sampling as a prime objective. Great care was taken to maintain the drill holes in a state suitable for microbial sampling. The Savannah River plant overlies the Atlantic Coastal Plain and has unconsolidated sediments to a depth of 300 metres. The sediments are then underlain by crystalline metamorphic rock or consolidated mudstone. There are some sandy aquifers interspersed between the clay and silt formations. A sample drill hole was bored first to determine the stratigraphy of the site. Then a sampling hole was drilled. The drilling fluid (sodium bentonite) was used to continuously flush the hole as it was drilled. To prevent contamination, the sampling container was lowered to a depth below the circulating drilling fluid. Autoclaved or steamed stainless steel core liners were used to collect the samples. The sediments were removed from the core liners in a N2-flushed glove bag to preserve anaerobic conditions. All transfers were done within 30 min. of sampling the drill hole. A number of different experiments were performed with these materials.

1. 2. 3. 4. 5.

Rates of incorporation of acetate into lipids Radioactive thymidine incorporation into DNA Aerobic mineralisation of acetate and glucose to carbon dioxide Anaerobic mineralisation of acetate and glucose to carbon dioxide Most Probable Number (MPN) counts of aerobic heterotrophs

Some typical Results: Microbial Activities and MPN counts of Aerobic Heterotrophic Microbial Populations from Deep Subsurface Boreholes. Depth metres Acetate to Lipids dpm/day Thymidine to DNA dpm/day MPN (log/g dry weight)

0 45 58 140 205 257 264

3.00 x 107 2.45 x 10 3.00 x 10 9.30 x 10 3.50 x 10

4 3 3 2

++++ ++ +++ ++ nd

7 4 6 2 2 4 4

4.30 x 102 2.54 x 10



Biology 447 ­ Environmental Microbiology

Aerobic and Anaerobic Mineralization of 14C-acetate and 14C-glucose to 14CO2 in Deep Subsurface Sediments. Depth (m) Aerobic Glucose (dpm/day x 103) Activity Acetate (dpm/day x 103) Anaerobic Glucose (dpm/day x 103) Activity Acetate (dpm/day x 103)

45 59 146 205 257 261

291 ± 165 720 ± 72 92 ± 25 49 ± 33 203 ± 72 864 ± 192

678 ± 60 165 ± 54 18 ± 6 12 ± 8 52 ± 47 1065 ± 288

nd nd nd 35 ± 2 17 ± 2 132 ± 51

nd nd nd 7±3 12 ± 3 1036 ± 4

Some general observations were: The numbers of culturable bacteria in the clay sediments were almost 3 to 5 orders of magnitude (1000 to 100000 times) less than in the shallow aquifers or the surface soils. The sandy water-bearing layers had the highest counts and the greatest microbial activities. Water-bearing sandy layers had higher numbers and activity than clay layers much nearer the surface; depth is not necessarily the limit to growth and activity. In another study, coliforms, sulfate reducers and methanogens were enumerated. Anaerobic metabolic activity was measured by monitoring the disappearance of lactate, formate and acetate and the production of methane and hydrogen sulfide. Although anaerobic microorganisms were present in the deep subsurface layers in the Savannah River site, the sediments in the area did not appear to be primarily anaerobic in nature. The anaerobes were 100 to 100000 times less abundant than aerobes. The anaerobes found were presumably growing in anaerobic microenvironments or were tolerant to oxygen levels found in the sediments. Most of the anaerobes were found in the water-saturated sandy zones where anaerobic degradation of acetate and benzoate and methane production were found in addition to the metabolism of lactate and formate that was found through the sediment. There was no phenol degradation. The numbers of coliforms dropped rapidly from the surface layers to the deeper layers. There was no evidence of coliforms in the unused drilling fluids, but coliforms were found in circulating drilling fluids. No fecal streptococci were found. All of these observations lead to the conclusion that recent contamination of the deep subsurface by surface coliforms or by sewage is unlikely, and that the subsurface may harbour a population of coliform bacteria. Another part of the investigation looked at denitrification in the subsurface. The acetylene blockage method was used to detect denitrification activity. All tested samples from all depths showed activity; it was highest at the surface and decreased with depth. It was also highest in the water-bearing sandy parts of the subsurface and lower


Biology 447 ­ Environmental Microbiology

in the clay sediments. Addition of nitrate enhanced denitrification in samples from immediately below the water table down to a sample depth of 289 metres.

How do these bacteria grow or survive at depths up to 1.7 miles below the surface ? Possibilities : 1. They were incorporated into the sedimentary rock materials during formation many millions of years ago and have survived on a "starvation" diet since then. 2. They enter through infiltrating groundwater from the surface (most likely with bacteria found in igneous rocks such as basalt or granite because of the very high temperatures during formation). 3. Some bacteria are growing slowly on inorganic energy sources and thus providing organic carbon to other microorganisms in the rock matrix (SLiME - Subsurface Lithautotrophic Microbial Ecosystems).


Biology 447 ­ Environmental Microbiology

Module Seven - Groundwater Microbiology


As an introduction to the subject, the groundwater section of the course will deal with the Evolution and Distribution of Groundwater, Groundwater Environments in general terms, the Microbiology of Groundwater, aspects of Groundwater Modelling and then with aspects of Groundwater Contamination, Remediation and Clean-up of contaminated sites. There is a great deal of overlap between the concepts in groundwater remediation and soil remediation, especially when BIOREMEDIATION (Module 8) is covered. For this reason, some parts of both topics are covered only once but apply to both areas.

General Introduction to Groundwater

Although groundwater is third in quantity behind the oceans and glaciers and permanent snow, it comprises about 69% of the world's fresh water and about 1.7% of the world's total water. However, its replacement time is over 1400 years, about half that of the world's oceans. Groundwater can exist in many different environments, but is important when it is in aquifers that we can access for our needs. The particular type, chemical content (some groundwater is extremely alkaline and/or saline) and depth below the surface depends on many factors including the rock materials or substrate it is in and the infiltration of water from other sources.

Reservoirs of the World's Water



Total volume (km3) 1 338 000 000 23 400 000 10 530 000 16 500 24 064 100 21 600 000 2 340 000 83 500 40 600 300 000 176 400 91 000 85 400 11 470 2 120 1 120 12 900 1 385 984 610 35 029 210

% of total 96.5 1.7 0.76 0.001 1.74 1.56 0.17 0.006 0.003 0.022 0.013 0.007 0.006 0.0008 0.0002 0.0001 0.001 100a 2.53a

% of fresh 30.1 0.05 68.7 61.7 6.68 0.24 0.12 0.86 0.26 0.03 0.006 0.003 0.04 100a

Replacement Period 2 650 yr 1 400 yr 1 yr 9 700 yr 1 600 yr 10 000 yr 17 yr 5 yr 16 d 8d

Annual volume recycled (km3) 505 000 16 700 16 500 2 477 25 30 10 376 2 294 48 400 600 000

World Oceans Groundwater (to 2000m) Predominantly fresh groundwater Soil Moisture Glaciers and Permanent Snow Antarctica Greenland Arctic Islands Mountain areas Ground ice (permafrost) Lakes Freshwater Salt water Marshes River Biological water Atmospheric water Total water Freshwater


Some duplication in subcategories and categories.

Most rocks near the earth's surface are in somewhat unstable condition; over long time periods they break down into smaller and smaller particles and form soils. Soils are redistributed by water transport, air movement, sedimentation, ice and gravity and eventually form new types of rock materials. Occurring over geologic time periods, these processes lead to the formation of the three main types of aquifers. Aquifers are water-bearing


Biology 447 ­ Environmental Microbiology

reservoirs capable of yielding usable quantities of water. The three types are alluvial, sedimentary and glacial. Igneous/metamorphic rocks are formed by volcanic activity or heat due to pressure and can contain water-bearing rock materials (aquifers). Alluvial Rivers and streams form groundwater reservoirs consisting of alluvial deposits. The rivers carry and deposit rock materials on the flood plain. These deposits are often of uniform grain size due to the action of the river currents sorting the particle sizes upon deposition. Some others show sharp gradations in particle size due to differential deposition onto a river bed at slower and faster regions of the current flow. Aquifers can be formed in these deposits when they are covered by other materials and buried. Sedimentary Deposition of sediments in marine and freshwater can lead to sedimentary rock materials being formed. If the land then rises due to continental movements or volcanic activities, these rock materials can then come to lie above current sea levels. If porous, they can be water-bearing. Glacial Glacial aquifers are present throughout much of the highly populated area of the US and Canada. In these cases, the underlying bedrock is igneous or metamorphic and has little water. If the bedrock does contain water in these areas, it is often of poor quality (brine). Glacial activity "grinds" rock materials and deposits them at a distance from their source. Rock material may be deposited at the edge of the glacier as it retreats, causing the formation of moraines. Some of the material in glaciers is released and moved as outwash as the glacier melts to water and forms rapidly flowing rivers. There have been numerous glaciation events in North America leading to the complex geological and aquifer formations of the Great Lakes area in particular.

Hydrogeology of Canada

Although surface water is abundant (about 24% of the surface fresh water supply of the entire world), about 10% of the water supplied by municipalities with populations of over 1000 is groundwater. Groundwater makes up an even greater proportion of the water used by individual houses because of the preponderance of dug and drilled wells in rural areas. Differences in climate and geology lead to the six regions of different hydrogeological conditions in Canada. The map below (from the United Nations, 1976) shows these regions; the Cordilleran, the Interior Plains, the Northern, the Canadian Shield, the St. Lawrence and the Appalachian regions.


Biology 447 ­ Environmental Microbiology

Briefly: The Cordilleran Region is mainly crystalline rocks with little surface deposit of materials. There is complex aquifer development in the river valleys and glaciated area. The Interior Plains Region is at the southern limit of the permafrost between the Rockies and the Canadian Shield region. The strata are nearly horizontal in arrangement with a thick layer of surficial deposits. There are some outwash type aquifers and some bedrock types where the underlying rock materials are water-bearing. In parts of Alberta and Saskatchewan, the aquifers contain water with very high salt concentrations The Northern Region is all of Canada north of the southern edge of the discontinuous permafrost limit. Rainfall and snowfall is low and the area is over a crystalline bedrock or sedimentary materials. Permafrost occurs everywhere and the aquifers can be on top, within or below the permafrost layer. The Canadian Shield Region is on mixed crystalline rocks with irregular surface deposits. The topography is very rugged. Groundwater aquifers are rarely used and are limited in extent. The St. Lawrence Region is covered with a thick surface deposit of glacial origins. Aquifer chemistry often reflects the limestone and dolomite rock materials; i.e. they contain calcium and magnesium bicarbonates. The Appalachian Region is characterised by flat sedimentary rocks, with thin surface deposits. The higher rainfall and short flow path leads to groundwater with lower levels of salts than the neighbouring St. Lawrence Region.


Biology 447 ­ Environmental Microbiology

The Groundwater Environment

Groundwater is part of the overall hydrologic cycle illustrated above. Groundwater can be present close to the surface (shallow aquifers) or at great depths. It can be in an UNCONFINED AQUIFER where the water in a porous layer (e.g. glacial till in the diagram below) or, if trapped between two impermeable rock formations can be a CONFINED AQUIFER. The recharge zone where water enters the groundwater can be at a distance. These recharge areas are now being protected where the aquifers are used as sources of drinking water (e.g the Region


Biology 447 ­ Environmental Microbiology

of Waterloo). In a typical system, surface water and groundwater interact; overland flows of water enter streams and rivers and the groundwater also can enter or leave the rivers and streams. The groundwater-containing zone is often called the "saturated zone" whereas the soil above it is the "unsaturated zone". The WATER TABLE is simply the top of the groundwater saturated zone.

If a confined aquifer happens to be present and the hydraulic pressure due to its topography is sufficient to drive the water to the surface by simply drilling into the aquifer, it is said to be an ARTESIAN WELL. Usually this happens because the recharge zone is higher than the lower land surface and the water is confined by the impermeable rock materials so that the hydraulic head is maintained.


