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Sindh University Research Journal (Science Series)

SURJ

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Published regularly since 1964, disseminating essentially the research output of the Faculty of Natural Sciences, SURJ also accepts manuscripts from other authors in all disciplines of science. On receipt and browse by the editor scientific sense the manuscript is issued an acknowledgement and sent for evaluation; as per remarks and recommendation of the evaluator the manuscript is revised and an acceptance letter is issued. SURJ readers keen to be on the evaluating panel can contact the Chief Editor, with their C. V.

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SINDH UNIVERSITY RESEARCH JOURNAL

SURJ

(Science Series)

ISSN 1813-1743

VOLUME 40

NUMBER 02

Editorial Board

Patron:

Mr. Mazhar-ul-Haq Siddiqui (S.I) Vice Chancellor

DECEMBER 2008

Chief Editor

Prof. Dr. G. M. Seehar Dean, Faculty of Natural Sciences

Members

Overseas

S. M. Rahmatullah, B.A.U. Bangladesh H. Suzuki, Kagoshima University, Japan H.P. Luterbach, University of Tubingen, Germany M. K. Kaminski, University College London, U. K. H.R. Esmaili, University of Shiraz, Iran A. El. S. Ismail, Nat. Agricult. Res. Centre, Giza, Eygpt F. M. Slater, University of Cardif, U. K. P. Qining, China Pharmaceutical University, China P. J. Houghton, Kings College, London, U. K. A. N. Mitra, I I T, Kolkata, India S. A. Telang, University of Calgary, Canada N. Bano, Dhaka University, Bangladesh

National

Ata-ur-Rehman, HEC, Islamabad M. Mazhar, Quid-e-Azam University, Islamabad T. M. Ansari, B.Z. University, Multan M. Ali, B.Z. University, Multan M. Javed, Agriculture University, Faisalabad Sh. A. Rasool, University of Karachi M. Qaisar, University of Karachi A. Rashid, University of Peshawar A. Dasti, B.Z. University , Multan Azra Khanum, Arid University, Rawalpindi Quddusi B. Kazmi, University of Karachi Nasira Khatoon, University of Karachi M. Sharif Mughal, G. C. University, Lahore Rana Khalid Naeem, University of Karachi M. Zahid, Quid-e-Azam, University, Islamabad M. Ali Maud, LUMS, Lahore Shamim A. Sheikh, University of Karachi M. Tahir Shah, University of Peshawar R.H. Baqri, PSF, Islamabad Shahida Parveen. Ahmed, University of Karachi M. Jamshaid, University of Punjab, Lahore M. Farid Khan, Gomal University, D. I. Khan

Biannual Publication: Dean, Faculty of Natural Sciences, University of Sindh, Jamshoro, Sindh, Pakistan

SINDH UNIVERSITY RESEARCH JOURNAL

(Science Series)

VOLUME 40

NUMBER 02

DECEMBER 2008

Biannual Publication: Dean, Faculty of Natural Sciences, University of Sindh, Jamshoro, Sindh, Pakistan

Sindh University Research Journal (Science Series) Contents of Volume 40 No. (2): December (2008)

PHYSICOCHEMICAL QUALITIES AND NUTRITIONAL ATTRIBUTES OF STABLE BREAD WHEAT VARIETIES REPRESENTING DIVERSE GENETIC ORIGIN S. I. H. Shah, K. A. Siddiqui, M. A. Sahito, S. Tunio and A. J. Pirzada ....................................................................... SMALLER FORAMINIFERAL ASSEMBLAGE OF THE KHIRTHAR FORMATION OF SUKKUR AND KOTDIJI AREA, SINDH P. A. Usmani, A. H. Markhand, M. K. Samoon. and S. A. Shaikh .............................................................................. NEW RECORD OF SOME PLANKTONIC CLADOCERAN FROM FISH PONDS OF JAMSHORO SINDH, PAKISTAN Mukhtiar Ahmed Mahar ........................................................................................................................................ INFLUENCE OF Y2O3 PARTICLES AND DEFORMATION ON RECRYSTALLISATION OF ODS STEELS MA956 AND MA957 M. Moazam Baloch..................................................................................................................................... FECUNDITY OF TWO TYPES OF PALLA (A+B), TENUALOSA ILISHA (HAMILTON) FROM RIVER INDUS SINDH, PAKISTAN P. K. Lashari, N. T. Narejo, M. Y. Laghari and B. Waryani ....................................................................................... APPLICATION OF QUEUEING THEORY TO SIGNALS SYSTEM AT TWO BUSY CROSS SECTIONS OF CITY HYDERABAD: A SIMULATION APPROACH Noor Ahmed Shaikh.................................................................................................................................... ECOLOGICAL STUDIES ON FRESHWATER GASTROPODS (SNAILS) OF INDUS RIVER AND ITS CANALS AT KOTRI BARRAGE SINDH, PAKISTAN G. H. Burdi, W. A. Baloch, F. Begum, A. N. Soomro and M. Y. Khuhawar ................................................................... OPTIMIZATION STUDIES OF YEAST FEED FOR ENHANCED GROWTH FOR ETHANOL PRODUCTION BY SHAKE FLASK METHOD A. A. Noor and M. U. Dahot ........................................................................................................................... INFLUENCE OF TRICHODERMA HARZIANUM AND AZOTOBACTER ON DECOMPOSITION OF ORGANIC SUBSTANCES OF STRAW AND FLAX OF WHEAT IN TATARISTAN, RUSSIA G. A. Mako and F. Alimova ........................................................................................................................... ARTICULATED PARTIAL SKULL, AND ITS ASSOCIATED AXIAL AND LIMB ELEMENTS OF MARISAURUS (BALOCHISAURIDAE, TITANOSAURIA, SAUROPODA) DISCOVERED FROM THE LATEST CRETACEOUS SULAIMAN BASIN OF PAKISTAN M. Sadiq Malkani ...................................................................................................................................... ELEMENTAL INVESTIGATION AND ANTI MICROBIAL ACTIVITY OF MEDICINAL PLANT NERIUM INDICUM MILL. AGAINST DERMATOPHYTIC FUNGI A. J. Pirzada, W. Shaikh, A. M. Jakhar, S. I. Shah and M. P. Pirzada ............................................................................ SEARCHING OF OPTIMAL VERTICES IN A HAPHAZARD ROUTES N. Khan, F. H. Khan and S. Inayat Ullah ........................................................................................................... COMMON FOLK MEDICINAL AND ETHNOMEDICINAL USES OF THIRTY MEDICINAL PLANTS OF DISTRICTS DADU AND JAMSHORO, SINDH, PAKISTAN. A.H. Memon, F.M.A. Rind, M.G.H. Laghari, U.R. Mughal, N. Memon, R.A. Gilal, M.Y. Khuhawar and F. Almani. .................. BPEL SERVICES COLLABORATION A. G. Memon, and M. S. Chandio ....................................................................................... CITRIC ACID FERMENTATION OF HYDROLYSED RAW STARCH BY ASPERGILLUS NIGER IIB-A6 IN STATIONARY CULTURE S. Anwar, S. Ali and A. A. Sardar .................................................................................................................... 89-108 85-88 79-84 55-78 51-54 41-50 37-40 31-36 27-30 19-26 15-18 05-14 01-04

109-118

Computer Composing Plus Technical Pasting by Mr. Iqbal Khan Pathan

Sindh Univ. Res. Jour. (Sci. Ser.) Vol: 40 (2) 01-04 (2008)

Sindh Univ. Res. Jour.

SURJ

PHYSICOCHEMICAL QUALITIES AND NUTRITIONAL ATTRIBUTES OF STABLE BREAD WHEAT VARIETIES REPRESENTING DIVERSE GENETIC ORIGINS S. I. H. Shah, K. A. Siddiqui* M. A. Sahito** S. Tunio*** and A. J. Pirzada** Seed Certification Officer, Federal Seed Certification and Registration Department Govt. of Pakistan, Bahawalpur, Punjab

(Received 14th June 2008 and Revised 17th September 2008)

Abstract

Ten commercial genetically improved wheat varieties namely: Punjab-96, Kiran-95, Mehran-89, Shahkar-87, Pirsabak-85, Parwaz-94, Tandojam-83, Abadgar-93, Chakwal-86 and Anmol-91, representing diverse genetic origins were investigated for physicochemical properties and nutritional attributes including moisture content, protein content, wet and dry gluten, gluten index, water binding in wet gluten content, near infrared reflectance (NIR), hardness and falling number, for assessing their end-product suitability and consumer's preference. Protein contents of all genotypes were found in the range 12.2% to 15.6%. Moisture contents of all genotypes ranged from 9.2% to 10.2%. Hardness of all samples ranged from score 48 to 58. Gluten index ranged from 38.8 to 75.3, depicting wide range of strength. Falling number of all genotypes was more than 400 seconds. This proved the absence of alpha-amylase activity. The results revealed that all the studied varieties are suitable for all types of breads including flat leavened and un-leavened bread. Keywords: Investigation of for physicochemical properties, moisture contents, protein contents, wet and dry Gluten, water binding, hardness and falling number.

1.