Biology 447 ­ Environmental Microbiology

The rate at which groundwater flows through the matrix materials is determined by the hydraulic pressure and the conductivity of the materials. This can be measured in gallons per day per square foot. Typical values are given in the diagram below for different matrix materials. Note: hydraulic conductivity is a log. scale

1. Groundwater Microbiology Overview The microbiology of groundwater has only recently received much attention from microbiologists. Early studies indicated a decrease in numbers with increasing depth, so it was assumed that groundwater in aquifers would essentially be sterile. After 1970, studies began to reveal the extent and complexity of microbial populations in groundwater and, more recently, deep subsurface environments (+2000 ft) have been studied and revealed substantial colonization (see the special issue of Microbial Ecology Vol. 16 (1988) and the symposium proceedings "Proceedings of the First International Symposium on Microbiology of the Deep Subsurface, 1989").


Biology 447 ­ Environmental Microbiology

Total microscopic counts of bacteria in a pristine (uncontaminated) shallow aquifer range from about 100,000 to 10,000,000 per gram dry weight. Viable counts range from essentially zero to 10,000.000 per gram. The lower numbers found with viable counting methods may reflect our ignorance of the conditions required for growth of the organisms. In deeper aquifers, the situation is more variable; some deep aquifer layers have almost no microorganisms while others have viable bacteria up to 100,000,000 per gram. Many of these bacteria seem to be growing under low nutrient levels; they have morphologies and cell sizes typical of "starved" organisms. Biomass measurements using ATP and membrane lipid determinations correlate reasonably with direct microscopic counts. Activity measurements based on respiration, metabolism of substrates are low but significant. The types of bacteria present vary with depth. The diversity of aquifer microbial populations is lower than at the surface in soils, but does not seem to decrease significantly with increasing depth. Twenty-four genera were found by Hirsch et al (In: Progress in Hydrochemistry. pp 311-325, Springer-Verlag, Heidelberg). The genera included: Pseudomonas Achromobacter Acinetobacter Aeromonas Alcaligenes Chromobacterium Flavobacterium Moraxella Caulobacter Hyphomicrobium Sphaerotilus Gallionella Arthrobacter Bacillus Gram negative bacteria predominated in sandy aquifers. Filamentous bacteria and spores have only rarely been seen. Many bacteria isolated could not be identified. This indicates that they are different from normal soil microbial populations. When analysed by DNA and RNA hybridisation studies, there was also a much greater degree of diversity than with bacteria from soils. Very few strains found were identical to surface strains. There are rare reports of eucaryotic organisms (algae, yeasts, fungi) in groundwater. Most are "invaders" that come from surface connections (flow paths, fractures, etc.). Protozoa have been detected more often from various depths. Some studies give numbers as high as 100 protozoa per gram).

Bacteria in Groundwater - Agriculture

From: The bacteria routinely measured in water quality testing are not necessarily those that cause disease. Their presence indicates the possibility that water may be contaminated by animal or human waste. They are targeted in testing because they are much easier to detect than the actual disease-causing organisms, which may be present in extremely small numbers and difficult to grow in the laboratory. The Canadian drinking water guidelines for coliform and Escherichia coli bacteria are therefore rather arbitrary values. Scientific evidence relating these limits to human health is scanty, because these organisms are not the actual causes of disease. A recent survey in Ontario found that a greater incidence of diarrhea in farm family members was associated with the detection of E. coli in their well water at some point during the study year. The combination of nitrate and bacterial contamination may be important, because methemoglobinemia has been associated with waters containing both. In general, water from drinking water wells in Canada is more likely to exceed guidelines for bacteria than for nitrate or pesticides.


Biology 447 ­ Environmental Microbiology

In Ontario's 1992 survey of groundwater quality, 34% of the wells tested exceeded the maximum acceptable number of coliform bacteria, and about 7% were contaminated with both bacteria and nitrate. The incidence of bacteria · · decreased with depth for dug or drilled wells but not for wells formed by a sharp point driven into sandy material (sandpoint wells) was higher in older wells than in younger wells.

Smaller numbers of bacteria were recorded for samples taken in the winter than in those taken in summer. Potential point sources of nitrate or bacteria contamination, such as septic or sewage disposal systems and feedlots or exercise yards, were also investigated during the survey. The distance from a well to the weeping bed or septic tank had no influence on the level of well contamination with nitrate or bacteria. Feedlots and exercise yards were identified as significant localized sources of groundwater contamination with bacteria. Multi-level field wells tested in the survey were expected to separate out point-source contamination. However, in only one case was a farmstead domestic well contaminated and the multi-level well on the same farm not contaminated. In all other cases, bacterial levels were similar in drinking water wells and multi-level field wells, which suggests that the bacteria came from agricultural fields as much as from point sources. An Ontario study using tracer bacteria showed that water flow through cracks and macropores in the soil can quickly move bacteria 100 metres or more from a septic system, manure storage, or solid beef manure. This phenomenon can result in high levels of bacteria reaching tile drainage outlets within a short time after spreading manure. Once in groundwater, bacteria appear able both to survive for several months because of the cool temperature and to multiply there. Compared with bacterial levels measured in wells in Ontario from 1950 to 1954, the number of wells with fecal coliform bacterial counts over the guidelines may have almost doubled over the past 45 years. In contrast, nitrate levels have remained much the same. In the 1995­1996 Alberta well survey, 2% of the 448 deep wells tested had concentrations of fecal coliforms above the Canadian drinking water guideline, and 10% had total coliforms above the guideline. Of 376 shallow wells tested, 5% exceeded the guidelines for fecal coliforms, and 19% for total coliforms. Some data for bacteria in groundwater in other provinces are given in Table 6-5.

Table 6-1 presents the results of groundwater surveys carried out in Ontario since the 1950s. The share of wells with nitrate levels of more than 10 milligrams per litre recorded in 1991­1992 did not differ significantly from that reported in 1950­1954. Surveys carried out between these dates indicated that about 5 to 20% of drinking water wells had levels of nitrate greater than the Canadian drinking water guideline. These results suggest that


Biology 447 ­ Environmental Microbiology

agricultural activity over the past 50 years has not significantly changed the amount of nitrate added to groundwater. Multi-level monitoring wells were also installed in farm fields and woodlots adjacent to the drinking water wells at 144 survey farms. Nitrate concentrations exceeded the Canadian drinking water guideline for more than half the sampling intervals at 23% of the field multi-level sites. The average concentration of nitrate­nitrogen in these wells decreased from about 10 milligrams per litre near the water table to 3 milligrams per litre at a depth of about 6.5 metres. The share of contaminated wells was similar for both the drinking water wells and field multi-level wells in the survey, indicating that contamination is caused as much by activities on the cultivated fields as by on-farm point sources of contamination.

Although well surveys lack precision in evaluating the effects of specific farming or cropping practices, it was observed that · · farms where manure was spread were more likely to have wells contaminated with nitrate­nitrogen and bacteria than other farms uncultivated conditions in the woodlots appear to have provided an environment in which nitrate, but not bacteria, was removed from groundwater.

Nitrate contamination is a more serious problem in groundwater than in surface water in Quebec. It has been particularly associated with areas of intensive potato production. This crop is produced on sandy soils using large amounts of nitrogen fertilizers. Nitrate concentrations over the Canadian guideline for drinking water have been found in the aquifers that supply drinking water to several municipalities. For example, 40% of wells in the regional municipality of Portneuf have had nitrate concentrations above this guideline. Recent measurements have indicated


Biology 447 ­ Environmental Microbiology

improvements in the situation, believed to be the result of using cover crops, reducing fertilizer nitrogen inputs, and applying smaller applications of fertilizer.

Ontario farm groundwater quality survey

In 1991 and 1992, Agriculture and Agri-Food Canada sponsored a survey of water quality in farm drinking-water wells. The survey was carried out by a consortium of the Ontario Soil and Crop Improvement Association, the universities of Guelph and Waterloo, and the Ontario ministries of Health; the Environment; and Agriculture, Food, and Rural Affairs. Four farm wells were chosen in each township where more than 50% of the land area was used for agricultural production. Elsewhere, one well per township was usually sampled. Within each township, the types of farming activity and dominant soils were additional criteria for selection. Each participating farm household completed a questionnaire about their well construction, distance to potential point sources of contamination (septic system weeping beds and tanks, feedlots or exercise yards, and manure storages), use of manure and fertilizers, cropping system, pesticide usage, and petroleum storage. The network included almost 1300 of the estimated 500 000 water wells in Ontario. None of the point sources investigated contributed significantly to the general level of contamination of farmstead domestic wells by nitrate­nitrogen. Using aggregated data, no relationship was detected between the distance separating the well from potential point sources and the incidence of nitrate contamination. The occurrence of contaminated groundwater was related to the type, depth, and age of the water well. Contamination occurred more frequently

· · ·

in dug and bored wells or shallow sandpoints than in drilled wells, regardless of depth at lesser depths in all wells in older wells, especially shallower, non-drilled wells. M.J. Goss and D.A.J. Barry,University of Guelph and D.L. Rudolph, University of Waterloo

Conclusion The impact of agriculture on groundwater quality is a concern worldwide. Elevated concentrations of nitrate have been observed in groundwater in intensively agricultural areas in many parts of the world, including the United States, United Kingdom, European Union, Australia, New Zealand, and Canada. In Canada, groundwater quality is generally within Canadian Water Quality Guidelines in most areas of the country, but nitrate levels are a continuing concern. Research and surveys have shown that intensive agricultural practices may increase both the risk and incidence of nitrate contaminating groundwater. Nitrate leaching results mainly from the mismatch between crop demand for nitrate and microbial activity in the soil and is associated with all agricultural practice. Bacterial contamination of groundwater is also observed, particularly in areas where large quantities of manure are applied. Pesticides have been detected in some groundwater, but concentrations that exceed water quality guidelines are uncommon and are usually associated with point sources, such as pesticide spills. The prospects for improving this situation depend largely on producers who apply environmentally sound management practices (recognizing the limitations of individual farms related to climate, topography, soil, equipment, finances, and time). Although many of these practices are easy to adopt, others require an investment of time and money.

Sampling Techniques

Soil sampling


Biology 447 ­ Environmental Microbiology

Sampling of shallow layers of soil is performed using standard sampling methods for soils. Deeper samples of the soil and vadose zone can sometimes be taken by digging deeper pits and sampling horizontally with borers or sampling tubes. Groundwater Sampling for Microbiological Assays. There are many problems associated with sampling groundwater for microbiological purposes. One of the main problems is that of ensuring representative samples of both the groundwater and the mineral matrix of the aquifer. Bacteria often adhere to particles and may not be equally distributed between the groundwater and the particles. The groundwater is also moving through the matrix at various rates depending upon the permeability of the matrix and this can complicate sampling techniques. The preferred method is to take core samples whenever possible so that both water and matrix material are disturbed as little as possible. In groundwater environments close to the surface (high water table), piston-driven cores can be forced into the aquifer material to obtain samples. A hammer drill or similar device is used to force the sampling tubes (cores) into the soil and aquifer material. In cohesive matrix materials, withdrawing the core also withdraws the material and the tube can be stored until sampled.

The (usually aluminum) cores can be stored refrigerated until sampled, and the tubes can be easily sectioned into smaller lengths. The outer peripheral layer of material that could have been in contact with the tube (and therefore could be contaminated) is not used. Samples are taken from the interior of the core. These can be diluted and plated or examined microscopically after staining (usually with fluorescent staining methods).


Biology 447 ­ Environmental Microbiology

These sampling techniques can be adapted to anaerobic systems by using nitrogen gas flows to "blanket" the sampling core, storing it in nitrogen filled containers for transport (always handling it in a nitrogen-filled anaerobic chamber environment during sectioning), peeling off the potentially contaminated outer layer of sample and sample taking. Some of these anaerobic chambers can now be taken into the field to carry out these anaerobic sampling processes. If groundwater samples are required from various depths in a drilled well, many different lengths of Teflon tube (an inert material)

can be inserted into the well after it has been drilled or bored into the aquifer. Many different techniques are available to drill the well, but these multi-level piezometer wells rely on being able to withdraw the well casing, leaving the tubes in place with their openings at different depths in the aquifer. Usually, many wells are drilled in an area to obtain a good coverage of the groundwater system. Groundwater can be withdrawn from the tubes as required to obtain a complete three-dimensional "sample" of the groundwater in the area sampled. One sampling system consists of a vacuum- or pump-driven, switchable manifold that allows the different tubes to be sampled and the water drawn into sterile sample bottles.


Biology 447 ­ Environmental Microbiology

This system is most useful for obtaining detailed "snapshots" of the distribution of natural or injected chemicals in an aquifer. It has been used extensively at Camp Borden, Ontario, to follow the migration and fate of injected chemicals in the Camp Borden aquifer (see later).

Detailed Sampling Systems One study [Barbaro, Albrechtsen, Jensen, Mayfield and Barker: Geomicrobiology Journal Vol 12 203-219 (1995)] has examined the distribution of bacteria over small distances in an aquifer (the aerobic zone of the Camp Borden aquifer). The microbial numbers were determined for 9 cores, 1.5 metres in length collected from the sand aquifer. They were from a zone that had not been used for other experiments, so it represented a "pristine" or normal condition. Viable cell counts, electron transport system activity, dissolved oxygen levels, dissolved organic carbon levels and hydraulic conductivity were determined for contiguous samples at each 10-cm interval in the cores. The cores were arranged in a Y-shape with the open end of the "Y" facing towards the groundwater flow direction. Maximum microbial occurrence and activity was at the top of the shallow aquifer and decreased rapidly with depth. The activity was correlated with oxygen level and depth (these were also related, as might be expected). Analysis also showed a correlation with dissolved organic carbon levels (these were low and only supported limited microbial growth) Growth was stimulated only when a source of nitrogen was added. This suggests that the limiting nutrient in the system was nitrogen. There was also a considerable difference between the various samples from similar depths in the 9 core samples and between contiguous samples in the same column. This clearly demonstrated the large degree of variation present in microbial distributions in this (relatively homogeneous) aquifer.

Microbial Processes

Chemical and Physical Conditions in Normal Groundwater

The range of physical and chemical properties in groundwater environments can vary widely. The particular properties will be dependent upon such things as: · · · · · the origin of the rock materials forming the matrix the chemical composition of materials earlier in the flow path the state of the fracturing or the weathering of the materials the grain size distribution the age of the formation (how long it has been eluted by groundwater)

The nutrient status of groundwater varies widely according to the variation in physical and chemical properties. To support microbial activities, the following must be present:


Biology 447 ­ Environmental Microbiology

· · · · · ·

carbon source for biomass production (and often energy production - by heterotrophic bacteria) source of energy (if not from carbon metabolism) electron acceptors major nutrients for biomass production (N, P, S) minor nutrients for enzyme activities other required growth factors (trace elements, organic cofactors, etc.) In terms of percentage dry weights, typical cellular values for various elements are: Element Carbon Oxygen Nitrogen Hydrogen Phosphorus Sulphur Potassium Sodium Magnesium Chlorine Iron All others Percentage of dry weight 50 20 14 8 3 1 1 0.5 0.5 0.5 0.2 0.3 (approx.)

These values should be present in the same approximate concentrations in any environment that allows microbial growth. As microbial growth occurs, oxygen depletion may occur and anaerobic conditions will be produced. This will most likely occur under conditions of high nutrient loading entering the groundwater from any source. Typical situations are where a landfill site leaches materials into the groundwater, or where organic materials enters from a contaminated or high nutrient status surface water body. Since oxygen diffuses 10,000 times more slowly in water than in air, and is sparingly soluble in water (typical values are in the low mg/L range), microbial activity can quite readily remove all available oxygen. This will cause a change in the Eh or pE (redox potential) of the groundwater. This redox potential will then be further changed (assuming oxygen is absent), by the presence and chemical equilibria of the ions dissolved in the water. Typically, growth in uncontaminated groundwater is limited by the low carbon and/or nitrogen levels present. The system is often C or N limited. Addition of these elements in an available form leads to increased microbial activity. This activity can then lead to oxygen depletion and production of anaerobic conditions and low pE values (measured in millivolts) Chemical Conditions in Groundwater Contaminated with Organics The number of contaminants entering groundwater is potentially very large. Any source of contamination that can enter groundwater through surface waters, disposal practices, septic systems, air transport, run-off, infiltration from streams, lakes or rivers can lead to effects on groundwater. Gasoline is a common contaminant of groundwater and forms a plume of soluble gasoline components in groundwater systems. The hydrocarbons of gasoline float on the surface and can move. The soluble components dissolve in the groundwater and migrate.


Biology 447 ­ Environmental Microbiology

Movement of materials in groundwater Groundwater moves at slow rates depending on the porosity and hydraulic conductivity of the medium, through which it flows. Any compounds dissolved in the groundwater also move, but the movement is complicated by the adsorptive processes of the compounds on the mineral and organic parts of the rock material in the aquifer. If the compound is not adsorbed at all (chloride or bromide ions are examples) then it moves at the same velocity as the water. This is ADVECTION.


website for animation)

Note that the water moves at the same speed as the advected material (chloride ions in this example) If the compound is adsorbed onto materials in the matrix, the effective movement will be slower than that of the groundwater; this is RETARDATION. Compounds that dissolve in lipids also tend to more soluble in the organic matter in soils and the groundwater matrix material. The sorption distribution coefficient (Kd) of the compound is a measure of the degree of retardation that can be expected.


website for animation)

Note that the retarded compound (the red "plug") moves at a slower rate than the water (the blue arrow). It is RETARDED compared to the speed and extent of water flow. This behaviour occurs because the retarded compound is adsorbed onto organic materials in the aquifer and then released. This effectively "slows down" the speed at which the material can move. If the compound is undergoing biological or chemical degradation as it travels, its actual volume may become smaller as the materials is used. Normally, however, without degradation, the volume of the material becomes larger (but less concentrated) as it moves because of the processes of DISPERSION and DIFFUSION


Biology 447 ­ Environmental Microbiology ANIMATION OF DISPERSION PROCESS (see

website for animation)

Note that the yellow "plug" of material is moving at the same speed as the water (the blue arrow), but that it is getting larger as it moves through the aquifer matrix. This is because it is "dispersing" or "diffusing" as it travels. Movement of contaminants


website for


Note the rather tortuous pathway that a water molecule takes as it passes through the water surrounding the "grains" of sand. Each water molecule may take a slightly different length path, causing the perceived dispersion of materials as they are transported through the aquifer. If the compound is a conservative tracer such as chloride ions (i.e. it is not retarded or adsorbed and is not biodegraded), then simple dispersion of the compound will occur.


(see website for animation)

In an experiment designed to show the fate of gasoline soluble components in groundwater, CHLORIDE, BENZENE and TOLUENE were injected into the site at Camp Borden through an injection well. The threedimensional movement of the resulting plume in the moving groundwater. The plan below shows the position and extent of the "slugs" of chloride, benzene and toluene (from left to right) after 3, 53 and 108 days. The slug at each of the three times is on the same diagram. They are separated on the diagram even though in the actual experiment they were all moving along the same flow path. In fact, no material was left at the injection wells at 53 and 108 days. The contour lines in each slug show the calculated three dimensional concentration of the materials reduced to 2 dimensions for plotting. The data for these plots was gathered from 20 depth samples at each of the sampling wells on the diagram as the "slugs" passed. The X and Y coordinates are in metres.

BTX Behaviour in a sandy aquifer These processes can be seen in the movement of "slugs" of benzene, toluene and chloride ions in a sandy aquifer. The toluene "slug" is being rapidly degraded as it moves and is becoming smaller as it moves slowly through the aquifer. The benzene is degraded more slowly and maintains a larger volume as it moves. The chloride is not degraded at all and so the mass remains constant, but the volume occupied becomes larger (and the concentration becomes lower). If, on the other hand, the compound IS absorbed (retarded) and IS biodegraded (such as toluene) - then the shape of the slug of compound will be very different. Even though dispersion is occurring, it may be masked by the actual disappearance of mass due to biodegradation and the "slowing" of the rate of transport of the slug due to retardation (by adsorption)


Biology 447 ­ Environmental Microbiology

Animation of movement through the Borden Aquifer (see

website for animation)

1. The CHLORIDE ions move by a process of advection and dispersion/diffusion. Note that the chloride plume

grows larger but less concentrated. There is no MASS LOSS but rather a movement and dispersion of the material through a larger volume. This kind of movement is typical of a CONSERVATIVE TRACER; no biodegradation, no retardation, simply advection and dispersion with no mass loss. 2. Both the BENZENE and TOLUENE are retarded (they move a smaller distance than the chloride ions). There is very little mass change in the benzene but there is a large change in the toluene. The toluene is being biodegraded and has essentially disappeared by day 108. 3. Note the spreading of the materials in the linear direction of movement. This is due to the fact that dispersion in the direction of flow is usually greater than dispersion in other directions (see the plume for chloride and benzene at day 53) 4. Note that the plumes of different materials are not uniform in concentration throughout their volume. Differences in dispersion rates due to the heterogeneous nature of the aquifer materials leads to this uneven distribution of concentration (see the plume for chloride at day 53).

Rates of Biodegradation of Organic Compounds in Various Environments in Groundwater One of the major concerns when examining the biodegradation of chemicals in groundwater is the rate of the processes. This is because of the fact that the movement of groundwater leads to migration of materials that can


Biology 447 ­ Environmental Microbiology

then cause problems at remote sites. This becomes a legal issue of responsibility for clean-up of those contaminants. The only "safe" situation is where the plume does not migrate past the property boundary of the company or person causing the pollution. It is then a matter of cleaning up that site and preventing any migration to another property. The issues of rate of biodegradation and microbial activity are obviously closely linked. It is important to establish which reactions are possible or probable and how fast they are likely to occur in a particular contaminated groundwater system. A typical groundwater contaminated with organic material (eg. leachate from a landfill site) shows a series of different zones over a distance in the flow path.

Redox Reactions Redox reactions are the most common type of reaction, modifying or removing compounds from groundwater environments. The groundwater environment often develops different redox potentials due to growth of microorganisms and consequent removal of oxygen followed by a progressive reduction in Eh or pE due to the growth and activities of other microorganisms (see evolution of groundwater). The response of different groups of microorganisms to chemical contaminants at different redox conditions is an important aspect of biodegradation. This response can be divided into two parts: 1. Energy Yield (Thermodynamic equations) 2. Rates of Degradation (Kinetics) These two are NOT the same; a reaction can be thermodynamically possible and actually yield energy, but it is so slow without the presence of "catalytic" enzymes from bacteria (for instance), that it will not be significant in the environment. 1. Energy Yield (Thermodynamics)


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Every redox reaction consists of two half reactions - an oxidation and a reduction. In theory, any set of half reactions can be combined and an energy yield calculated; this does NOT mean that pairs of half reactions that yield energy will necessarily be fast enough to be significant, only that at equilibrium the energy yield will be "X" kcals. The speed at which the reactions reach equilibrium is a function of the kinetic, not the thermodynamic, equations. One way to examine these half reactions is to look at a series of oxidation and reduction reactions and calculate the energy yields by pairing them. If the energy yield is positive, then the reaction is thermodynamically possible without the input of external energy - i.e. it is an energy-yielding reaction. If growth is to occur, then energy yielding reactions are required. We can calculate Free Energy for various half reactions important in groundwater environments and present the results as a "delta G [DG] in kilocalories per mole of reactants". That is the change in free energy in kilocalories per mole. These half reactions can then be combined to calculate (algebraically) the delta G for the combined reactions. This will provide the energy yield for that set of reactions. Half Reaction 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 DG - kcals/mole EH0 volts 0.966 0.808 0.770 0.743 0.408 0.357 -0.220 -0.250 -0.286 -0289 -0.289 -.0329 -0.331 -0.341 -0.370 -0.419 -0.432 -0.497

1/3 NO3- + 4/3 H+ + e= 1/6 N2 + 2/3 H2O -22.263 + 1/4 O2 + H + e = 1/2 H2O -18.675 Fe3+ + e= Fe2+ -17.780 + 1/5 NO3 +6/5 H + e = 1/10 N2 +3/5 H2O -17.128 1/2 NO3- + H+ + e= 1/2 NO2- + 1/2 H2O -9.425 + + 1/8 NO3 + 5/4 H + e = 1/8 NH4 + 3/8 H2O -8.245 2+ 1/8 SO4 +19/16 H + e = 1/16 H2S +1/16 HS- +1/2 H2O 5.085 1/8 CO2 + H+ + e= 1/8 CH4 +1/4 H2O 5.763 + 1/8 CO2 + 1/8 HCO3 + H + e = 1/8 CH3COO- +3/8 H2O 6.609 + 15/92 CO2+ 1/92 HCO3 + H + e = 1/92 CH3(CH2)14COO + 31/92 H2O 6.6657 1/7 CO2 + 1/14 HCO3- + H+ + e= 1/14 CH3CH2COO- + 5/14 H2O 6.664 + 1/8 CO2 +H + e = 1/12 CH3CH2OH + 1/4 H2O 7.592 1/6 CO2 + 1/12 HCO3- + 1/12 NH4+ + H+ + e- = 1/12 CH3CHNH2COOH + 5/12 H2O 7.639 1/6 CO2+ 1/12 HCO3- + H+ + e= 1/12 CH3CHOHCOO- + 1/3 H2O 7.873 + 1/5 CO2+ 1/10 HCO3 + H + e = 1/10 CH3CHOCOO + 2/5 H2O 8.545 H + + e= 1/2 H2 9.670 + 1/4 CO2 + H + e = 1/24 C6H12O6 + 1/4 H2O 10.020 + 1/2 HCO3 + H + e = 1/2 HCOO + 1/2 H2O 11.480