Introduction Bread wheat (Triticum aestivum L.) has remained the central theme of self- sufficiency programme in Pakistan. Although, Pakistan was the first country in Asia to achieve selfsufficiency in wheat after the advent of Green Revolution, the situation in 2008 has altogether changed in respect of supply and demand of bread wheat. (Hussain and Qamar, 2007). Experimental endeavours to develop efficient and sustainable wheat production have been highly successful (Siddiqui, 2002). Most of the wheat is grown for human food and about 10 % is retained for seed and industrial use (for production of starch, paste, malt, dextrose, gluten). Wheat grains contain all essential nutrients; kernel contains about 12 % water, including carbohydrates (60-80% mainly as starch), protein (8-15%), containing adequate

amount of all essential amino acids (except lysine, tryptophan and methionine, fats (1.5-2%), minerals (1.5-2%), vitamins (such as B complex, vitamin E) and 2.2% crude fibers (Hussain and Qamar, 2007). Siddiqui, (1997), has provided programmatic solutions to the major problems of food and agriculture in the environmental context of 21st century. Nevertheless, new problems are also emerging in the wake of green biotechnology, nanobiotechnology, globalization, poverty alleviation and food sovereignty. Wheat is the first important and strategic cereal crop for the majority of world's population. It is the most important staple food of about two billion people (36% of the world population), (Siddiqui, 2007). Worldwide, wheat

* Department of Biotechnology, Genetic Eng. and Environmental Sciences, Centre of Excellence in Analytical Chemistry, Univ. of Sindh ** Institute of Botany, University of Sindh, Jamshoro ***Sindh Agriculture University, Tandojam

S. I. H. Shah, et al.,

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provides nearly 55% of the carbohydrates and 20% of the food calories consumed globally. It exceeds in acreage and production than every other grain crop (including rice, maize, etc.) and is therefore, the most important cereal crop of the world, which is cultivated over a wide range of climatic conditions. Generally wheat quality refers to its suitability for a particular end-use. Botanical, subjective and objective criteria are used to assess the quality. Objective criteria are based on physical, chemical and nutritional properties of wheat grain and are more reliable in evaluation of wheat quality, hardness; protein content and gluten strength are the basic quality attributes. Protein content is a key quality factor (Siddiqui, 1972; Siddiqui and Doll, 1973, Siddiqui, et .al, 1975 and Siddiqui, 2005), that determined the suitability of wheat for a particular type of product as it affects other factors including mixing tolerance, loaf volume and water absorption capacity. Wheat is unique among cereals and other protienaceous plants since its milled product "flour" alone is capable of forming the dough due to its gluten content. The dough retains the gas evolved during fermentation (Bushuk and Wrigley, 1974; Hoseney, 1998), and on baking yields a light, well-aerated loaf of bread. This unique characteristic of wheat is derived from its proteins that are gliadin and glutenin and upon hydration results in gluten; the actual substance that imparts the property of gas retention to dough. Bread (or hard wheat) flour has high gluten content, and is therefore good for yeast raised bread, which requires an elastic framework. On the other hand, weak wheat flour is best suited for cakes and for biscuit making (Gaines, 1991 and Souza et al. 1994). Moisture content of wheat flour is very important in relation to storage, milling and handling properties of wheat. Hardness score is an important criterion for conformity to the market place. Falling number (FN) test gives an indication of the amount of sprout damage that has occurred within a wheat sample. The falling number test does not directly measure amylase activity, but measures changes in the physical properties of the starch protein of the wheat

kernel caused by the enzymes during the test. As the enzyme activity increases, the FN value decreases. The objective of the present study is to evaluate the physicochemical qualities at nutritional attributes of stable bread wheat varieties representing diverse genetic origins. 2. Materials and Methods Seeds of ten wheat varieties were collected from four Research Institutes viz. Ayub Agricultural Research Institute Faisalabad, Cereal Crop Research Institute Pirsabak (NWFP), NIA Tando Jam and Wheat Research Institute Sakrand. The individual samples were cleaned manually and mixed through precisian Electronic Divider. These samples were then milled to whole wheat flour using Perten Laboratory Mill 3100, installed with 0.8 Sieve and then mixed thoroughly. Protein content, moisture content and hardness were determined through near infrared reflectance (NIR). Gluten contents on wet as well as dry weight basis and water binding in wet gluten were determined according to AACC (2000) method No.38-12. Falling number was determined according to AACC (2000) method No.56-81 B. All the studied parameters were taken in triplicate and with standard deviation that was statistically analyzed in accordance with (Zar, 1974). 3. Results and Discussions Ten wheat approved varieties by Federal Seed Certification and Registration Department, Government of Pakistan, Islamabad were grown across different agro-ecological locations viz. TandoJam, Sakrand, Sukkur, Rahimyar Khan, Khanewal, Sahiwal, Dokri, Jacobabad and Quetta during the Crop Year (1999-2000). The selected varieties were evaluated for following physicochemical characteristics. Moisture Contents of these wheat varieties ranged from 9.2% to 10.2% (Table-1). This indicated that these wheat varieties can be easily stored due to low moisture content, and being less prone to microbial attack. At low moisture content the grains tended to be brittle and could break during the commercial handling operations. However this is usually the case for wheat, which is artificially dried to lower the moisture content (Zenleny, 1991).

Physicochemical qualities and nutritional... Table ­ 1. The Physicochemical Characteristics and properties of different wheat varieties

Name of Genotype Punjab-96 Kiran-95 Mehran-89 Shahkar-87 Pirsabak-85 Parwaz-94 TandoJam-83 Abadgar-93 Chakwal-86 Anmol-91 Moisture Content (%) 9.4+0.05 10+0.05 9.2+0.15 10+0.11 10.2+0.05 9.4+0.11 10+0.05 9.5+0.05 10+0.11 9.2+0.05 Protein Content (%) 14.4+0.05 12.4+0.25 12.6+0.15 14.15+0.11 12.2+0.05 15.4+0.05 15.2+0.05 15.6+0.05 14.3+0.11 15.6+0.05 Wet Gluten (%) 32.5+0.51 27.4+0.45 26+0.11 38.5+0.5 24.5+0.5 30+0.11 35.5+0.05 30.5+0.5 28.5+0.5 39.5+0.5 Gluten Index 75.3+0.57 41+1 64+1 44+0.57 54.9+1.52 56.7+1 45.5+1 60.2+1.73 45.2+1 38.8+0.57 Dry Gluten (%) 10+0.57 8+0.11 9.8+0.28 12.1+0.17 8+0.11 10.1+0.17 12+0.05 10.1+0.15 8.6+0.57 12.3+0.57 Water Binding in Wet Gluten (%) 22.5+0.47 19.4+0.45 16.2+0.25 26.4+0.54 16.4+0.51 19.9+0.05 23.4+0.5 20.3+0.47 19.8+0.28 27.2+0.34 Hardness 51+0.57 54+0.57 50+0.57 50+0.57 57++0.57 52+0.57 53+0.57 58+0.57 56+0.57 48+0.57

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Falling Number (sec.) 650+5 551+2 630+10 552+9 670+4 615+3 606+4 408+5 450+5 556+5

Average of triplicate analysis, + Denote Standard Deviation

Protein Content of Parwaz-94, TandoJam-83, Abadgar-93, and Anmol-91 were ranged from 15.2% to 15.6% which was higher than other wheat varieties. This indicated their stability for hard wheat products like yeast-leavened bread. The dough made from this type of varieties had greater dough strength, resulting in increased loaf volume after baking (Inone and Bushuk, 1992). High protein levels are related to undesirable cookie texture (Posner and Hibbes, 1997). Protein content of Punjab-96, Kiran-95, Mehran-89, Shahkar-87, Pirsabak-85 and Chakwal-86 ranged from 12.2% to 14.4% and characterize as semi-hard wheat. Dough from these varieties were strong, stretchable, elastic and non-sticky that were suitable for un-leavened bread like chapati. All varieties used in this study were found to have medium to very strong gluten. High gluten index, interpreted strong gluten matrix, which increases the viscosity of cookie dough during baking, Thus is un-desirable because cookie spread is restricted (Hoseney, 1994), but it is good for bread. Variety Punjab-96 the highest gluten index, which enabled it to form the strongest gluten network suitable for very hard wheat products like pasta. Variety Punjab-96 had 14.4% protein content and 75.3% gluten index, while Abadgar-93 had 15.6% protein content and 60.2 gluten index. This revealed the highest protein content of flour is indicative of its strongest gluten strength. Thus, quantities of protein as well as quality are both important parameters for evaluation of their end product suitability. Hardness of tested varieties ranged from 48-58 score. Hard wheat flour is suitable for bread and very hard for products like pasta. Grain hardness is negatively correlated with cookie diameter because damage starch increases water absorption capacity and viscosity, which hinders cookie spread (Monsalve-Gonzalex and Pomeranz, 1993). Falling Number of all varieties was found more than 400 seconds, which implied that the wheat varieties included in this investigation are free of sprout damage that is a desirable character (Bushuk and Rasper, 1994). The wheat varieties showing better quality can be used by the wheat breeders in the future wheat improvement programmes. The information obtained from this study will be useful for different stake-holders such as plant breeders, farmers as well as the end-users. 4. Conclusion The physiochemical characteristics of wheat vary considerably with wheat variety, which in turn influences their quality with respect to their end product suitability. All seed samples used in the study were found to be hard to very hard. So they are all suitable for hard wheat products

S. I. H. Shah, et al.,

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like traditional bread i.e. chapati as well as yeast leavened bread. As no wheat genotype was found to fall into soft wheat category, so none of them is suitable for products like biscuits and cookies. Protein content, gluten index, falling number and hardness score were found to be the basic physiochemical characteristics to evaluate their end product suitability. References American Assoe. of Cereal Chemists. (AACC) (2000) Approved Methods of the AACC, 10th ed. Method 38-12; 56-81B. The Assoc. St. Paul, Minnestoa Bushuk, W., and C.W. Wrigley. (1974) Protein: Composition, Structure and Function. 119-145 In Wheat: Prod. And Utilization. G.E. Inglett, ed. VI Pub Co. Inc.: Westport, Connecticut. Bushuk, W., and V.F. Rasper. (1994) Wheat production, properties and quality. Blackie Academic and Professional, Glasgow, UK. 239. Gaines, C.S., (1991) Instrumental measurements Of the hardness of cookies and crackers. Cereal Foods World. (36): 989-996. Hoseney, R.C., (1994) Principle of cereal science and technology. 91-94. Minnestoa, USA The American Assoc. of Cereal Chemists. Hoseney, R.C., (1998) Principles of cereal science and technology. 2nd Ed. Amer. Assoc. Cereal Chem. St. Paul, Minnestoa. Hussain and Qamar, (2007) wheat genomics challenges and alternative strategies. Proc. Pakistan Acad. Sci. 44 (4): 305-306. Inone, Y., and W. Bushuk, (1992) Studies on Frozen doughs. II. Flour Quality Requirements For Bread Prod. From Frozen Doughs. Cereal Chem. (69): 423-428. Monsalve-Gonzalex, A., and Y. Pomeranz. (1993) Effect of spring and winter growth habitate on Compositional, milling and baking characteristics of winter wheats. Cereal Chem. (70): 354-359. Posner, E., and A. N. Hibbes, (1997) Wheat flour milling. Am. Assoc. Cereal chem. St. Paul, Minnestoa. Siddiqui, K.A., (1972) Protein content and quality of wheat chromosome substitution lines. Heriditas (71): 157-160. Siddiqui, K.A., (1997) Solving the major problems of food and agriculture in the environmental context of the 21st century. In: Science for Sustainable Development in Latin America and the Caribbean, 217-227, TWAS/TWNSO/IFS, Rio de Janeiro, Brazil Siddiqui, K.A., (2002) Experimental endeavours to develop efficient sustainableagriculture in Pakistan in the environmental context of 21st century, Proc. 2nd Int. Conf. on Sustainable Agriculture for Food, Energy and Industry, 524-529, Beijing, China. Siddiqui, K.A., (2005) Elucidation of the inheritance of protein patterns in man mademulti genomic (AADDMVMV) plant species. In Protein Structure-Function Relationships, Ed. Atiya Abbasi and Syed Abid Ali, ISBN 969-8113-08-8. Siddiqui, K.A., (2007) Green biotechnology at the crossroads of nanobiotechnology, globalization, poverty, alleviation and food sovereignty, Indian Jour. Crop Science 2: 1-4.