It is now possible to combine the half reactions to calculate the energy yields from the various reactions:

Electron Donor Acetate

Electron Acceptor O2

Half Reactions Combined (14) - (2)

Complete Reaction

DG kcal/electrons

1/8 CH3COO- + 1/4 O2 = 1/8 CO2 +1/8 HCO3- + -25.28 1/8 H2O


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Acetate Acetate Acetate


(14) - (5) (14) - (10) (14) - (12)

1/8 CH3COO- + 1/4 NO3- = 1/8 CO2 +1/8 HCO3-16.03 + 1/8 H2O + 1/2 NO21/8 CH3COO- + 1/4 SO42- + 3/16 H+= 1/8 CO2 +1/8 HCO3- + 1/8 H2O + 1/16 H2S + 1/16 HS1/8 CH3COO- + 1/8 H2O = 1/8 CH4 +1/8 HCO3-1.52 -0.85

Reactions in Series: It is also possible to use the same concepts and calculations to examine the situation where a series of reactions occur in sequence (leading to a series of intermediates that are then metabolised to other compounds). Each step in the process can be assigned a set of reactions that will, in total, sum to give the overall reaction of the entire process. An example is the process of denitrification occurring with methanol as the substrate: Step 1. 0.067 CH3OH + 0.2 NO3- = 0.067 CO2 + 1.33 H2O + 0.2 NO2(nitrate to nitrite) Step 2. 0.100 CH3OH + 0.2 NO2- + 0.2 H+= 0.1 CO2 + 0.3 H2O + 0.1 N2 (nitrite to nitrogen gas)

Overall: 0.157 CH3OH + 0.2 NO2- + 0.2 H+ = 0.1 CO2 + 0.3 H2O + 0.1 N2


of nitrate to nitrogen gas)

Overview: If this general process is carried out for the various combinations of organic substrates and electron acceptors, the following summary graph is obtained:


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Greater energy is represented by greater negative values (-22 means more energy release than -10) Nitrite is the most efficient electron acceptor - more efficient than oxygen. There is decreasing energy availability for ALL electron donors as the electron acceptor changes from nitrite to oxygen to nitrate to sulfate to carbon dioxide (in that order). The compounds listed as electron donors (methane to formate) are typical compounds found in organic matter entering groundwater or produced in situ in groundwater with high organic carbon input. They are typical metabolic products of microbial activity in groundwater. Combination of electron donors with any electron acceptor leads to increasing energy yields from methane to acetate to benzoate to succinate to ethanol to lactate to glycine to pyruvate to methanol to glycerol to glucose to formate (in that order). Relationships to Groundwater Evolution Process: The order of decreasing energy availability is the same as the order of biochemical reactions observed in a groundwater plume. The most "available" or "utilised" electron acceptors are those that yield the highest energy per mole under the particular environmental and Eh conditions. Removal of oxygen leads to utilisation of nitrate as electron acceptor. Removal of nitrate leads to utilisation of sulfate as electron acceptor. Removal of sulfate leads to utilisation of carbon dioxide as electron acceptor. Not all organic compounds are utilised as electron donors under all conditions. Some are only utilised by certain groups of bacteria.


The kinetics of biodegradation are a set of empirically derived rate laws. Three suffice to describe most biological reactions: dCA/dt = -k0 Zero order dCB/dt = -klCA First order dCB/dt = -k2CACB Second order


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k0, k1, k2 = rate constants mol/1-sec, /sec, 1/mol-sec, respectively CA, CB = some reacting species This can be applied to the reaction of the compounds with a surface such as a metal catalyst, a soil surface or an enzyme. Two extremes of concentration can be delineated; the first is when there are few molecules of reactant (CA) and many of the surface. In this case, few of the available sites will be covered, so the reaction rate dCA/dt is proportional to the concentration of A (first order reaction above). Secondly, when CA is so large that every site is saturated with A, the rate is constant (zero order reaction above). The combined function of these reactions can be written; - k0 CA dCA/dt= k' + CA

Where k' = ko/kl This is the very common biological form of the equation for growth on a substrate as the concentration of the substrate is increased. It leads to Michaelis-Menton (or Monod-) type kinetics. The saturation coefficient (Ks) is the concentration of substrate equal to half that causing saturation of the enzyme sites (zero order). It is that same as adsorption onto a surface-area-limited substrate. The enzyme sites or the adsorbing sites are "saturated". The enzyme cannot operate faster, and the adsorbing substrate cannot adsorb any more material. Download "Hyper" (Website)- a software program for calculating and graphing reaction rates Bacterial growth kinetics are slightly more complex and follow the classical "Monod-type" kinetics. In this case, the rate of substrate utilisation is proportional to the concentration of the microorganisms present [X] and is a function of the substrate concentration. The Monod bacterial growth kinetics are traditionally written as:

k[X][S] d[S] = dt y[Ks]+[S]

Where: [S] = substrate concentration k = maximum utilisation rate for the substrate per unit mass of bacteria


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[X] = concentration of bacteria [Ks] = half-velocity coefficient for the substrate y = yield coefficient = d[X]/d[S] OR, in graphical terms ; (see Hyper software program for data analysis using Michaelis-Menton kinetics)

Example above from default data set (4) of Hyper software program Ks (or Km in graph above) typically ranges from 0.1 to 10.0 mg/L. Groundwater systems therefore usually operate in the range where Ks is more than [S]. In this particular case, the equation reduces to second order kinetics;




= K' [X][S]

dt yKs

Where K' = k/yKs


Biology 447 ­ Environmental Microbiology

If substrate concentrations are low, the reaction becomes first order with respect to both substrate and bacterial population size. This has been confirmed experimentally in many sites and with many systems. There are really three kinds of kinetic models used in describing biotransformations in soils and groundwater systems. The first, BATCH model kinetics, are those described above. They deal with the utilisation and biotransformation of the substrate and the growth of bacteria over time in a closed system. The second, CONTINUOUS model kinetics deal with a more-or-less constant flow of the substrate through or into a known volume system. These models are useful for predicting results of slow but continuous processes. The third is that of BIOFILM model kinetics (see page on Biofilms). It is based on the theory that the bacteria are attached to solid particles in the subsurface environment and behave accordingly. This last model still uses Monod-type kinetics but extends the model to include the effects' of biofilm thickness and diffusion of substrate into and out of the biofilm. More than likely the actual "biofilms" in the field situation are so sparse as to simply constitute a random distribution of individual cells attached to mineral or organic matter particles. They cannot be considered ''biofilms'' in the engineering sense. In particular, subsurface environments where the substrate content and concentration is very high (landfill site leachate?), some degree of biofilm may be present, but calculations of population densities and actual direct observations should always be done to confirm this possibility. The model below shows a series of simplified Monod-type kinetic models developed by Simpkins & Alexander (1984) to describe some simple systems with a single type of organism and a single substrate using only substrate concentration and cell density as the variables. To generate these models, a mass balance equation is substituted into the Monod equation. The population density is B and is expressed as equivalent biomass [X], which is the amount of substrate required to produce a population density B and has units of concentration;

Where Y is the yield and is assumed to be independent of biomass and substrate concentrations. Since it is based on a closed batch system, the sum of the substrate and equivalent biomass at any time must equal the initial substrate concentration [SO] added to the initial equivalent biomass [X0];

X + S = X0 + S0

Then, by combining the Monod equation and the equation above, the Monod equation with growth can be derived (see the Table below). Another five equations can be derived depending on the initial substrate concentration and initial equivalent biomass. Three of the models are based on the initial substrate concentration being much smaller than the initial equivalent biomass (Zero order, Monod with no growth, and first order). For zero order and logarithmic growth, the initial substrate concentration far exceeds the half saturation constant while for logistic growth the reverse is true. The six models for mineralisation kinetics from Simkins & Alexander (1984) Zero order Differential form Integral form Derived parameter Necessary conditions Monod - no growth Differential form Integral form -dS/dt =klS/(Ks + S) Ks In (S/S0) + S - S0 = -klt -dS/dt = kl S = S0 - klt kl = m max X0 X0 > > S0 and S0 > > Ks


Biology 447 ­ Environmental Microbiology

Derived parameter Necessary conditions First order Differential form Integral form Derived parameter Necessary conditions Logistic Differential form Integral form

kl = m max X0 X0 > > S0

-dS/dt = k3S S = S0 exp (-k3t) k3 = m max X0/Ks X0 > > S0 and S0 < < Ks

-dS/dt = k4S (S0 + X0 - S) S = S0 + X0 1 + (X0/S0)exp[k4(S0 + X0)t]

Derived parameter Necessary conditions Monod with growth Differential form Integral form Derived parameter Necessary conditions Logarithmic Differential form Integral form Derived parameter Necessary conditions

k4 = m max/Ks S0 < < Ks

-dS/dt = [m max S(S0 + X0 - S)]/(Ks + S) Ks In(S/S0) = (S0 + X0 + Ks)ln(X/X0) -( S0 + X0)m max t None None

-dS/dt = m max(S0 + X0-S) S = S0 + X0 [1 - exp (m max t)] None S0 > > Ks

If this is graphed:


Biology 447 ­ Environmental Microbiology


Co-metabolism and Secondary Substrate Utilization There are a number of compounds in the environment which are transformed by microorganisms, yet it has been difficult or impossible to find organisms that can use them as a source of carbon and/or energy. The compounds may be transformed sequentially by a series of bacteria or other microorganisms such that no organisms gained energy sufficient to allow growth or cell division, from the reactions It is necessary to have an alternate or primary substrate for growth under these conditions. A good definition of this co-metabolism is


Biology 447 ­ Environmental Microbiology

" the transformation of a non-growth substrate in the obligate presence of a growth substrate or another transformable compound'. Some examples of co-metabolism are: Organism Methylomonas Nocardia Growth substrate Non-growth substrate Products methane hexadecane hexadecane Achromobacter benzoate ethane toluene p-xylene m-chlorobenzoate naphthalene anthracene cyclohexane limonene p-cymeme ethanol, acetaldehyde, acetic acid 2,3-dihydroxybenzoic acid, a-methyl muconic acid 2,3-dihydroxytoluic acid, p-toluic acid 4-chlorocatechol, 3-chlorocatechol salicyclic acid 2-hydroxy-3-naphthoic acid cyclohexanone perillic acid, perillyl alcohol cumic acid

Corynebacterium hexadecane glucose AIycobacterium Pseudomonas Nocardia propane glucose hexadecane

A more comprehensive example comes from the work of Dalton & Stirling (1982) who examined the enzyme methane monooxygenase (MMO). This enzyme catalyzes the NAD(P)H-driven insertion of oxygen into a wide variety of compounds such as n-alkanes. haloalkanes, alkenes, ethers and aromatic, alicyclic and heterocyclic compounds. They found that MMO, of 31 compounds oxidized, 5 were only oxidized by resting cells and 7 were oxidized only in the presence of 4mM formaldehyde. None of the compounds were able to support growth and replication at the normal growth temperature in a period of 10 days. Substrate methane ethane chloromethane methanol dimethyl ether carbon monoxide ethene propene benzene toluene pyridine ammonia Products methanol, formaldehyde ethanol, acetaldehyde formaldehyde formaldehyde methanol, formaldehyde carbon dioxide epoxyethane epoxypropene phenol, hydroquinone benzyl alcohol, p-cresol pyridine-N-oxide hydroxylamine, nitrite

There are a many differences between what would be observed for a compound that acts as a substrate for growth, versus one that does not. The first case would follow some type of normal kinetic curve depending upon the Ks and the concentration of the substrate (see above). Not a substrate for growth (cometabolism) Organism will not use as sole C source Accumulation of intermediate

Topic Characteristics

Growth substrate Organism will use C source No lag after second addition of substrate


Biology 447 ­ Environmental Microbiology

products likely. No difference in pattern between first and subsequent additions. Kinetics Exponential; Low Km, high k First order, Constant E., Generally high Km low k,saturation may not be seen. No anomalous behaviour, first order kinetics apply.