Siddiqui, K.A., Rajput, M.A. and Arain, U.A. (1975) Induced eed protein mutants of Triticum aestivum. Naturwissenschaften. (62): pp393. Siddiqui, K.A., and Doll, H. (1973) Screening for improved protein quality mutants in wheat. Z.Pflanzenzucht (70): 143-17. Souza, E., M. Kruk and D.W. Sunder man, (1994) Association of sugar-snap cookie quality with High molecular weight glutenin alleles in soft white spring wheats. Cereal Chem. (71): 601-605. Zar, J. H., (1974) Biostatistical Analysis. Prentic Hall. Englewood Cliffs, New Jersey. Zenlney, L., (1991) Criteria of wheat quality. 19-45 in Wheat Chemistry and Technology. Pomeranz, ed. Am. Assoc. Cereal Chem. St. Paul. Minnestoa. Y.

Sindh Univ. Res. Jour. (Sci. Ser.) Vol: 40 (2) 05-14 (2008)

SURJ

Sindh Univ. Res. Jour. SMALLER FORAMINIFERAL ASSEMBLAGE OF THE KHIRTHAR FORMATION OF SUKKUR AND KOTDIJI AREA, SINDH P. A. Usmani, A. H. Markhand, M. K. Samoon. and S. A. Shaikh

[email protected]

Centre for Pure and Applied Geology, University of Sindh, Jamshoro

(Received 24th March 2008 and Revised 15th September 2008)

Abstract

Khirthar Limestone, the most prominent ridge-forming rock unit that has derived its name from the north-south stretching range of hills, the Khirthar Ranges, that attracted Blanford (1876) to name this well developed rock unit as "Khirthar Limestone", later formalized to Khirthar Formation. It extends towards the north east to Sulaiman Range and South Waziristan. The unit is about 1270m thick in the type section, the Gaj river section, but tapers to 15-30m towards south. However, inspite of its wide-spread occurrence no effort has been made at age-dating this unit and it is vaguely placed between 'Middle to Upper Eocene to Lower Oligocene'.

As such the present study was initiated and to start with, detailed study of the smaller foraminifera of the Khirthar rocks exposed in Sukkur and Kotdiji areas was carried out with a view to determine the character of the faunal assemblage and to find out whether these rocks can be age-dated and correlated more accurately.

The investigation enabled to identify 43 species, belonging to 15 genera. These include 15 Planktonic forms. Among the species identified 17 benthonic species and 7 planktonic species have been earlier reported from various Eocene rocks of Pakistan.

The study reveals that foraminifera in rocks of Sukkur and Kotdiji areas probably represent reworked fauna derived intra-basinally. They include forms typical to both Eocene and Basal Oligocene. Further, the age of this outcrop of Khirthar Formation extends upto Lowest Oligocene. Keywords: Khirthar rocks exposed in Sukkur, Kotdiji areas Khirthar Formation.

1.

Introduction Studies of foraminifera in Pakistan region had their beginning as early as 1861 (Carter) followed by work of other investigators. Nuttal's studies presented first a systematic account of the foraminifera of the Kirthar Series (Nuttal, 1926). This account was restricted to the larger forams. Later accounts of the foraminifera included studies by Davies (1926), Eames (1952), among others, mainly on larger foraminifera. However, elsewhere it was realized that smaller foraminifera are far more significant particularly in interpretting Paleoecology.

Haque (1959) carried out systematic examination of the smaller foraminifera from the Eocene rocks of Sor Range. This was followed by another significant contribution on the Paleocene-Eocene smaller foraminifera of Nammal Gorge (Haque, 1962) and "On the smaller foraminifera of the basal Paleocene" (Haque, 1964). Later Samantha (1973) produced a systematic account of the Paleocene-Eocene Planktonic foraminifera of Rakhi Nala Section, Pakistan. These advances in the study of smaller and Planktonic foraminifera were few and far between and left many more voids in the systematic micropaleontology particularly with

P. A. Usmani, et al.,

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respect to Sindh where comparatively undisturbed Cenozoic sequences of shallow marine origin with rich assemblages of larger as well as smaller foraminifera from the bulk of the outcrops. These larger forams have been undeniably used in subdividing the Cenozoic succession in series; however, the absence of well defined sedimentational breaks has rendered it difficult to define Formational boundaries precisely. This is particularly the case with the Kirthar Formation of the Lower Indus Basin which is reported to range from Mid Eocene to Early Oligocene (Jones, 1960) without observable sedimentational break. But earlier workers have placed it in Mid-Upper Eocene (Blanford, 1876; Noetling, 1903). Jones (1960) distinction of Eocene Kirthar member and Oligocene Gorag member in Blanford's Kirthar Series is based on mixed fauna, mainly larger foraminifera. The areas of Sukkur and Kotdiji were selected for present study because of their easy accessibility and secondly, because of the fact that these outcrops represent deposition on a depositional high the Jaccobabad ­ Khairpur high and mixed fauna, particularly planktonics bearing evidence on its diachronous nature are likely to be met here. Location and accessibility The Studied areas are easily accessible through metalled roads, and are at following latitude and longitude:

Sukkur Area Latitude 27º 41' 60'' N Longitude 68º 50' 60'' E Kotdiji Area Latitude 27º 20' 24'' N Longitude 68º 41' 24'' 2. Geology of the area Sukkur Outcrop: A beautiful outcrop of Limestone is present in the north-east of the New Pind Sukkur. The exposure is due to the querying of Limestone for cement factory. The Limestone is light grey in color and contains chert nodules in the upper part of the outcrop. The beds are mostly 9" to 1' thick. In this section the Limestone appears unfossiliferous in hand specimen. There is 6" thick bed of clay after each 20 feet. The total thickness of the unit is about 80 feet in Sukkur area. At the base of this section the limestone is white coloured hard and unfossiliferous. After 5 feet the color changes from light brown to dark brown. The upper beds are similar in lithology to that at the bottom. But after 15 feet the color changes from brown to milky white. Limestone is compact containing sparsely dispersed chert nodules. About 30 feet above the base there is change in color and lithology; one feet thick bed of clay is present which is deep yellow in color. Above this clay bed the lithology is similar to the other beds, i.e. limestone is white in color, slightly soft. After 50 feet the limestone is comparatively hard and more compact than the underlying units; it is dark in color nodular and contains the chert nodules. Kotdiji Outcrop: At this locality (KTD) the rocks are exposed along a road section in the west of the village of Kotdiji. The beds are almost horizontal and the thickness exposed here is about 110 feet. The limestone is brown, grey in color, soft and highly fossiliferous in the basal part of the outcrop; 20 feet above the color changes to cream and the limestone becomes comparatively hard. This lithology again changes at about 35 feet above this level, and,

Fig: 1. Location map of the studied area.

Smaller foraminiferal assemblage...

07

the limestone becomes very fine-grained. The rocks here are very thinly bedded to laminated. At about 45 feet above the base the limestone is soft, nodular and fossiliferous. From 45 to 80

Lithological Section of Sukkur Area (Thickness in ft.)

feet; the lithology is similar but it changes after 80 feet the limestone becomes brown in color, hard and nodular, and is unfossiliferous upto the top of the outcrop.

Fig: 2. Lithological sections of the studied area

Fig. 3. Geology map of the studied area

P. A. Usmani, et al.,

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3.

Materials and Methods Samples were collected from the outcrops of the Kirthar Limestone in Sukkur and Kotdiji areas. Kirthar Limestone is exposed in a long range of hill starting from Sukkur in the North to South of Kotdiji. In this range only the Kirthar Limestone is exposed, the lower contact of the Formation is not exposed at any place in this area. The Limestone is generally grey, cream, buffor white, light brown, brown or pinkish white in color. It is soft, marly and at places nodular, hard, and splintery beds are also present. The beds are mainly 1-10 feet thick, massive or laminated. The lithology differ at both the localities. The Samples were collected at the interval of 1-5 ft. in thin bedded rocks and upto 10 ft. in thick bedded units. The samples were numbered locality wise prefixing the abbreviated locality notation. 1. Sukkur (SK) 2. Kot Diji (KTD) The photography of micro-forams was carried out with the Zeiss Ultra microscope using magnification of 60x. 4. Classification In this work R.C. Moore's system of classification has been followed. It is most widely accepted classification. He asserted that classification of foraminiferida as of other animal groups should be based on complete morphological data obtained by all known suitable techniques. The classification adopted by Moore (1964) is mainly based upon the life, habit, habitat and geologic ranges. He regards wall composition and microstructure of the test as of primary importance for classifying the foraminifera. Foraminifers found (Plate 1,2,3, and 4 show the microphotographs of these foraminifers). Family: NONIONIDAE Genus: Florilus Transversus Genus: Nonion Nonion Planatum Nonion c.f. N. Scaphum Nonion Brunatus Hofker Nonion Acutidorsatum Genus: Nonionella Nonionella wilcoxensis Nonionella Kirthari

Nonionella Insecta Nonionella Basipinata Family: GLOBOROTALIDAE Genus: Globorotalia Globorotalia Renzi Bolli Globorotalia Chapmani Parr Globorotalia Compressa Globorotalia Bolivariana Globorotalia Cerro-Azulensis Globorotalia Menardii D'Orbigny Globorotalia Planoconica Subbotina Family: GLOBIGERINIDAE Genus: Globigerina Globigerina Triloculinoides Globigerina Officinalis Family: CIBICIDAE Genus: Planulina Planulina Ariminensis Genus: Cibicides Cibicides Punjabensis Haque Cibicides Cushmani Nuttal Cibicides Lobatus Cibicides Multifarous (Schwager) Cibicides Mensilli (Schwager) Var, Nammalensis Haque Cibicides Natlandi Olequaensis Beck Cibicides Hilgards Garrett Cibicides Praecursorius Stichocibicides Cubensis Family: HANTKENNINIDAE Genus: Globoanomalina Globoanomalina Micra Family: ANOMALINIDAE Genus: Anomalina Anomalina Acuta Anomalina Castiana Anomalina Chirana Anomalina Grazaensis Family: ALABAMINIDAE Genus: Gyroidina Gyroidina Tayyabi Gyroidina Lottensis Family: DISCORBIDAE Genus: Discorbis Discorbis Cocoaensis Cushman & Garrett Discorbis Lamark sp. Nov. Family: SPHAEROIDINIDAE Genus: Sphaeroidina Sphaeroidina Bulloides D'Orbigny Family: DISCORBIDAE Genus: Valvulaneria

Smaller foraminiferal assemblage...