Behaviour at low Ks Acclimation

Possible anomalous behaviour due to threshold for induction

Major effect; lag may be variable due Often no effect; rarely causes to "patchiness" and low density of induction, may increase resistance to degrading organisms and also low toxic chemical. growth rates of organisms in situ. Probably not valid; probably habitat specific and "patchy" due to low density. General: expect eventual degradation. Problems of growth kinetics and acclimation effects. Generally fast rates. Environmental parameters (pH pO2, temperature etc.) of less importance. Diauxic pattern May be valid since activity is related to biomass (often proportional). Generally slower rates, more difficult to measure accurately. Environmental parameters more important.

Relation of degradation kinetics to total activity or biomass (e.g. B for second order rate expression) Extrapolation

Effect of added carbon

Generally effect is proportional to population unless specific C source induces or represses/inhibits activity being investigated Passive transport more likely; likely slow and so rate-limiting

Transport into cell

Active transport more likely

Contaminated Groundwater The most common contaminants found in groundwater are derived from activities involving production or use of synthetic organic compounds such as organic solvents, pesticide and other chemical production facilities, fuels and fuel additives. dye production, plastics production and use, and various chemical feedstock operations. In addition, plants such as wood treatment plants can introduce metal contamination (arsenic and copper), oil refineries can introduce metal catalyst residues, and disposal practices can introduce many other metal forms. Generally, these chemicals are introduced during production, transport, storage, utilisation, as feedstocks in other processes or loss by dispersion during use, spillage, accident and improper disposal. The most common organic contaminants are: · · · · · · · · chloroform trichloroethylene carbon tetrachloride tetrachloroethylene 1,1,1-trichloroethane dichloroethylenes dibromochloropropane methylene chloride

and, in lower amounts; · toluene


Biology 447 ­ Environmental Microbiology

· · · · · ·

benzene xylene phthalates chlorinated aromatics PAHs pesticides (about 40 have been detected in North America at concentrations ranging from 0.1 to 5 mg/L).

The most commonly found pesticides are herbicides (typically alachlor, 2,4-D and atrazine) and soil fumigants or sterilants (such as 1,2-dichloropropane and EDB). This reflects the heavier use patterns for these compounds compared to the insecticide group of pesticides. The presence of metal ions in groundwater is dependent on the pE and pH of the environment. The solubility of elements such as aluminum, manganese, iron, cobalt, and others depends very much on the pH of the system. For instance, aluminum is much more soluble at lower, acidic, pH levels. (See the abstract on acid mine drainage and metal mobility - Website)The redox state of the environment determines to a large extent the valance state of the element in solution. As an example of a survey of contaminated groundwater, see the file from BC Environment (Website) on the extents and sources of groundwater contamination in that province. Also, see the full report from the BC Ministry of the Environment (Website) on Groundwater in BC.


Biology 447 ­ Environmental Microbiology

Groundwater Modelling

There are two aspects to groundwater modelling that are of interest to environmental microbiologists. 1. 2. The modelling of groundwater flow and environmental conditions in aquifers Modelling the activities (including biodegradative) of microorganisms in groundwater.

When the two are linked, it will be possible to predict the fate and transport of contaminants in groundwater systems. This is not an easy set of problems to solve, nor is it easy to get the data and information required to construct robust models of either of these two main areas of interest. 1. Modelling of groundwater flow and environmental conditions in aquifers.

According to Freeze and Cherry (1979) in their book "Groundwater", there are 4 main steps in modelling groundwater: 1. Examination of the physical problem 2. Replacement of the physical problem by an equivalent mathematical problem 3. Solution of the mathematical problems using accepted mathematical techniques 4. Interpretation of the mathematical results in terms of the physical problem

The overall view of this is that the main difficulty in modelling (of ALL types) is a problem of interpretation and interconversion between "real" physical problems and the mathematical interpretations of those problems. This is compounded by a lack of data and understanding about certain aspects of the problems (e.g. the actual kinetic rates of biodegradation in field conditions). The processes that have to be considered in physical models of GROUNDWATER FLOW and ENVIRONMENTAL CONDITIONS are


Advection Dispersion Molecular Diffusion Filtration Facilitated Transport Volatilisation Gas Phase Physical decomposition (e.g. surface catalysis)


Radioactive Decay Dissolution/Precipitation Co-precipitation Redox effects Complexation Sorption Biodegradation Biotransformation Chemical decomposition

2. Modelling the activities (including biodegradative) of microorganisms in groundwater Example Model An example model will be used to demonstrate the concepts involved. It is a simplified model in that it does not deal with all of the parameters listed above. It is a realistic model in that it does deal with a real physical problem. The site is Camp Borden, Ontario - but it is extended to predict results in other types of aquifers. The problem addressed is groundwater flow and oxygen concentrations in flow regimes in that aquifer. The characteristics used


Biology 447 ­ Environmental Microbiology

for the physical nature of the groundwater sediment is based on Borden sand, a silty sand and a coarse sand. The properties of these are:

Parameter Silty Sand Borden Sand Coarse Sand

Velocity (m/day)








Longitudinal dispersitivity




Transverse dispersivity




Organic carbon (%)




BTEX diffusion coefficient (cm2/d)




Oxygen diffusion coefficient (cm2/d)




When the model for BTEX movement and oxygen concentration in the various aquifer materials is run, the following results were obtained (Sudicky, et al. Earth Sciences, University of Waterloo). Animation of benzene-oxygen relationships at Camp Borden Aquifer (Website)

Other Groundwater Models

Groundwater Flow Models: Some simple models that deal with the flow of groundwater according to the topography of the water table are available as a web-enabled modeling system. They are from the USGS and are freely downloadable. The first is ParticleFlow (Website) and it simulates flow in a rectangular domain. A key purpose of the ParticleFlow model is to illustrate how heterogeneities in hydraulic properties cause the spatial spreading of fluid particles. This spreading is analogous to macro-scale solute dispersion.

Run the ParticleFlow Model (Website)

The rectangular flow domain (above) is assumed to be bounded on the left and right sides (AD and BC) by specified head boundaries, and on the top and bottom (AB and DC) by no-flow boundaries. Assuming that the head along AD is higher then the head along BC, the average flow is from left to right. It shows the path of particles through the system and demonstrates the effects of diffusion and hydraulic conductivity The other is Topodrive and is a topography-driven flow system is one in which ground water flows from higher-elevation recharge areas, where hydraulic head is higher, to lower-elevation discharge areas, where hydraulic head is lower. This type of flow system is commonly encountered in ground-water basins. The main factors that control ground-water flow are basin geometry, shape of the water table, and the distribution of hydraulic properties. A key purpose of the TopoDrive model is to enable the user to investigate how these factors control groundwater flow.

Run the Topodrive Model (Website)


Biology 447 ­ Environmental Microbiology

Complex Models

Image was created by the PICS Visualization Tool - G3D ­ developed by University of South Carolina

Key Points ­ Groundwater Microbiology

1. Groundwater Introduction & Environment Hydrologic cycle and its importance to groundwater Concept of aquifer and groundwater flow Confined and unconfined aquifers Recharge and discharge zones Water table and hydraulic head Hydraulic conductivity 2. Groundwater Microbiology General types of bacteria present in groundwater environments Sampling problems and processes for water and matrix material 3. Microbial Processes Chemical and physical processes in normal groundwater C and N limitation Movement of groundwater Movement of contaminants by advection, dispersion, retardation and biodegradation. Effects on observed distances of movement


Biology 447 ­ Environmental Microbiology

4. Rates of Biodegradation Typical groundwater regime when contaminated with organic compounds. Zones of activity - anaerobic, denitrification, sulfate reduction and methanogenesis Redox reactions and their place in groundwater microbiology Energy yields, thermodynamics and relative efficiency for different carbon substrates 5. Kinetics Definitions of zero order, first order and second order kinetics Types of responses obtained under those different types of kinetics when organic materials are biodegraded in groundwater 6. Mechanisms causing variation in Groundwater plume conditions Overview of the microbial mechanisms responsible for the observed conditions in a groundwater flow path contaminated with organics The four main reasons why the processes occur in the order observed. 7. Groundwater Modelling Parameters of importance in groundwater modelling - physical and biochemical/chemical 8. Summary and Integration Factors to be considered in groundwater studies of contaminated groundwater (and normal groundwater) environments Geochemical nature of the volume of groundwater Bioenergetics of the processes Biotransformations or biodegradation processes Site conditions


Biology 447 ­ Environmental Microbiology

Biological Treatment of Soil and Groundwater Overview

Bioremediation is the application of microorganisms or microbial processes or products to remove or degrade contaminants from an area. A more rigorous definition is: Bioremediation is the intentional use of biological degradation procedures to remove or reduce the concentration of environmental pollutants from sites where they have been released. The concentrations of pollutants are reduced to levels considered acceptable to site owners and/or regulatory agencies. A bioremediation project or program should consider many aspects of the site, the contamination, the microorganisms, the environment, the goals and regulatory limits on contaminants set by the appropriate agencies (MoEE, EPA, etc.) and features that would impact on the successful outcome of the project. One way to look at this is through a "flow diagram" of items and features that should be examined.


Biology 447 ­ Environmental Microbiology

There are two basic questions to be answered before bioremediation starts: 1. Where will the contaminants be metabolized - and what factors will determine this ? The possible types of bioremediation activities fall into two main categories: ex-situ and in-situ. In-situ bioremediation occurs in the soil. groundwater or other environment without removal of the contaminated material. In contrast, ex-situ bioremediation entails the removal of all or part of the contaminated material for treatment. 2. How aggressive will the site remediation be - and what factors will determine this ? Intrinsic bioremediation is passive - it relies on the capacity of microorganisms or other organisms in the system to metabolize, remove, reduce or inactivate the pollutants. It is by definition "in-situ". Another type is Engineered Bioremediation where the operators take an active role in promoting or carrying out the bioremediation process. This can be either in-situ or ex-situ. Some processes originally designed to carry out remediation through chemical or physical means are now known to, at least partially, involve bioremediation processes. Typical of these is bioventing where air is passed through a soil or groundwater to volatilize contaminants. It is now thought that the microbial activities in the volume being treated is enhanced by this process.

Classification of Bioremediation Options:

There are certain chemicals and groups of chemicals that have been found more amenable to bioremediation. Generally, compounds susceptible to bioremediation show the following characteristics: They are water soluble, have a simple molecular structure, are not sorbed, are non-toxic, and serve as substrates for growth for many aerobic organisms.


Biology 447 ­ Environmental Microbiology

Bioremediation Prospects - Classified by Chemical Class Prospect for Frequency of Partitioning Bioremediation Occurrence*** characteristics**** (1999)


Chemical Class

Biodegradability* Mobility**

Aerobic/Nitrate Reducing/Anaerobic

Hydrocarbons BTEX Low MW;gasoline, #2 Fuel oil High MW: oil, PAH Creosote Oxygenated Hydrocarbons Low MW alcohols, ketones, esters, ethers. Halogenated aliphatics Highly chlorinated Less chlorinated Halogenated aromatics Highly chlorinated Less chlorinated PCBs - Highly chlorinated PCBs - less chlorinated Nitro-aromatics A4, A2, N5, AN2 A2, A3, N2, AN2 A4, N5, AN2 A2, A1, N5, AN4 A2, N5, AN2 L M L L M C C I I C S M S S M Emerging Emerging Emerging Emerging Emerging A4, A3, N5, AN2 A2, A3, N5, AN2 M H F F M M Emerging Emerging A1, N5, AN3 H C W Established A1, N2, AN2 A1, N3, AN2 A2, N4, AN4 A1, N2, AN4 H M L L F F C I M M S S Established Established Emerging Emerging

* Numeric rating under A (aerobic), N (nitrate reducing) or AN (anaerobic) biodegradability is: 1=readily mineralized as growth substrate 2=biodegradable under narrow range of conditions 3=metabolized via co-metabolism when second substrate is available 4=resistant 5=insufficient data

Compounds resistant to microbial metabolism are: insoluble (or slightly soluble) in water, have strong sorptive reactions, are toxic and do not support microbial growth Only a few (mainly petroleum hydrocarbon derived) chemicals have established processes and are accepted widely as suitable candidates for bioremediation. Others have an "emerging" status where more information is being gathered and more tests are under way. An overview of the "generic" process that is commonly used to plan and initiate a bioremediation in a contaminated site shows that a number of decisions have to made at the various stages.