09

Valvulaneria Stainforthi Hoffer Valvulaneria Eocenica Cushman Family: EPONIDIDAE Genus: Eponides Eponides Anconeusis Eponides Umbonatus Lenticuline Degolyeri Family: TEXTULARIIDAE Genus: Textularia Textularia Gertrodema Davis 6. Discussion. There are some interesting aspects of the distribution of species in the samples studied. The distribution is not uniform. 1. No smaller forams could be retrieved from the Sukkur outcrop samples although larger forms are profuse and described fully by Nuttal (1926). In all 16 samples were analyzed. However as the material was hard and compact thin section study only was carried out. 2. Study of Kotdiji samples yielded most of the forms in samples KTD-1 to KTD-4 only, with maximum concentration in KTD-4. The treatment of the samples above this level did not yield any identifiable larger or smaller form. The sequence above this level is barren consisting mostly of soft micritic carbonate sediments. It may be noted that the lithology of Sukkur outcrop and associated fauna suggest shelf facies. The lithology of the rocks of Kotdiji outcrops and absence of larger foraminifera in them may point to a bank to basin transition. They comprise of 33 genera and their 70 species. Many of the species described herein have also been reported by other workers from the Paleogene rocks of Pakistan subcontinent and elsewhere in the world. Some of the forms are however anomalous in terms of their stratigraphic position, among them, is the genus described here as Pseudohyalinea after the genus Hyalinea described from the Miocene-Recent (Hofker 1951) with which it compares favorably but the stratigraphic position does not justify its assignment to Hyalinea and hence the modified generic name. Similarly the form named here as Pseudoholmanella is treated as distinct in

occurrence only from its compareable form genus Holmanella described from Miocene (Leoblich and Tappan, 1962). Among the forms described here, 24 species have already been described from the various Eocene horizons of Pakistan. The species under report that have also been described from the Khirthar Formation of Sor Range area, the Paleocene-Eocene of Nammal Gorge and the Meting Shale unit of Lower Eocene of Sindh by Haque (1956, 1959), and the Paleocene rocks of Rakhi Nala section (Samanta, 1973). These include 17 benthonic and 7 planktonic species. Among the benthonic forms Nonionella Kirthari, N.c.f.N. ansata, Nonion acutidorsatum, cibicides punjabensis, C. Hilgardi Garrett, Gyroidina Tayyabi, Eponides anconensis, are typical Middle-Late Eocene species. Also the planktonic species Globorotalia renzi bolli, G. Chapmani and Globigerina officinalis are again typical forms restricted from Middle to Late Eocene. This comparison of the forms reported here with those earlier described from Pakistan and outside lends sufficient justification to support Middle to Late Eocene age of these rocks. 7. Conclusion Keeping in view that the Sukkur outcrop of the rocks with associated larger foraminifera represent shelf conditions, it can be concluded that towards south of this shelf on the high, basinal conditions prevailed in which synchronous intra basinal micritic carbonate sediments derived from Paleocene and Eocene sediments developed. To this carbonate assemblage typical planktonic forms of basal Oligocene were also added. However, the deposition came to a halt following Oligocene regression (Powel & Conaghan, 1973). This interpretation would support Jones (1960) contention extending the age of Kirthar Formation, his Brahui limestone, to lower Oligocene. This interpretation would, however, have to await confirmation by a thorough investigation of not only the smaller foraminifera of the Kirthar Formation from the type area but

also detailed Petrographic examination of Kirthar Formation. This was outside the

scope of present study but would form a suitable topic for future research.

P. A. Usmani, et al.,

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Smaller foraminiferal assemblage...

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P. A. Usmani, et al.,

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Smaller foraminiferal assemblage...

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References Bandy, O. L., (1944) "Eocene foraminifera from Cape Blanco, Oregon"J. Paleon., (18): 366-377. Blanford, W. T., (1876) "On the geology of Sindh". Geol. Surv. India, Rec. 9 (1). Cushman, J. A., and R.M., Stainforth, (1951) "Tertiary foraminifera of the coastal Ecuador, Part I Eocene": J. Paleon, (25): 129-164; pls. 25-28. Cushman, J. A., (1950) "Foraminifera, their classification and economic uses". Hyderabad Uniniversity Press. (1950). Davies, L. M., (1926) "Remarks on Carter's genus conulites-dictyoconoides nuttal with descriptions of some new species from the Eocene of north-west India". India, Geol. Survey, Rec., vol. (59): pt.2, 237-253, pls.16-20. Eames, F. E., (1952) "A contribution to the study of the Eocene in West Pakistan and western India: A. The geology of the standard sections in the western Punjab in Kohat district". Quaterly J. of the Geological Society, London 107 (1952) (20): 159-200. Ellis, B.F., A. R., Messina, R., Chamataz, and L.E., Ronai, (1969) "Catalogue of Index smaller foraminifera". vol. (2): special pub. Amer. Museum of Nat. Hist. (Index) Farshori, M. Z., (1972) "The Geology of Sindh". Sindh Uni. Press. Hyderabad, Pakistan 3436. Haque, A.F.M.M., (1956) "Foraminifera of the Ranikot and the Laki of the Nammal Gorge Salt Range"; Paleontologica Pakistanica; 1. 1-300. Haque, A.F.M.M., (1966) "Some late Tertiary foraminifera from Makran,W.P." Paleontologica Pakistanica, 2. 51-65. Hunting Survey Corporation ltd. (1950) "Reconnaisance Geology of part of Pakistan". A report published by Government of Canada. 85-90 and 180-182. West

Moore, R.C., (Ed.) (1964) "Treatise on invertebrate Paleontology Part C, Protista 2 (1)". Geol. Soc. Amer., C-510 ap. Nagappa, Y., (1959) Foraminiferal biostratigraphy of the Cretaceous-Eocene succession in the India-Pakistan-Burma region: micropaleontology, vol.(5): No. (2): 145-192. Noetling, F., (1903) "Ubergang zwsorchen kredi and Eocan in Baluchistan". Central B.I Miner. Geol. Paleont. Noetling, F., (1905) Vorlanfige Mitteilung uber die Entwickelung und die Gliederung der Tertiar formation im westlichen Sind (Indien). Zbl. Min. Geol. Palaont., 129-137. Nuttall, W.L.F., (1926) The zonal distribution of the larger foraminifera of the middle and lower Kirthar Series (middle Eocene) of parts of western India: Rec. Geol. Survey India, vol. (59): 115164. Samanta, Bimal K., (1973) "Planktonic foraminifera from the Paleocene-Eocene succession in the Rakhi Nala Sulaiman Range, Pakistan". Bull. British Museum. (Natural History) pp22.

Shah, S.M.I., (Ed.) (1977) "Stratigraphy of Pakistan" vol. (12): Geological Survey of Pakistan. Vredenburg, E.W., (1906) "A review of Tertiary Sequence of Sindh". Pal. Indica, New Ser.; 3 Pt.1.

Sindh Univ. Res. Jour. (Sci. Ser.) Vol: 40 (2) 15-18 (2008)

SURJ

Sindh Univ. Res. Jour. NEW RECORD OF SOME PLANKTONIC CLADOCERAN FROM FISH PONDS OF JAMSHORO SINDH, PAKISTAN Mukhtiar Ahmed Mahar [email protected] Department of Fresh Water Biology and Fisheries, University of Sindh, Jamshoro, Sindh, Pakistan

(Received 14th August 2008 and Revised 05th November 2008)

Abstract

Five species of planktonic cladoceran Bosminopsis deitersi (Richard), Ceriodaphnia setosa (Matile), Ilyocryptus bhardwaji (Battish), Moina chankensis (Ueno), and M. rectirostris (Leydig) belonging to four families have been recorded for the first time from Pakistan, during the survey period of 2007- 2008 in fish ponds of Jamshoro thermal power plant effluent. Keywords: Cladocera, zooplankton, Jamshoro thermal power plant.

Introduction Cladoceran are considered to be the ecological indicators of the water bodies; these are the major group of zooplankton community. Cladoceran forms exist in lakes, reservoirs, natural depressions, ponds and irrigation canals. Most of the genera are occurring in less saline water bodies. Lot of studies have been carried out on the zooplankton forms in Pakistan by Arora (1931), Baqai and Ishrat (1973), Baqui et al. (1974a and b), Baqui and Iqbal (1976), Baig and Khan (1976), Mahoon and Zia (1985), Iqbal and Kazmi (1990), Tasneem and parvaiz (1994), Jafri et al. (1999), Leghari et al. (1999), Baloch et al. (2000), Mahar et al. (2000), Baloch et al. (2004), Mahar et al. (2007), Mahar et al. (2008) and Baloch et al. (2008). Present study provides the information about the presence of five new recorded cladoceran species which would be the addition in the taxonomy of cladoceran zooplankton and their distribution up to Pakistan. 2. Materials and Methods Collections of the cladoceran were carried out during October 2007 to May 2008 after noon. Specimens were collected by plankton net No. 25 (mesh size 55µ) and preserved in 5% formalin solution on the spot in transparent plastic bottles and brought to the laboratory. The specimens were identified with the help of taxonomic keys and illustrations, published by Ward and Whipple (1959), Edmondson, (1959); Hutchinson, (1967); Mizuno and Takahashi, (1991); Battish, (1992), Yunfang; (1995) Pennak, (2001). Photographs were taken at magnifications ×100 and ×400 with the help of digital camera model DCM-35 fitted on the trinocular microscope (Swift M. 3300D). 3. Results Five species of cladoceran i.e Bosminopsis deitersi (Richard, 1895), Ceriodaphnia setosa (Matile, 1890), Ilyocryptus bhardwaji (Battish, 1981), Moina chankensis (Ueno) and M. rectirostris (Leydig, 1860) are newly recorded from Pakistan (Tab.1). The detail description is given below. (i). Bosminopsis deitersi (Richard, 1895)

1.

M. A. Mahar

16

Body carapace is clearly transparent, and semi ovate. A very long pointed curved rostrum leads by head; diverging laterally near apex with long point at clove. Antenna with three jointed rami, basal part of antennules united with each other. Post abdomin tapering to point at claws, 1 large spine near claws and several small spinules anterior to it. Length near about 0.34 mm. this species was recorded in winter season in association with rotifers (Fig. 1).

Fig.1. Bosminopsis deitersi Richard

(ii). Ceriodaphnia setosa (Matile, 1890) The carapace more or less quadrate, body is transparent with an orange tinge and lack of posterior spines. Head ovate anteriorarily and produced below into an acuminate rostrum. The anterior oval portion of head provided a large eye. Antennules are small and weak do not extend up to the tip of rostrum. Ocellus is present at the base of antennules. Post abdomen is elongated, truncate with more than two abdominal setae. The claws are smooth, pointed and no pectin is found. The intestine is semi linear. Total length of the body is 0.76 mm recorded during spring months (Fig. 2).

Fig. 2. Ceriodaphnia setosa Matile

(iii). Ilyocryptus bhardwaji (Battish, 1981) Body shape oval-triangular with small head forming apex. Ventero- posterior margins of the carapace bears several concentric lines and plumore fixed setae occur. Eye prominent, ocellus smaller, antennules long and freely movable. Post abdomen very large, broad and compressed with eight pre-anal spines. Total body length of the species recorded 0.53mm and height of the body 0.43 mm (Fig. 3). This species was recorded from weedy area during August.

Fig. 3. Ilyocryptus bhardwaji Battish

(iv). Moina chankensis (Ueno) Transparent body carapace and head are rounded without any depression and also having sinus above the eye. The eye large with numerous

lenses but ocellus is absent. Antennules are long with censory cilia. Total length measured from head to posterior portion of carapace recorded about 1.02mm and from dorsal to ventral side 0.92 mm (Fig.4). This species was collected from open water during summer season in planktonic form.

Fig. 4. Moina chankensis Ueno

(v). Moina rectirostris (Leydig, 1860)

New record of some planktonic ... 17

Head depressed and little linear rounded. Carapace ball shaped antero-posteriorly without spine and horn; eye and ocellus are quite prominent and surrounded by numerous lenses. Antennules are long with terminal setae and bearing cilia distally in middle of the antennule. Body is colourless transparent, total length of the ball shaped body is 1.28 mm from head to posterior end (Fig.5). This species was commonly reported in association with copepod and rotifers during winter period.

Fig. 5. Moina rectirostris Leydig

Suborder: Cladocera Family: Alonidae 1. Alona guttata Sars 2. A. rectangula Sars 3. A. quadrangularis O.F. Muller 4. Leydigia acanthocercoides Fischer Family: Bosminidae 5. Bosmina coregoni Baird 6. B. longirostris O.F. Muller Family: Chydoridae 7. Chydorus barroisi Richard 8. C. globosus Baird 9. C. ovalis Kurz 10. C. poppei Richard 11. C. sphaericus O.F. Muller 12. Dunhevedia crassa King 13. Leydigia acanthocercoides Fischer 14. Oxyurella tenuicaudis Sars 15. Pleuroxus denticulatus Birge Family: Daphniidae 16. Daphnia ambigua Scourfied 17. D. dubia Herrick 18. D. galeata Sars 19. D. longispina O.F. Muller 20. D. longiremis Sars 21. D. lumholtzi Sars 22. D. magna Straus 23. D. pulex Leydig 24. D. psittacea Baird 25.D. hyaline Leydig 26. Camptocerys sp. 27. Ceriodaphnia cornuta Sars 28. C. laticaudata P.E. Muller 29. C. megalops Sars 30. C. pulchella Sars 31. C. reticulate Jurine 32. C. rigaudi Richard 33. Scaphloleberis kingi Sars 34. Simocephalus exspinosus Koch 35. S. vetulus O.F. Muller Family: Leptodoridae 36. Leptodora kindti Focke

Discussion Five species of cladoceran i.e. Bosminopsis deitersi (Richard, 1895), Ceriodaphnia setosa (Matile, 1890), Ilyocryptus bhardwaji (Battish, 1981), Moina chankensis (Ueno) and M. rectirostris (Leydig, 1860) are reported for the first time from Pakistan (Tab.1). The earlier studies on cladoceran including those of Arora (1931) documented fifteen species of caldoceran from Lahore. Baqai et al., 1974a, b, who recorded 16 species of cladocera and Baig and Khan (1976) who described four genera. Preliminary observations on seasonal abundance of cladoceran genera in Keenjhar lake were noted by Iqbal and Baqai (1976). Iqbal and Kazmi (1990) reported 15 species of cladocera from Hub lake. Nine species were recorded from Chotiari reservoir by Leghari et al., (1999). Baloch et al., (2000) reported 6 species from Indus river. In another study 3 species were documented by Baloch et al., (2005) from Rawal lake. Report on seasonal variation and species composition of cladoceran zooplankton was reported by Mahar et al., (2008) with new record of a cladoceran species Dunhevidea crassa (King). Baloch et al., (2008) reported 5 species of cladocera in association with rotifers from Kheer Thar hill torrents. In all 49 species have already been reported from Pakistan (Tab. 2), the present new record of 5 species from ponds of waste water of Jamshoro Thermal Power Plant Sindh is an addition to already known freshwater Cladoceran zooplanktons fauna of Pakistan.

4.

Table -1. Cladoceran recorded from fish ponds of

Jamshoro Thermal Power Plant, during present study. Suborder: Cladocera Family: Bosminidae 1 .Bosminopsis deitersi (Richard, 1859) Family: Daphnidae 2. Ceriodaphnia setosa (Matile, 1890) Family: Macrothricidae 3. Ilyocryptus bhardwaji (Battish, 1981) Family: Moinidae 4. Moina chankensis (Ueno) 5. M. rectirostris (Leydig, 1860) Table -2. List of Cladoceran species recorded (previously) from Pakistan. Source: Arora(1931), Iqbal (1990), Kazmi (2002) and Baloch (2004, 2005 and 2008).

M. A. Mahar

18

Family: Moinidae 37. Mediomoina elliptica Arora 38. Moina affinis Birge 39. M. brachiata Jurine 40. M. flagellate Hudendroff 41. M. irrasa Brehm 42. M. macrocopa Straus 43. M. micrura Kurz 44. Moinodaphnia sp. Family: Macrothricidae 45. Macrothrix laticornis Fischer 46. Macrothrix rosea Birge Family: Sididae 47. Diaphanosoma excisum Sars 48. D. brachyurum Sars 49. Sida crystalline O.F. Muller

References Arora, G.L., (1931) Fauna of Lahore Deptt. Zool. Punj. Univ. Lahore. Vol. (1):

2-Entomostraca (water-Fleas) of Lahore. Bull. 62-100.

Baqai, I. U., and R., Ishrat, (1973) Seasonal fluctuation of freshwater copepods of Keenjhar lake, Sindh and its correlation with physico-chemical factors. Pakistan. J. Zool., 5, (2): 165-168. Baqai, I.U., P.A., Siddiqui, and M., Iqbal. (1974a) Limnological studies of Haleji lake. Agr. Pakistan 25 (4): 321-344. Baqai, I.U., V. A., Zuberi, and M., Iqbal (1974b) Limnological studies of Kalri lake. Agr. Pakistan 25 (2): 119-135. Baig, N. A., and M.Y., Khan, (1976) Biological and chemical conditions of Manchhar lake (Distt. Dadu). Pakistan J. Sci. (28): 33- 40. Battish, S.K., (1992) Zooplankton of India. Oxford and IBHP Publishing Co., 66 Janpath, New Delhi, p.233. Baloch, W.A., (2000) Occurrence of planktonic communities in River Indus at Kotri Barrage, Sindh. Pakistan. Proc. Pakistan Cong. Zool., (20): 77-83. Baloch, W.A., A.N., Soomro and S.I.H., Jafri, (2004) Zooplankton of highly saline water body near Hyderabad, Sindh. Sindh Univ. Res. J. (Sci. Ser.) (36): 25-28. Baloch. W.A., S.I.H., Jafri, and A. N., Soomro (2005) Spring zooplankton composition of Rawal lake, Islamabad. Sindh Univ. Res. J. (Sci.Ser) (37): 41-46. Baloch. W. A., A.N., Soomro and G.H., Buledi (2008) Zooplankton, especially rotifer and cladoceran communities of the spring and rainwater streams (Nai) in Kirthar range, Sindh, Pakistan. Sindh. Univ. Res. J. (Sci. Ser.) (40): (2) 17-22. Chaudhari, I.I., M., Maqsood and A. A., Ghauri (1986) A preliminary study of flora and fauna of Nullah Deg algae and crustaceans. Biologia special supl. 121- 135. Edmondson W.T., (1959) Fresh Water Biology. 2nd edition. John Willey and Sons. New York, 127-169. Hutchinson, G.E., (1967) A treatise on limnology. Vol. (2): John Wiley and Sons, Inc, New York, p. 245. Iqbal, M., and I.U., Baqai, (1976) Seasonal abundance of cladoceran genera in Keenjhar (Kalri) lake. Pakistan J. Sci. (3): 74-77.

Iqbal, M., and M. A., Kazmi (1990) Cladocera of Hub lake with notes on species and size composition. Sarhad. J. Agri. (6): 85-88. Jafri, S.I.H., M.A., Mahar, and S.M., Leghari (1999) Diversity of fish and plankton in Manchhar lake (Distt. Dadu) Sindh, Pakistan. Proc. Semi. Aq. Biodiv. Pakistan. (eds. Q.B. Kazmi and M. A. Kazmi), 63-70. MRC and Department of Zoology, Univ. of Karachi. Kazmi, Q.B., (2000) Invertebrates of Indus Delta Eco-region. Proc. Consult. Workshop on "Indus Delta Eco-region (IDER). WWF Pakistan. 87-105. Mizuno, T., and E., Takahashi, (1991) An illustrated guide to freshwater zooplankton in Japan. Tokai Univ. press. P. 532. Mahar. M.A., S.I.H., Jafri, and W.A., Baloch. (2008) Seasonal variation and species composition of crustacean zooplankton (Order:Cladocera) in Manchhar Lake, Sindh. Pakistan. Pakistan J. Zool. (40): (2) 65-68. Pennak, R.W., (2001) Freshwater Invertebrates of the United State. 2nd Ed., John Willy and Sons, New York, U.S.A., p. 803. Tasneem, A.S., and A.S., Parvaiz, (1994) Seasonal fluctuation and species composition of freshwater plankton in keenjhar lake. Zoologica Pakistan (4): 31- 42. Ward, H. B., and G.C., Whipple (1959) Fresh Water Biology. 2nd ed., John Willey and Sons, London. 587-656.