Strategy for Implementing a Bioremediation Project


Biology 447 ­ Environmental Microbiology

1. Preliminary Site Investigation to determine site geochemistry, environmental characteristics, soil conditions,

hydrology, hydrogeology, concentration and distribution of contaminants.

2. Propose intrinsic microbial reactions that can and have affected concentration and distribution of 3. 4. 5. 6. 7. 8.

contaminants Detailed site investigation to confirm these proposals. Use information from steps above to decide if intrinsic or engineered bioremediation processes could be used to remediate site to required conditions. This is done in relation to the regulations applying to the site and to the proposed use for the site. Implement intrinsic or engineered program OR investigate alternative remediation options Monitor site for efficacy of program Modify program on the basis of on-going analyses and any other developments Terminate program when "clean-up" goals are reached - completely document the entire process

If bioremediation is a viable option at Step 4 above, then further more complicated decisions can be made on which type of bioremediation should be employed: For example -

Soil and Groundwater Bioremediation: Decision Tree Diagram

The diagram below attempts to present a rationale and a process to decide on whether or not to try bioremediation to treat contaminated soil. It does NOT attempt to give answers, only to present questions that should be answered and a series of decision points that can be used to "estimate" the likelihood of bioremediation being successful. In addition, it attempts to list the various kinds of information that should be taken into account when making decisions. Start with the "Chemicals" box in the top right-hand corner and work through the flow diagram. You see a "Questionnaire" section that indicates that the "Environmental Conditions" present at the site, the "Microbial Processes" that would be involved in bioremediation and the "Regulations and Legal Issues" present should be considered. These Legal and Regulatory issues could include legal liabilities, standards for cleanup in the jurisdiction (how clean is clean ?), and similar issues should be considered. If the decision is that bioremediation can be considered, then the choice of "In situ Treatment", "Bioaugmentation", Intrinsic Bioremediation" or "Engineered Solutions" can then be made. If, at any point, bioremediation is ruled out as an option, then other non-bioremediation options can be considered. The main reason to try bioremediation first is that it is often the cheapest solution. If this is NOT the case, then non-


Biology 447 ­ Environmental Microbiology

bioremediation options are considered at the beginning of the process. Whatever option is chosen, the treatment must be "Monitored and Verified"; by this we mean that the efficacy of the bioremediation treatment must be proven and the progress of the treatment must be monitored over time.

Or: Intrinsic Bioremediation of Groundwater: Decision-making Outline


Biology 447 ­ Environmental Microbiology

A classification and examples of the various types of remediation processes including bioremediation is given in the VISITT database. A summary of parts of that database (below) shows the various types of remediation processes with examples of demonstration projects and companies selling the procedures.


Active links are to Bioremediation-related technologies from the VISITT database of US EPA (Now called listed technologies. See also the Bioremediation in the Field Search System (BFSS) Version 2.1 computer program (website)


Biology 447 ­ Environmental Microbiology

Commentary on Bioremediation Technologies




* = Involve some form of bioremediation activity

Choices and Processes

Bioremediation can be the major type of remediation occurring in a particular technology or it can be a consequence of another technology or an integral part of that technology. For instance, pumping air through soil to


Biology 447 ­ Environmental Microbiology

improve volatilization of organic contaminants (Bioventing) leads to an increase in the rate of biodegradation in the soil. Similarly, pumping air into groundwater forming bubbles that "strip" organic contaminants (Air Sparging) also leads to increases in oxygen supply in the groundwater and often leads to increased rates of biodegradation. The main groups of technologies employing biodegradation as part or all of their remedial strategy are: Primary focus is Bioremediation · · · · Bioremediation - in situ in soil Bioremediation - in situ in groundwater Bioremediation - ex situ in "heaps" of soil Bioremediation - ex-situ in reactors of various kinds

bioremediation may be enhanced · · · · · Soil vapour extraction Air Sparging of groundwater Bioventing Surfactant enhanced recovery Soil washing

Site Characterization: What Should We Measure, Where (When?), and How?

-Michael J. Barcelona Department of Civil and Environmental Engineering, University of Michigan, Ann Arbor, MI

Abstract Site characterization represents the initial phase of the active monitoring process that occurs as part of intrinsic organic contaminant bioremediation efforts. Initial characterization work sets the stage for evaluating the progress of the natural transformation of contaminants. The following have frequently been observed: parent compound disappearance, active microbial populations with biotransformation capabilities, and the appearance or disappearance of organic and inorganic constituents that provide evidence of bioremediation at contaminated sites. Quantitative evidence is lacking, however, for net removal of toxic compounds from complex mixtures solely by biological processes. This is due largely to the reliance on monitoring well samples for evidence of biological activity, rather than on identifying the mass of contaminants (and total reactive organic carbon) and estimating the net removal/transformation of reactive compounds over time. A dynamic approach to quantitative site characterization is needed that recognizes intrinsic bioremediation as an active cleanup approach. Careful attention must be paid to the identification of the three-dimensional distribution of contaminant mass. Then the correspondence between contaminant distribution and favorable physical, geochemical, and microbial conditions in the subsurface over time provides a basis for net contaminant-removal estimates. Mere adaptations of detective ground-water monitoring networks are insufficient for quantitative evaluation of intrinsic bioremediation technologies. Introduction The practice of site characterization for remediation of subsurface organic contaminants has evolved slowly in the past decade. Early guidelines (1-3) for minimal ground-water contamination detection monitoring (i.e., monitoring wells upgradient and downgradient) have been applied to many sites of potential concern from detection through remedial action selection phases. This minimal approach has been applied widely, regard-less of the physicochemical characteristics of contaminant mixtures or the complexity of hydrogeologic settings. With soluble inorganic constituents, this approach may be adequate for detection purposes, but assessment efforts require substantially more comprehensive approaches. For organic contaminant assessment efforts (i.e., determinations of the nature and extent of contamination), wells alone have been found to be inadequate monitoring tools. Recognition of the value of subsurface soil vapor surveys for volatile organic components of fuel and solvent mixtures has generated a flurry of modified site characterization approaches based on monitoring wells (4). These approaches to site characterization and monitoring net-work design suffer also from a failure to identify the total mass of contaminant in the subsurface.


Biology 447 ­ Environmental Microbiology

This failure occurs for three main reasons. First, al-though volatile organic compounds (VOCs) are mobile in ground water and are frequently early indicators of plume movement (5), their detection in vapor or well samples and their apparent aqueous concentration distribution do not identify the total mass distribution of organic contaminant (6). Second, efforts to correlate observed soil vapor or ground-water VOC concentrations with those in subsurface solid cores have often been unsuccessful, because current bulk jar collection/ refrigeration at 4 o C guidelines for solid core samples for VOC analyses lead to gross negative errors (7). Third, "snapshots" (i.e., one-time surveys) of back-ground and disturbed ground-water chemistry conditions have been interpreted as "constant," ignoring temporal variability in subsurface geochemistry. |Top of Page| The unhappy result of the slow improvement in site characterization and monitoring practices has often been the very low probability of detecting the source of mobile organic contaminants. This outcome may be followed by the misapplication of risk assessment or remediation models and fiscal resources. Nonetheless, good reasons exist for a more optimistic view of the future reliability of site-characterization and monitoring efforts. The shortcomings of previous contaminant detection and assessment efforts have been recognized. New guidelines and recommendations for network design and operations will lead to more comprehensive, cost-effective site characterization (7,8) in general. Also, excellent reviews of characterization and long-term monitoring needs and approaches in support of in situ remediation efforts should guide us in this regard (9,10). Site characterization efforts provide a basis for long-term monitoring design and actually continue throughout the life of a remediation project. Advanced Site Characterization and Monitoring How do we estimate the potential for subsurface intrinsic bioremediation success and track its performance into the future? Clearly, we should seek to design technically defensible characterization and monitoring networks that will provide reasonable estimates of in-place contaminant distributions over time. Therefore, a dynamic, ongoing sitecharacterization effort includes the following objectives: · · Identify the spatial distribution of contaminants, particularly their relative fractionation in subsurface solids, water, and vapor, along potential exposure pathways, recognizing that the mass of contaminants frequently resides in the solids. Determine the corresponding spatial distribution of total reactive organic matter (e.g., degradable normal, aliphatic, and aromatic hydrocarbon compounds), be-cause overall microbial activity and disruptions in subsurface geochemical conditions (and bioremediation indicators) are due to the total mass of reactive organic carbon. ·background, source, and down-gradient zones during the first year of characterization and monitoring. Derive initial estimates of net microbial transformations of contaminant-related organic matter over time that may be built into an efficient long-term monitoring network design.

· ·

The first three objectives establish the environment of major contamination and the conditions under which bioremediation may occur. The latter two objectives are vitally important, because evaluating the progress of intrinsic bioremediation processes depends on distinguishing compound "losses" due to dilution, sorption, and chemical reactions from microbial transformations. This approach has been suggested emphatically by Wilson (9) and was recently developed into a draft technical U.S. Air Force (USAF) protocol by Wiedemeier et al. (10). |Top of Page| The latter reference focuses directly on the implementation of intrinsic remediation for dissolved fuel contamination in ground water. The general approach is shown in Figure 1, which has been modified from the original work. The draft USAF protocol (10) has as its goals the collection of data necessary to support: · · · Documented loss of contaminants at the field scale. The use of chemical analytical data in mass balance calculations. Laboratory microcosm studies using aquifer samples collected from the site.


Biology 447 ­ Environmental Microbiology

These data, if collected in three dimensions for an extended period, should be sufficient to implement intrinsic remediation successfully (11). The data collected in the initial site characterization effort (Figure 1) support the development of a site-specific conceptual model. This model is a three-dimensional representation of the groundwater flow and transport fields based on geologic, hydrologic, climatologic, and geochemical data for a site. The conceptual model, in turn, can be tested, refined, and used to determine the suitability of intrinsic remediation as a risk-management strategy. The validity of the conceptual model as a decision tool depends on the complexity of the actual hydrogeologic setting and contaminant distributions relative to the completeness of the characterization database. The draft USAF protocol is quite comprehensive in identifying important parameters, inputs, and procedures for data collection and analysis. The major categories of necessary data are listed in Table 1 from the draft protocol (10). Ongoing work on the protocol has revised some of the detailed guidance it provides on sampling and analytical protocols for these critical parameters; thus, recent drafts of the protocol should be even more useful to practitioners. Typical detective monitoring data sets available prior to in-depth site characterization are more likely to contain contaminant-related information rather than the three-dimensional aquifer property, hydrogeologic, or geochemical data needed to formulate a conceptual model. A recognition of the variability inherent in these parameter distributions is critical to site characterization efforts. Sampling in Space The initial site characterization phase should be de-signed to provide spatially dense coverage of critical data over volumes corresponding to 10-yr to 100-yr travel times along ground-water flow paths. If the flow path intersects a discharge zone in less than 100 yr, then the volume should be scaled accordingly. For example, if the flow path discharges after 10 yr, the critical volume would be 1 yr of travel time. The "volume-averaged" values of the contaminants, hydrogeological and geochemical parameters within zones along the flow path(s), should be derived from data sets that are large enough to permit estimation of statistical proper-ties (e.g., mean, median, correlation distance, and variance). In general, this means that the data sets for derived mass loadings of contaminants, aquifer proper-ties, and geochemical constituents (Table 2) derived from spatial averages of data points must include approximately 30 or more data points (12-14). Indeed, this minimum data-set size strictly applies to points in a plane. |Top of Page| Table 1. Site-Specific Parameters To Be Determined During Site Characterization Fractionation and Spatial Extent of Contamination Extent and type of soil and ground-water contamination · · Location and extent of contaminant source area(s) (i.e., areas containing free- or residual-phase product) Potential for a continuing source due to leaking tanks or pipelines

Hydrogeologic and Geochemical Framework Ground-water geochemical parameter distributions (Table 2) · · · · · · · · · · Regional hydrogeology, including: Drinking water aquifers Regional confining units Local and site-specific hydrogeology, including: Local drinking water aquifers Location of industrial, agricultural, and domestic water wells Patterns of aquifer use Lithology Site stratigraphy, including identification of transmissive and nontransmissive units Grain-size distribution (sand versus silt versus clay)


Biology 447 ­ Environmental Microbiology

· · · · · ·

Aquifer hydraulic conductivity determination and estimates from grain-size distributions Ground-water hydraulic information Preferential flow paths Location and type of surface water bodies Areas of local ground-water recharge and discharge Definition of potential exposure pathways and receptors

Table 2. Target Constituents for Site Characterization in Support of Intrinsic Bioremediation Contamination Area Apparent/Geochemical Redox Zone Contaminant Mixture Inorganic Constituents Intrinsic Constituents Source Reducing Fuels Chlorinated solvents O2 , CO2 , H2S; pH, Fe2+ , HS -/S= , NO2 , NH3 , alkalinity Organic carbons, CH4 , organic acids, phenols As above and: chlorinated metabolites, ethylene, ethane Downgradient Transitional/Suboxic Fuels Chlorinated solvents O2 , CO2 , H2S; pH, Fe2+ , , alkalinity, NO2, NO3 , NH3 , HS-/S= Organic carbon, CH4 organic acids, phenols As above and: chlorinated metabolites, ethylene, ethane Upgradient/Far-field downgradient Oxic Fuels Chlorinated solvents O2 , CO2 , H2S; alkalinity, Fe2+ , NO3 , NO2-, NH3 Organic carbon, CH4 , organic acids, phenols As above and: chlorinated metabolites, ethylene, ethane

Two major decisions must be made with regard to how spatially averaged masses of contaminants, electron donors (e.g., organic carbon, Fe 2+ , S = , and NH3 ), and electron acceptors (e.g., O2 , NO3 -, NO -, Fe and Mn oxides, and SO4=) are to be estimated. The first question deals with identification of the media in which the bulk of the constituent's mass resides. For aquifer properties (e.g., grain size and laboratory esti-mates of hydraulic conductivity), the answer is simple. In this case, the solids are clearly the media of interest. For constituents, particularly VOCs, which are sparingly water soluble, the bulk of the mass may in fact reside in the solids, though both solids and water samples must be collected carefully. |Top of Page| The second question pertains to the depth interval over which "planar" data points might be averaged. With fuelrelated aromatic contaminants, the depth interval above and below the capillary fringe/water table inter-face typically exhibits order-of-magnitude differences in solid-associated concentrations. In this situation, aver-aging data points over depths greater than 0.5 m could easily lead to order-of-magnitude errors in estimated masses for a site. Continuous coring of subsurface sol-ids and close interval (i.e., <1 m) sampling of water should be considered in many VOC investigations. To approach this level of depth detail in sampling, "push" technologies and/or


Biology 447 ­ Environmental Microbiology

multilevel sampling devices present very useful tools for site characterization. Push technologies rely on hydraulic or hammer-driven, narrow diameter (i.e., <2 in.) probes for solid or water sampling. These technologies have the potential to provide greater spatial coverage of the subsurface at less cost than drilling techniques. The approach to site characterization for chlorinated hydrocarbons is significantly more difficult. Very few models of site characterization for these contaminants have estimated mass loadings in specific media. Many of the previously referenced methods may work satisfactorily. Free-phase detection, assessment, and quantitation, however, may be more a matter of luck and exhaustive sampling than intuition based on experience. Sampling Over Time VOC compounds (e.g., aromatic hydrocarbons and chlorinated solvents) are among the target contaminants that are considered constituents of concern in remedial investigations. Their aqueous solubility and demonstrated association with aquifer solids require sampling of these media during the site characterization phase. This suggestion also applies to organic metabolites of complex organic mixtures (e.g., ethylene, vinyl chloride, aromatic acids, and phenols). Aqueous plumes that develop subsequent to the release of these organic mixtures and byproduct compounds have received the most attention in the past. The fact that the mass of these contaminants frequently resides in the solids strongly suggests that the solids should receive the most attention in the initial site characterization effort. This should also be the case for the physical, geochemical, and microbial determinations. Initially, conventional nested monitoring wells with screened lengths of 1 m or more will be useful for estimating the spatial extent of the dissolved plume, for delineating apparent geochemical zones, and for providing data on water level and aquifer property (e.g., slug-and pump-test derived hydraulic conductivity estimates). Semiannual or annual sampling of wells, particularly multilevels appropriately designed and completed, should be quite useful over the course of the long-term monitoring program. Sampling should track the downgradient progress of riskassociated target compounds and permit testing predictions of intrinsic bioremediation effects on risk reduction. Proof of the effects of the net removal of specific solid-associated contaminants due to intrinsic bioremediation, however, will depend on solid sampling and analysis at annual or greater intervals, because solid-associated concentrations may be expected to change slowly. Unless biotransformation can be shown to be a major loss mechanism for contaminants mainly in solids over ex-tended periods, it will remain an area of research rather than practice. Because very few contamination situations have been monitored intensively for periods exceeding several years, it is difficult to define specific sampling frequencies for the range of hydrogeologic and contaminant combinations that may be encountered. The adoption and future refinement of recently developed, technically defensible protocols will improve intrinsic remediation approaches to risk management in subsurface contamination situations. Acknowledgments The author would like to express his gratitude to the following individuals who aided in the preparation of the manuscript: Dr. Gary Robbins, Mr. Todd H. Wiedemeier, Dr. John T. Wilson, Dr. Fran Kramer, and Ms. Rebecca Mullin. References 1. Scalf, M.R., J.F. McNabb, W.J. Dunlop, R.L. Cosby, and J.S. Fryberger. 1981. Manual of ground-water sampling procedures. National Water Well Association. 2. Barcelona, J.J., J.P. Gibb, J.A. Helfrich, and E.E. Garske. 1985. Practical guide for ground-water sampling. Illinois State Water Survey, SWS Con-tract Report 374 U.S. Environmental Protection Agency, Ada, OK. 3. U.S. EPA. 1986. RCRA technical enforcement guidance document, OSWER-9950.1. Washington, DC.


Biology 447 ­ Environmental Microbiology

Overview and Summary

There is a complex set of interactions between the types and numbers of microorganisms in groundwater, their biodegradative abilities, the groundwater environment and the resulting bioremediation activities. This can be better understood by looking at the geochemistry of a particular region of groundwater, determining the most likely microorganisms that would be active in those areas and predicting the kinds of biodegradative activities that those organisms could have. This is still an inexact science - no-one can predict exactly what will happen to a given contaminant or group of contaminants in a particular environment. The factors that should be considered are:

1. 2. 3. 4. 5.

The geochemical nature of the volume of groundwater The bioenergetics of the process The biotransformation or biodegradation processes Site conditions for bioremediation processes Design, operation and monitoring of the processes

1. The geochemical nature of the volume of groundwater. This can be best summarized by considering the relative levels of the different geochemical species at individual points in the groundwater profile as it evolves due to microbial activities.

Note: At any particular position along the flow path, the concentrations of the various geochemical species are different, define the redox conditions and control which microorganisms are able to grow at that position and are a result of microbial activities. 2. The bioenergetics of the process There will be different amounts of free energy associated with the various reactions at the different positions:


Biology 447 ­ Environmental Microbiology

REACTION Aerobic respiration Denitrification Manganese(IV) reduction Fe(III) reduction Sulfate reduction Methanogenesis

Electron Acceptor oxygen nitrate manganese(IV) Fe(III) sulfate Carbon dioxide

Free Energy change -29.9 -28.4 -23.3 -10.1 -5.9 -5.6

For example, if a well is drilled and finds high MN(II) levels, low Fe(II), decreased sulfate levels and small quantities of hydrogen sulfide, the position on the flow path and the redox conditions can be calculated. The actual extent of overlap between the various conditions is not completely understood and may vary at different sites. Note: The key feature is that it is MUCH easier to measure the specific ions than to carry out a full microbiological investigation of a groundwater system. If the ion concentrations can be correlated with particular microbial habitats, then biodegradative activities could perhaps be predicted. An interesting suggestion is that the hydrogen (H2) concentrations in sediments vary with the physiological characteristics of the microorganisms consuming the H2 and will follow the order: methanogens > sulfate-reducers > Fe(II)-reducing > Mn(IV)-reducing > nitrate-reducing 3. The biotransformation or biodegradation processes The redox conditions strongly influence the electron acceptor for microbial activities and thus the biotransformation or biodegradation of chemicals. Examples of this are: · · · · · · · · · · Oxidation of halogenated solvents by methanotrophs Biotransformations with nitrate Reductive dehalogenation of halogenated compounds Sequential aerobic/anaerobic transformations of halogenated organics Chemical reactions mediated by microorganisms Oxidation of halogenated solvents by methanotrophs Biotransformations with nitrate Reductive dehalogenation of halogenated compounds Sequential aerobic/anaerobic transformations of halogenated organics Chemical reactions mediated by microorganisms

4. Site Conditions for Bioremediation Processes (modified from Bouwer, 1992)

Favourable Factors or Conditions Chemical Characteristics · Small number of organic compounds

Unfavourable Factors or Conditions


Numerous contaminants


Biology 447 ­ Environmental Microbiology


Complex mixture of inorganic and organic compounds

· · ·

Appropriate electron acceptors present

· · ·

Toxic concentrations of compounds

Diverse and varied microbial population

Sparse microbial activity and numbers

pH 6 to 8

Extremes of pH

Hydrogeological Characteristics · · · · · Granular, highly porous media · · · · · Fractured rock

High permeability

Low permeability

Homogeneous mineralogy

Complex mineralogy

Homogeneous media

Heterogeneous media

Saturated media

Unsaturated to saturated conditions

5. Design, operation and monitoring of the processes Assuming that the site characterization process has shown potentially favourable results from bioremediation as a treatment option, the processes to implement bioremediation activities should be as follows: · · · · · Characterization of site hydrological and contaminant conditions in terms of the microbiological processes Removal of any gross contamination (e.g. leaking tanks or other contaminant source) and any separate immiscible phase (e.g. gasoline pools on surface of groundwater). Feasibility Studies including sorption studies System design and operation Monitoring system performance

Bioremediation Models:

There are many different models that are used to simulate and predict bioremediation activities and outcomes under various conditions. Many are from the US-EPA and are available for downloading from their websites. Typical of these are:


Biology 447 ­ Environmental Microbiology

CSMoS Ground-Water Modeling Software

Free Public Domain Ground-Water and Vadose Zone Models All Links are Web Links CSMoS distributes various public domain ground-water and vadose zone models. Press here to view a short description of all models. Models and manuals are available through various different options. You can: · · · Click the links in the table below to view installation instructions, system requirements, model descriptions and files available for download. Send CSMoS blank CDs, ZIP disks, or 3.5" floppy diskettes to CSMoS. CSMoS will copy the requested models or manuals to disks and return the disks to you. Contact Mark Stacy at [email protected] or (580) 436-8724 for other arrangements.

CSMoS provides free technical support for many of the models listed in the table below. Join the CSMoS e-mail list to receive news about new releases of public domain ground-water models, version updates, bugs, fixes, and other important items. CSMoS also maintains an on-line database of commercially available ground-water modeling software. Model Release Date & Platform


BIOCHLOR is a screening model that simulates remediation by natural attenuation of dissolved solvents at chlorinated solvent release sites. BIOCHLOR can be used to simulate solute transport without decay and solute transport with biodegradation modeled as a sequential first-order process within one or two different reaction zones. BIOCHLOR is an easy-to-use screening model that simulates remediation by natural attenuation (RNA) of dissolved solvents at chlorinated solvent release sites. The software, programmed in the Microsoft Excel spreadsheet environment and based on the Domenico analytical solute transport model, has the ability to simulate 1-D advection, 3-D dispersion, linear adsorption, and biotransformation via reductive dechlorination (the dominant biotransformation process at most chlorinated solvent sites). Reductive dechlorination is assumed to occur under anaerobic conditions and dissolved solvent degradation is assumed to follow a sequential first-order decay process. BIOCHLOR includes three different model types: · · · Solute transport without decay Solute transport with biotransformation modeled as a sequential first-order decay process Solute transport with biotransformation modeled as a sequential first-order decay process with two different reaction zones (i.e., each zone has a different set of rate coefficient values)

June 2002 Windows 95/98/NT, Excel

Local Link: Download Biochlor for Microsoft Excel 97


Biology 447 ­ Environmental Microbiology

Biochlor Manual for Excel 97 Local Link: Download Biochlor for Microsoft Excel 2000 Biochlor Manual for Excel 2000


See Download and Installation Instructions and Manuals for Bioplume III BIOPLUME II is a simulation that computes concentrations of dissolved hydrocarbon under the influence of oxygen-limited biodegradation in an aquifer. The model solves the solute transport equation for both hydrocarbon and oxygen, assumes an instantaneous reaction between oxygen and hydrocarbon, and combines the two plumes using the principle of superposition. Computations account for convection, dispersion, mixing, and biodegradation effects. Also, the program can simulate slow hydrocarbon plumes undergoing biodegradation and can simulate in-situ biorestoration schemes such as the injection of oxygenated water. Moreover, the model can simulate reaeration and anaerobic biodegradation as a first-order decay in hydrocarbon concentrations.