Sindh Univ. Res. Jour. (Sci. Ser.) Vol: 40 (2) 19-26 (2008)

SURJ

Sindh Univ. Res. Jour. INFLUENCE OF Y2O3 PARTICLES AND DEFORMATION ON RECRYSTALLISATION OF ODS STEELS MA956 AND MA957 M. Moazam Baloch Department of Metallurgy and Materials Engineering, Mehran University of Engineering and Technology, Jamshoro, Sindh, Pakistan.

(Received 23rd August 2008 and Revised 19th October 2008)

Abstract

Commercialised mechanically alloyed yttria dispersion strengthened alloys exhibit unusual recrystallisation behaviour i.e. instead of recrystallising in an equiaxed grain structure they recrystallise in a manner which closely resembles that obtained by directional solidification, with the elongated columnar grains aligned along a particular sample axis. In spite of their large stored energy content, they tend to recrystallise at temperatures close to melting. The recrystallised microstructure is often very coarse and highly anisotropic, characterised by columnar grains. In the present paper influence of dispersed particles and deformation on recrystallisation behaviour of oxide dispersion strengthened MA956 and MA957 are investigated. It was found that degree of deformation prior to recrystallisation and manufacturing process, which tends to align the dispersoid particles along the principal fabrication direction, encourages recrystallisation to be directional. Keywords: Dispersion strengthened, recrystallisation, deformation, columnar grains, mechanical alloying.

Introduction Some of the most successful mechanically alloyed materials include the oxide-dispersion-strengthened (ODS) alloys are based on iron or nickel Capdevilas, et al., (2003) the mechanical alloying process has been described in detail else where, Benjamin, Gessinger (1984) and relatively recently by Bhadeshia (1997) Briefly, mechanical alloying involves the severe deformation of mixtures of powders until they form the most intimate of atomic solutions. Inert oxides can also be introduced to form a uniform dispersion of fine particles which strengthened the consolidated product Bhadeshia (1997). After mechanical alloying and processing into bulk form, the ODS ferritic stainless steels posses an ultra fine microstructure containing sub-micrometer sized grains of ferrite and a dislocation density of about1015m-2 Capdevila, (2001) and Bhadeshia.

1.

(2000) Chao, (2004) and Gonzalez the hardness in this condition is unacceptably high Chou (1993) and Bhadeshhia (2000) and contains very high levels of stored energy. Therefore, it has to be recrystallised into a coarse columnar grained microstructure before use Capdevila, and Bhadeshia. However, the recrystallisation behaviour of iron based ODS alloys is peculiar in that they recrystallise into a grain structure which resembles that obtained by directional solidification, with coarse, columnar grains having their longest axis along the extrusion direction Baloch, (1991) and Bhadeshia (2000). and Furthermore, recrystallisation usually does not occur until temperatures close to melting are reached Baloch, Elongated grains are desirable where the creep resistance at high temperatures is of paramount importance and iron based oxide-dispersion-strengthened alloys exhibit a high creep strength at temperatures from 900 to 1100°C Miller etal., and Wasilkowaska et al., (2003).

M. Moazam Baloch

20 for alloy MA957 specimen were cut in the rectangular shape with the final dimensions of 2.5×4×20mm, with the length direction parallel to the rolling direction and others were prepared with the long dimension perpendicular to the rolling direction.

Alloy MA956 MA957 Cr 0.014 19.88 0.01 14.29 Al 4.52 1.01 Ti 0.04 O 0.21 Y2O3 0.52 0.27 Balance Fe Fe

Oxide dispersion strengthened Fe-based alloys are the only materials presenting jointly two important properties: (a) a high dimensional stability under irradiation, i.e. high resistance to swelling and irradiation creep, due to their bcc structure, and (b) a potential high temperature strength resulting from the homogeneous dispersion of nanometric oxide particles Almo et al,(2004). and Haken et al., (2007) in the case of oxide dispersion strengthened ferritic alloys, the main difficulty could be the anisotropy in mechanical properties resulting from the anisotropy of microstructure developed during fabrication. In the present work, the effect of yittrium oxide particles and deformation is examined in as received/deformed condition and after recrystallisation at various temperatures. The microstructural investigations were performed using transmission electron microscope and light optical microscope. 2. Material

Oxide dispersion strengthened ferritic stainless steels Incoloy MA956 and MA957 were supplied by INCOMAP (Wiggin Alloys Ltd. United Kingdom) in "hot-rolled" condition, their chemical composition is given in Table 1. The alloys were fabricated by charging three primary powders (elemental iron, prealloyed metallic alloys, and yittria) into a water cooled vertical attritor for the mechanical alloying. The consolidation of the resultant powder was achieved by extrusion at 1000°C while packed in a mild steel can. This was followed by a rolling at 1000°C, with a reduction in diameter from 54mm diameter to 25mm diameter (an 54% reduction in area) being achieved in one pass and from 54mm diameter to 9.5mm diameter (an 82% reduction in area) being achieved in one pass for oxide-dispersion-strengthened Incoloy MA956 and MA957 respectively. The extrusion and rolling are not to be thought of as "hot-working" processes, since the microstructure in as received condition is in the cold deformed state with a grain size of 0.45µm and 0.4µm for MA956 and MA957 respectively. For further work specimens for alloy MA956 were cut in the form of square rods of dimension 5×5×20mm and

C

Table 1. Chemical composition (wt%) of Inconel Alloys MA956 and MA957. 3. Experimental techniques 3.1 Zone annealing Zone annealing experiments were carried using a crystal grower as the way of achieving directional recrystallisation. The specimens were placed in a silica tube, mounted on a carriage driven by a variable speed electric motor. A Pt/Pt-13wt%Rh thermocouple was used to measure the temperature during operation, with one end of thermocouple in the middle of the specimen and the other end attached to a Comark (a device which converts electrical potential to temperature) for measuring the temperature during the movement of the specimen through the coil. The zone annealing experiments were performed in air. A schematic diagram of the crystal grower adapted and used is shown in (Fig. 1).

Fig. 1. Schematic diagram of crystal grower, used for zone annealing experiments.

Influence of Y2O3 particles and deformation 3.2 Microscopy Mechanically polished samples were etched for optical microscopy using a mixture of 2g CuCl2 in 40ml HCl and 40-80ml ethanol. Thin foils for transmission electron microscopy (TEM) were prepared from 0.25mm thick discs machined from the sample concerned. After reducing the thickness to 0.05mm by abrasion on SiC paper, the samples were electropolished using a Fishione twin jet unit. The polishing solution consisted of 20 vol-% perchloric acid in methnol and was used at ambient temperature with the polishing potential set to 25-30 V. Unless otherwise stated, all the microscopy reported on the zone annealed samples was conducted on the longitudinal section, where the anisotropy of grain structure is most obvious. The hardness tests were performed using Vicker's hardness testing machine. A load of 10kg applied for each indentation. 4. Results The manufacturing process of oxide dispersion strengthened alloys MA956 and MA957 as described in section 2 involves rolling at a temperature of 1000ºC, with a reduction in diameter from 54 mm to 25 mm (an 54% reduction in area) and from 54 mm to 9.5 mm diameter (an 82% reduction in area respectively, being achieved in one

21 pass. On examining the thin foils, a true cold deformed structure was observed with a very high dislocation density in the as received condition, as shown in (Fig. 2). From (Fig. 2a), which was recorded during the examination of the thin foils prepared from the transverse section of the bar of alloy MA956, an equiaxed grain structure (consistent with the circular section of the extruded bar) can be seen, by contrast, (Fig. 2b) longitudinal section revealed an elongated grain structure, with tangled dislocation net works. Where as thin foils examined for alloy MA957 from transverse and longitudinal sections exhibited slightly anisotropic grain structure in the transverse section of the bar (Fig.2c) but, highly elongated grains for the longitudinal section (Fig.2d). The microstructure was found to be consistent with a cold-deformed state, with an obviously high density of dislocation.

The hardness measured in the as received condition was also found to be very high i.e. 350 and 410 HVN (10kg) for alloys MA956 and MA957 respectively, again consistent with a cold deformed microstructure; fully annealed iron can have a hardness of less than 200 HVN (10kg).

a.

b.

c.

d.

Fig. 2.Transmission electron micrographs reveals the microstructure of alloys MA956 and MA957 in the as-received condition. a and b are from transverse and longitudinal M. Moazam Baloch sections of MA956 and c and d are from transverse and longitudinal sections of MA957. 22

Experiments were performed to directionally recrystallise the samples of MA956 at various

peak temperatures (TP) ranging from 1150, 1180 to 1380ºC in a temperature gradient using crystal grower as a means to achieve columnar grain structure with various specimen travel speeds (0.2, 0.4, 0.8, 1.4, 3.2, 5.0, 7.7 and 10.0 mm/min). Zone annealing at TP 1150ºC failed to produce any substantial change in the optical microstructure (Figure 3) but only a very small amount of directional recrystallisation could be seen at even the lowest travel speed (0.2mm/min) utilized, and the overall hardness showed a little change relative to the hardness before annealing (322 HVN (10kg). This observation suggested that either a further lowering of specimen travel speed (i.e., less than 0.2mm/min, which was not possible with our equipment) or an increase in temperature can successfully produce columnar grains in ODS MA956.

grains were found to be straight along the length of specimen and as long as the sample size. Zone annealing experiments were also performed on an identical ODS ferritic stainless steel MA957 at peak temperatures (TP) ranging from 1150 to1350ºC using various specimen travel speeds (0.2, 0.4, 0.8, 1.4, 3.2, 5.0, 7.7 and 10.0 mm/min).