September 1997 Windows 95/98


BIOSCREEN is a screening model which simulates remediation through natural attenuation of dissolved hydrocarbons at petroleum fuel release sites. The software, programmed in the Microsoft Excel spreadsheet environment and based on the Domenico analytical solute transport model, has the ability to simulate advection, dispersion, adsorption, and aerobic decay as well as anaerobic reactions that have been shown to be the dominant biodegradation processes at many petroleum release sites. Includes three different model types: (1) solute transport without decay, (2) solute transport with biodegradation modeled as a first-order decay process (simple, lumped-parameter approach), and (3) solute transport with biodegradation modeled as an instantaneous biodegradation reaction with multiple soluble electron acceptors including dissolved oxygen, nitrate, and sulfate. The model is designed to simulate biodegradation by both aerobic and anaerobic reactions. Local Link: Download BIOSCREEN (right-click and "Save Target as..." ) from this Website, and execute "bioscrn.exe" Local Link: Download Bioscreen Manual in pdf format

July 1997

Windows 95/98/NT, Excel


HSSM simulates flow of the LNAPL phase and transport of a chemical constituent of the LNAPL from the surface to the water table; radial spreading of the LNAPL phase at the water table, and dissolution and aquifer transport of the chemical constituent. September 1997 The HSSM model is one-dimensional in the vadose zone, radial in the capillary fringe, two-dimensional vertically averaged analytical solution of the advectiondispersion equation in the saturated zone. The model is based on the KOPT, OILENS and TSGPLUME models. Windows 95/98


Biology 447 ­ Environmental Microbiology


Application: Originally a three-dimensional finite difference model for multiphase flow, transport and chemical flooding, the UTCHEM code has been modified to transform it into a general purpose NAPL simulator. Appropriate physical, chemical and biological process models have been incorporated into the simulator to create a 3D multiphase multi-component model capable of simulating the fate and transport of NAPL's in the saturated and unsaturated zones of aquifers. The model can be used to simulate the actual field operation of remediation activities such as surfactant remediation or bioremediation as well as laboratory experiments with large-scale aquifer models.

February 1999 Windows/UNIX

WhAEM 2000

Application: Originally a three-dimensional finite difference model for multiphase flow, transport and chemical flooding, the UTCHEM code has been modified to transform it into a general purpose NAPL simulator. Appropriate physical, chemical and biological process models have been incorporated into the simulator to create a 3D multiphase multi-component model capable of simulating the fate and transport of NAPL's in the saturated and unsaturated zones of aquifers. The model can be used to simulate the actual field operation of remediation activities such as surfactant remediation or bioremediation as well as laboratory experiments with large-scale aquifer models.

December 2000 Windows 95/98/NT


Biology 447 ­ Environmental Microbiology

Resources Lists for Assignment

Manure Nutrient Management (se Website for direct links) General & Ontario

· · · · · · · · Manure Nutrient Management Overview - Manure management in Ontario Ontario Legislation Consultation on Draft Regulations - Aug 2002 Ontario - New Draft Regulations New Nutrient Management Plan Protocols Aug 2002 BC- Farm Practices - Manure Use 1996 Manure Nutrient Management - Agriculture Canada

Canadian Provincial Ministries

Agriculture · Alberta Agriculture, Food and Rural Development British Columbia Ministry of Agriculture, Food & Fisheries Manitoba Agriculture and Food New Brunswick Dept. of Agriculture, Fisheries and Aquaculture Newfoundland and Labrador Agriculture Nova Scotia Agriculture and Fisheries Ontario Ministry of Agriculture, Food and Rural Affairs Environmental Management Prince Edward Island Department of Agriculture and Forestry Minist. de l'Agric., des Pêcheries et de l'Alimentation du Québec Saskatchewan Agriculture, Food and Rural Revitalization

Environment · · Alberta Environmental Protection


BC Ministry of Sustainable Resource Management Manitoba Conservation New Brunswick Department of the Environment Newfoundland Environment Nova Scotia Department of the Environment Ontario Ministry of the Environment and Energy Environmental Commissioner of Ontario Prince Edward Island Dept. of Fisheries, Aquaculture & Environment Minist. de l'Environnement et de la Faune du Québec Saskatchewan Environment and Resource Management

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Biology 447 ­ Environmental Microbiology

USA Government Links

· · · · · · · Environmental Resource Program NC Hog Site - Carolina Health and Environment Community Center (CHECC) - extensive listing of interesting sites on the hog industry in North Carolina. Extension Nutrient and Pest Management Program - Univ. of Wisconsin National Center for Manure and Animal Waste Management National Pork Producers Council National Biosolids Partnership Pork - The Business Magazine for Pork Producers - Product & Services Guide Sustainable Agriculture Network (SAN) - SAN is the communications and outreach arm of the Sustainable Agriculture Research and Education (SARE) program. SARE is a U.S. Department of Agriculture-funded initiative that sponsors competitive grants for sustainable agriculture research and education in a regional process nationwide. [991203] Swine and Pig Internet Resources - Swine and Pig Index; Market Information; Commodity Markets; Weather; Trade Information; Govt. Regulations -- Grade Standards. Soil-Plant-Nutrient Research links National Food and Energy Council (NFEC) - Green Power Opportunities - Agricultural Methane Recovery United States Dept. of Agriculture Natural Resources ConservationService (NRCS) - Animal Feeding Operations Agricultural Research Service (ARS) Cooperative State Research Education and Extension Service (CSREES) Economic Research Service (ERS) National Agricultural Statistics Service (NASS) U.S. Environmental Protection Agency (1200 Pennsylvania Avenue, NW, Washington, DC 20460) Laws & Regulations Offices - Office of Research and Development Office of Water Water Quality Standards Criteria and Methods Drinking Water and Ground Water Protection Programs Listing of Projects and Programs AgSTAR Program (Methane and Sequestration Branch) Endocrine Disruptors Research Initiative Great Lakes Information Network Global Warming Program

· · · ·


USA Agriculture Extension Links

· · · · · · · Alabama Animal Waste Management (Alabama Cooperative Extension System) Colorado State University Cooperative Extension - Publications Iowa State University Extension - Agriculture & Natural Resources - Manure/Odor Management Publications - Iowa Manure Management Action Group (IMMAG) Maryland Department of Agriculture - Nutrient Management in Maryland Michigan State University Extension - Michigan Manure Resources Network NCR-183 - Utilization of Animal Manure and Other Organic Wastes in Agriculture (regional committee of University and USDA/ARS soil fertility faculty from 10 north central states committed to improving access to quality information on managing manure and other organic wastes in agricultural systems. North Carolina North Carolina Cooperative Extension Service - NC State University (College of Ag & Life Sciences) - Waste Management Programs - Water Quality and Waste Management - Land Application of Manures Biosolids and Septage; - Biological & Agriculture Engineering Extension; Animal Waste Management


Biology 447 ­ Environmental Microbiology

· · · · · · · · · · · · · ·

Ohio State University Extension - Agriculture & Natural Resources - Ohio Livestock Manure And Wastewater Management Guide Oregon State University Extension Service - Farm Publications - Publications & Videos Penn State University Coop Extension - Penn State College of Agricultural Sciences Purdue University Extension - Agriculture & Natural Resources - Publications - Manure Management Planner Software Texas A&M University Animal Manure Management Issues - Publications Washington State University Coop Extension - Publications West Virginia University Extension Service - Waste Management University of Florida - College of Agricultural and Life Sciences - Department of Animal Sciences University of Georgia - College of Agricultural and Environmental Sciences Publications - Biological & Ag Publications University of Minnesota Extension Service - Manure & Odor Education Research - Environment and Natural Resources University of Missouri Extension - Explor (Extension Publications Library) - Agricultural Engineering Publications - Missouri Manure Management Action Group (MoMMAG) University of Nebraska Cooperative Extension - Manure Matters Web Site - Institute of Agriculture and Natural Resources University Of Vermont - Extension - Nutrient and Manure Management University of Wisconsin - Biological Systems Engineering Extension - Coop Extension Pub.


· · Inventory of Canadian Agri-food Research (ICAR) - a product of the Canadian Agri-Food Research Council (CARC) gNIC (Agriculture Network Information Center) (USA) - a distributed network that provides access to agriculture-related information, subject area experts, and other resources. It was established by an alliance of the US National Agricultural Library, land-grant universities, and other organizations committed to facilitating public access to agricultural and related information. - What's New? Notable Additions to AgDB a very extensive resource. US National Dairy Database - Waste Management and Water Quality Publications. The National Dairy Database is presented on InforM via cooperation between the University of Maryland and the USDA National Agricultural Library. The ADDS Center - Agricultural Databases for Decision Support Environmental Management & Production Agriculture Milk Composition InfoBase National Goat Database National Pig Information Database US National Water Quality Database - an information management tool for locating water quality and waste management educational resources created by the 50 State Cooperative Extension Services. National Dairy Manure Management Database - UC Davis, California - (4614 references, Nov., 1996) Search Online or Download free copy of software (874 KB) - The National CREES Dairy Manure Management Committee has developed a citation database for dairy manure documents. Numerous databases were searched to obtain these citations: Agricola, Eureka, water quality abstracts, Wordcat, and the Kerr Foundation's summary of livestock and environmental abstracts. Search CADDET Energy Efficiency Web site - CADDET Energy Efficiency collects, analyses and enhances the international exchange of impartial information on new, cost-effective, energy-saving technologies.

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Biology 447 ­ Environmental Microbiology

Resources for Assignment

Biosolids ­ Sewage Sludge Disposal (see Website for links)

· · · · Ontario - Biosolids - Sewage Sludge Disposal Ontario - Biosolids Management Ontario - Land Application of Biosolids Analytical Results, Findings, and Recommendations of the 1995 OMAFRA Sewage Biosolids Field Survey EPA Biosolids Page EPA Biosolids LibraryGeneration, Use and Disposal in the US EPA Biosolids Publications (selected) · · · · · · · · · · · · · · · · · · Guide to Field Storage of Biosolids Land Application of Sewage Sludge: A Guide for Land Appliers on the Requirements of the Federal Standards for the Use or Disposal of Sewage Sludge, 40 CFR Part 503 [PDF] A Plain English Guide to the EPA Part 503 Biosolids Rule A Guide to the Biosolids Risk Assessments for the EPA 503 Rule Biosolids Recycling: Beneficial Technology for a Better Environment Use of Reclaimed Water and Sludge in Food Crop Production Nomination Guidance 2002 Biosolids Exemplary Management Awards Program EPA Domestic Septage Regulatory Guidance document [PDF] National Pollutant Discharge Elimination System (NPDES) A Guide to the Biosolids Risk Assessments for the EPA Part 503 Rule Guide to Field Storage of Biosolids and Other Organic By-Products Used in Agriculture and for Soil Resource Management Biosolids: Generation, Use and Disposal in the USA

· · ·

Australia: Sustainable Use of Biosolids (Sewage Sludge) in Plantation Forests Pennsylvania - Biosolids - Characteristics and Treatment City of Toronto - Biosolids programs Water Environment Association of Ontario: Fate and Significance of Selected Contaminants in Sewage Biosolids Applied to Agricultural Land Through Literature Review and Consultation with Stakeholder Groups [PDF] The SafeWater Group Canadian Infectious Disease Society


Cryptosporidium and Giardia (pdf) Escherichia coli (pdf) Pfiesteria and Algal Blooms (pdf) Pathogens in Agricultural Watersheds (pdf)



General Overview & Introduction

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