Fig. 4. Optical micrographs recorded after zone annealing MA956 at 1280ºC. a. 0.8mm min-1 b. 1.4mm min-1 c. 3.2mm min-1 d. 5.0mm min-1 e. 7.7mm min-1 f. 10.0mm min-1

Fig. 3. Optical micrographs recorded after zone annealing specimens at TP 1150ºC. The micrographs are from

the regions where TP was measured. a. Specimen travel speed 0.2mm min-1. b. Specimen travel speed 5.0mm min-1 (Alloy MA956) c. Specimen travel speed 0.8mm min-1. d. Specimen travel speed 5.0mm min-1 (Alloy MA957)

In order to resolve the ambiguities further zone annealing experiments were performed at higher peak temperatures i.e. TP 1280 and 1380ºC. The optical microstructure recorded after these experiments are shown in figures 4 and 5. From Figures 4 and 5, it can be clearly seen that the specimens of alloy MA956 are fully directionally recrystallised at minimum specimen travel speeds used (0.4 and 0.8 mm/min at 1280 and 1380ºC respectively) and the hardness values measured dropped significantly i.e. 288 and 248 HVN(10kg) at 1280 and 1380ºC respectively and the columnar

Fig. 5. Optical micrographs recorded after zone annealing MA956 at 1380ºC. a. 0.8mm min-1 b. 1.4mm min-1 c. 3.2mm min-1 d. 5.0mm min-1 e. 7.7mm min-1 f. 10.0mm min-1

The minimum peak temperature applied to directionally recrystallise the samples in a temperature gradient (i.e. TP 1150ºC) does not seems to alter the microstructure as can be seen

Influence of Y2O3 particles and deformation

23

from Figure 3B, and the hardness measured turn out to be 374HVN (10kg). Since the optical microstructure at TP 1150ºC resembles the microstructure obtained in the as-received / deformed condition but slightly lower hardness value as mentioned above suggest some kind of recovery in the material at this temperature. As the zone annealing experiments performed at relatively higher temperatures (TP 1250 and 1350ºC) and the micrographs recorded show complete directional recrystallisation with a specimen travel speed of 0.2 mm min-1 at 1250ºC, with a substantial drop in measured hardness value (i.e. 178 HVN 10kg) but at relatively faster specimen travel speeds (0.4 and 3.2 mm min-1) partial directional recrystallisation was observed (Figure 6A). As the temperature raised to maximum peak temperature (TP 1350ºC), used to zone anneal the specimens of ODS alloy MA957 found to directionally recrystallised at even faster specimen travel speeds (Fig. 6B) and hardness value measured were decreased further as161, 164, 167, 167, 201 and 293 HVN (10kg) for specimen travel speeds of 0.8, 1.4, 3.2, 5.0, 7.7 and 10.0 mm min-1 respectively.

Fig. 6B. Optical micrographs recorded after zone annealing MA957 at 1350ºC. a. 0.8mm min-1 b. 1.4mm min-1 c. 3.2mm min-1 d. 5.0mm min-1 e. 7.7mm min-1 f. 10.0mm min-1

The optical microstructure recorded from the samples zone annealed at various peak temperatures exhibit columnar grains with an appearance of cast ingot type grain structure, an effect which was thought to be due to dominance of grains growing from the surface It may be argued, that the unidirectional of the sample during zone annealing. grain growth takes place during directional recrystallisation in oxide dispersion strengthened ferritic steels MA956 and MA957 is a consequence of the fabrication process. An attempt was made to analyse the effect of yittrium oxide (Y2O3) particle alignment on the recrystallisation of MA956. Alignment that can be seen in (Fig. 7), which was taken from an unetched specimen zone annealed at TP 1280ºC and 1.4 mm min-1. It is apparent that some of the particles are aligned along the deformation direction, the spacing between adjacent rows of aligned particles being approximately equal to the maximum lateral spacing observed between two adjacent grain boundaries in directionally recrystallised specimens.The transmission electron micrograph shown in (Fig. 8A), further confirms the effect

Fig. 6A. Optical micrographs recorded after zone annealing MA957 at 1250ºC. a. 0.2mm min-1 b. 0.4mm min-1 c. 0.8mm min-1 d. 1.4mm min-1 e. 3.2mm min-1 f. 5.0mm min-1

M. Moazam Baloch

24

of particles on the columnar grain growth process for MA956. The micrograph further reveals that very fine and relatively coarser Y2O3 particles are aligned along the grain boundary, which was found after considerable effort because of large grain size obtained after zone annealing.

An examination of thin foils prepared from the longitudinal section of directionally recrystallised sample of ODS MA957, revealed tendency for particle alignment along the working direction (Figure 8B), although the tendency for alignment appeared less than in alloy MA956. This could be due to the relatively small additions of Y2O3 particles (0.27 wt%) (Table 1), compared with MA956 which contains 0.52 wt% Y2O3 particles. There is an obvious tendency for grain boundary to be pinned by the particles, although the pinning is not adequate to completely stop recrystallisation. However, the effect of the Y2O3 particles was evident in the observation of small regions of unrecrystallised material pinned and left behind as the remainder of the recrystallisation front advanced relatively rapidly (Fig. 9).

Fig. 7A. Showing alignment of particles along the rolling direction, recorded from an unetched sample of MA956.

A.

B.

Fig. 8. (A) Transmission electron micrographs of the MA956 samples zone annealed at 1280ºC at a specimen travel speed of 3.2mm min-1. (B) Transmission electron micrographs of the MA957 samples zone annealed at 1250ºC at a specimen travel speed of 0.2mm min-1. Note the degree of particle alignment along the rolling direction in both MA956 and MA957.

Influence of Y2O3 particles and deformation

25

As completely different grain structure i.e. cast ingot type and straight and long grains extending to the length of specimen, was observed for two identically processed ferritic steels MA957 and MA956 respectively, and heat treated under almost similar conditions, it was thought necessary to measure the activation energy, stored in the material in the form of defects (i.e. dislocations).

Fig. 9. Transmission electron micrographs of the MA956 samples zone annealed at 1280ºC at a specimen travel speed of 3.2mm min-1. Streams of unrecrystallised grains are seen to be trapped between two fully recrystallised, "dislocation free" grains.

Using experimental data obtained after zone annealing experiments at two different peak temperatures and a range of specimen travel speed for both the materials individually, the activation energy Q required to directionally recrystallise was measured. The measured value of activation energy for alloy MA957 was found to be 654257 J mol-1 which is much larger than that of alloy MA956 (402791 J mol-1). This significant difference in the measured activation energies of two identically processed alloys (MA957 and MA956) may be due to the amount of deformation applied to each alloy 82% and 54% reduction respectively. Although MA957 has a lower oxide content, the tendency for particle alignment, as discussed above, is smaller, so that grain boundary mobility along the rolling direction may be more effectively impeded. Conclusions The influence of dispersed yittrium oxide particles and deformation on directional recrystallisation behaviour of two oxide dispersion strengthened ferritic steels MA956 and MA957 processed in identical conditions, were investigated and following conclusions were made: 1. The recrystallisation behaviour of alloys MA956 and MA957 is highly influenced by the fabrication process. 2. Strong alignment of Y2O3 particles along the rolling direction for MA956, was found to boost the grains to grow unidirectionally, whereas, smaller particle alignment tendency was found to appear in MA957. 3. The activation energy measured is MA957 (654257 J mol-1) was much larger than that of alloy MA956 (402791 J mol-1) which may be due to the amount of deformation applied to each alloy 82% and 54% reduction respectively. References Alamo, A., V. Lambard, X. Averty, and M. H. Mathon, (2004) "Assessment of ODS-14% Cr ferritic alloy for high temperature applications". Journal of Nuclear Materials, vol. (329): 333-337. Benjamin, J. S., (1970) "Dispersion strengthened superalloys by mechanical alloying". Metallurgical Transactions, vol. (1): 2943-2951. Bhadeshia, H.K.D.H., (1997) "Recrystallisation of Practical Mechanically Alloyed Iron-Base and Nickel-Base Superalloys". Materials Science and Engineering, A233, 64-77. 5.

Bhadeshia, H.K.D.H., (2000) "Directional Recrystallisation and its Exploitation in Mechanically Alloyed Metals". In Proceedings of the 21st Riso Symposium on Materials Science, eds. N. Hansen, X. Huang, D. Juul Jensen, E. M. Lauridsen, T. Leffers, W. Pantleon, T. J. Sabin and J. A. Wert, published by Riso International Laboratory, Denmark, 15-28.

M. Moazam Baloch 26

Baloch, M. M., and H.K.D.H. (1991) Bhadeshia, "Directional recrystallisation in a nickel-base ODS superalloy". Materials Science and Technology, vol. (6): 1236-1246. Baloch, M. M. (1989) "Directional Recrystallisation in Dispersion Strengthened Alloys" Ph. D. Thesis, University of Cambridge. UK. Capdevila, C., Y. L. Chen, A.R Jones, and H.K.D.H. (2003) Bhadeshia, "Grain Boundary Mobility in Fe-Base Oxide Dispersion Strengthened PM2000 Alloy" ISIJ International, vol. (43): 777-783. Capdevila, C., and H.K.D.H Bhadeshia,. (2001) "Manufacturing and Microstructural Evolution of Mechanically Alloyed Oxide Dispersion Strengthened Superalloys". Advanced Engineering Materials, vol. (3): 647-656. Chao, J., and J.L.Gonzalez-Carrasco, (2004) "On the differences of the 475ºC age hardening between as-hot rolled and recrystallised MA956 alloy". Scripta Materiala, (50): 1457-1460. Chou, T.S., and H.K.D.H Bhadeshia,. (1993) "Grain control in mechanically alloyed oxide dispersion strengthened MA957 steel". Materials Science and Technology, vol. (9): 890-897. Capdevila, C., and H.K.D.H. Bhadeshia, (2000) "Deformation and Recrystallisation of Yittria Dispersion Strengthened, Mechanically Alloyed Iron (MA957)". In Proceedings of the 21st Riso Symposium on Materials Science: Fundamental Aspects and Relations to Deformation Microstructure, editors: N. Hansen, X. Huang, D. Juul Jensen, E. M. Lauridsen, T. Leffers, W. Pantleon, T. J. Sabin and J. A. Wert, published by Riso International Laboratory, Roskilde, Denmark, 1-6. Gessinger, G. H., (1984) "Powder Metallurgy of Superalloys", Butterworth and Co., London. Hakan Ates, Mehmet Truker and Adem Kurt, (2007) "Effect of friction pressure on the properties of friction welded MA956 iron-based superalloy". Materials and Design, vol. (28): 948-953.

Miller, M. K., D. T. Hoelzer, E. A Kenik, and K.F. Russel, (2005) "Stability of ferritic MA/ODS alloys at high temperatures". Intermetallics, Vol. (13): 387-392. Wasilkowska, A.,M. Bartsch, U. Messerschmidt, R. Herzog, and Czyrska-Filemonowicz, (2003) "Creep mechanism of ferritic oxide dispersion strengthened alloys". Journal of materials Processing Technology, vol. (133): 218-224.

Sindh Univ. Res. Jour. (Sci. Ser.) Vol: 40 (2) 31-36 (2008)

SURJ

Sindh Univ. Res. Jour. APPLICATION OF QUEUEING THEORY TO SIGNALS SYSTEM AT TWO BUSY CROSS SECTIONS OF CITY HYDERABAD: A SIMULATION APPROACH Noor Ahmad Shaikh Institute of Mathematic and Computer Science, University of Sindh, Jamshoro

(Received 23rd April 2008 and Revised 11th August 2008) Abstract This paper attempts to analysis a traffic system and adjustment of signal timings at Thandi Sarak, Hyderabad and then it suggest a signal timing at another cross section of road (next to it) situated at National Highway and Wadhoo Wah road intersection near Rani Bagh. A program in GPSS is developed in order to simulate a queuing system depicting signal timings during rush hours. Each simulation run is essentially an experiment on the system. The advantage of simulation is that these experiments can be completely controlled and completely observed. Effective signal timings are suggested to reduce queue length (traffic congestion). Keywords: application of queuing signals system reduce queue length

1.

Introduction Simulation is a very powerful method for solving traffic flow problems because of its wide applicability and because it provides a laboratory to study systems without the costs of building or modifying the real system. The objective of this paper is to analysis an existing traffic signal system and suggests the traffic light system at another cross section of the road to effectively control/manage the traffic flow there. To achieve this goal, two simulation programs are written in General Purpose Simulation System (GPSS) and obtained results from these programs are mainly discussed. Queuing theory one of the diversified field of applicable mathematics that is widely used in many areas including Operation Research, telecommunications, production systems, computer science and Road Management/Engineering. In 2005 an American study revealed that there might be seven root causes of congestion on roads, poor signal timing is one of them. Various authors studied traffic analysis both analytical as well as with simulation approach such as Brigham (1995), Bhatt and Raos (1972), Hoover and Perry (1989), Lo and Chow (2004), this paper is organized as follow

Description is given in sections 2. Sections 3 describe some aspects of GPSS as a simulation language. Simulation program of two cross sections (Round about) of the Road in GPSS are given in section 4 and 5. Results are discussed in section 6. 2. Description (Fig. 1) shows the first round about, where as (Fig. 2) is the representation of second round about, where currently there is no signals system available. Due to the absence of signals drivers have to face little difficulty, when they cross this round about. (Table-1 and Table-2) shows the statistical data for both models respectively. (Fig. 3) shows the whole model connecting both rounds about and suggests the signals position at second round about. Second round about is situated at National Highway between Wahdat Colony round about and Qasim Chouk round about. At this round about there is no traffic light system. Due to this drivers have to face lot of trouble, when they cross this intersection. There is lot of risk of accidents due to the absence of traffic signals. (Table-1) shows the statistical data of vehicles arriving at this intersection from various points within one minute. If a traffic light system is established, the risk of accidents can be reduced and as well as the time of journey can be reduced for those who are crossing this intersection.

N. A. Shaikh

32

Below is given the statistical data collected through observations. (Table-1 and Table-2) shows data for above both models

Date/day Time Wahdat Col to Gul Center (Street-1) 60 to 80 veh in 18 sec. 40 to 50 30 to 45 50 to 70 Qasimabad to Wahdat Colony/Kotri 15 To 20 Veh in 10 sec. 10 to 15 10 to 15 12 to 18

respectively. Simulation programs use that data to generate results.

Table-1. Shows the statistical data on first round about

Qasimabad to Latifabad 30 To 35 Veh in 12 sec. 20 To 30 20 to 30 25 to 30 Latifabad to Qasimabad 30 to 40 Veh in 12 sec. 15 to 25 15 to 25 30 to 40 G.O.R to Qasimabad 8 To 12 in 8 Sec. 5 to 10 5 to 10 10 to 15 Jamshed S. Munawar Ali Shah Amjad Ali Noonari Observer

03-01-2008 Thursday 09-01-08 Wednesday

6:15 to 6:50

Mahboob Ali

6:45 to 7:20 8:30 to 9:10

16-01-08 Wednesday

6:30 to 7:00

Table-2. Shows the statistical data on second round about

Date/day Time Shahbaz Bld to Naseem Nagar 30 to 40 veh cros tm: 1 min 35 to 45 veh cros tm:1 min 0.5 to 1.5 sec/vehicle 12 sec Naseem Nagar to Shahbaz Bld 25 to 30 veh cros tm: 1 min 30 veh cros tm: 1 min 1 to 3 sec/vehicle 12 sec Qasimabad/Wahdat Col to Qasim chok 40 to 50 veh cros tm: 1 min 40 to 45 veh cros tm: 1 min 0.5 to 1.5 sec/vehicle 15 sec Qasim Chok to wahdat colony/qasimabad 40 to 50 veh cros tm: 1 min 35 to 45 veh cros tm: 1 min 0.5 to 1.5 sec/vehicle 15 sec Observer

05-01-2008 Saturday 07-01-2008 Monday Arrival rate Proposed Signal duration

5:40 PM 7:15 to 8:15

Maqsood Ali Jalbani Ayaz Ali

Fig. 1. This figure represents the traffic flow environment at the round about situated between Shahbaz Building and Rani Bagh in Hyderabad city.

THANDI SARAK THANDI SARAK

G.O.R ROAD GOES TO/COMES FROM LATIFABAD

ROAD GOES TO QASIMABAD/NASEEM NAGAR

G.O.R ROAD GOES TO/COMES FROM LATIFABAD ROAD COMES FROM QASIMABAD/NASEEM NAGAR

THANDI SARAK

THANDI SARAK

Application of queueing theory to signals system... Fig. 2. This figure represents the traffic flow environment at the round about situated at National Highway ­ between Shahbaz Building round about and Naseem Nagar round about.

33

WADHOO WAH ROAD COMES FROM SHAHBAZ BUILDING

ROAD COMES FROM WAHDAT COLONY

ROAD GOES TO WAHDAT COLONY

WADHOO WAAH

WADHOO WAH ROAD GOES TO SHAHBAZ BUILDING

ROAD GOES TO NASEEM NAGAR / BY PASS

WADHOO WAAH

ROAD COMES FROM NASEEM NAGAR / BY PASS

Fig. 3. This figure represents the traffic flow environment at both the round about.

N. A. Shaikh

34

3. General Purpose Simulation System (GPSS): The GPSS[6] is chosen as the simulation language, because of its availability and ease of handling simulation problems similar to one under investigation. GPSS has much of the underlying logic of system simulation built-in. some of the basic features of GPSS are as follows: 1. The ability to sample from all well known distributions. 2. The ability to define and sample from empirical distributions. 3. A built-in simulated clock. 4. Automatic generation of queueing logic and statistics at points defined in the system. GPSS programs for both models are written according to the statistical data obtained during several observations. Below is given the code and description of first GPSS programs that is written for figure-1 model above. 4. Computer programme (for fig. 1) simulate ***** Light Segment ****** generate ,,,1 ***Signal lights for street-1. begn1 seize grenS1 advance 18 release grenS1 seize grenR1 advance 12 release grenR1 ***Signal lights for street-2. seize grenS2 advance 12 release grenS2 seize grenR2 advance 10 release grenR2 ***Green light for street-3 for both straight and right turn. seize grnSR3 advance 8 release grnSR3 goto begn1 **************************************** ************ Street 1 segment ************* **************************************** *** Vehicles to go straight from street1 *** generate 2,1 arrive strt1S if grenS1=nu,waitS1

*time consumed by vehicle to cross the intersection (signal area) advance 2,1 goto joinS1 waitS1 waitif grenS1=nu *time consumed by vehicle to cross the intersection, if it waited for green signal advance 4,2 joinS1 depart strt1S terminate ***vehicles to turn right from street1*** *vehicle arrive every 10 to 20 sec to turn right generate 1 arrive Strt1R if grenR1=nu,waitR1 *time consumed by vehicle to turn right from intersection advance 3,1 goto joinR1 waitR1 waitif grenR1=nu *time consumed by vehicle to turn right from intersection, if it waited for green signal advance 4,2 joinR1 depart Strt1R terminate **************************************** *********** Street 2 segment ************** **************************************** ***vehicles to go straight from street2*** *vehicle arrive every 5 to 9 sec to cross straight generate 1 arrive strt2S if grenS2=nu,waitS2 *time consumed by vehicle to cross the intersection (signal area) advance 2,1 goto joinS2 waitS2 waitif grenS2=nu *time consumed by vehicle to cross the intersection, if it waited for green signal advance 4,2 joinS2 depart strt2S terminate ***vehicles to turn right from street1*** *vehicle arrive every 15 to 25 sec to turn right generate 2,1 arrive Strt2R if grenR2=nu,waitR2 *time consumed by vehicle to turn right form intersection advance 2,1

Application of queueing theory to signals system...

35

goto joinR2 waitR2 waitif grenR2=nu *time consumed by vehicle to turn right from intersection,if it waited for green signal advance 4,2 joinR2 depart Strt2R terminate **************************************** *********** Street 3 segment ************** **************************************** ***vehicles to go straight from street2*** *vehicle arrive every 10 to 20 sec to cross straight or turn right generate 2,1 arrive str3SR if grnSR3=nu,watSR3 *time consumed by vehicle to cross the intersection (signal area) advance 4,2 goto jonSR3 watSR3 waitif grnSR3=nu *time consumed by vehicle to cross the intersection, if it waited for green signal advance 5,2 jonSR3 depart str3SR terminate ***** stop segment ***** *simulation continues for 4 hours generate 14400 terminate 1 start 1 end Below is given the code and description of second GPSS programs that is written for figure-2 model as mentioned above. 5. Computer Programme (for fig.2) simulate ********* Light Segment ********* generate ,,,1 ***Green Signal for Wahdat Colony to Qasim Chouk/Shahbaz Building begn1 seize grenS1 advance 16 release grenS1 ***Green Signal for Qasim Chouk to Wahdat Colony/Naseem Nagar seize grenS2 advance 16 release grenS2 ***Green Signal for Shahbaz Building to Naseem Nagar/Qasim Chouk

seize grenS3 advance 12 release grenS3 ***Green Signal for Naseem Nagar to Shahbaz Building/Wahdat Colony seize grenS4 advance 12 release grenS4 goto begn1 ************************************* ********** Street 1 segment ************ ************************************* *** Vehicles to go straight/Right turn from Wahdat Colony To Qasim Chouk*** generate 1,0.5 arrive stret1 if grenS1=nu,waitS1 *time consumed by vehicle to cross the intersection (signal area) advance 0.5 goto joinS1 waitS1 waitif grenS1=nu *time consumed by vehicle to cross the intersection, if it waited for green signal advance 1,0.5 joinS1 depart stret1 terminate ************************************* *********** Street 2 segment ********* ************************************* *** Vehicles to go straight/Right turn from Qasim Chouk to Wahdat Colony*** generate 1,0.5 arrive stret2 if grenS2=nu,waitS2 *time consumed by vehicle to cross the intersection (signal area) advance 0.5 goto joinS2 waitS2 waitif grenS2=nu *time consumed by vehicle to cross the intersection, if it waited for green signal advance 1,0.5 joinS2 depart stret2 terminate **************************************** *********** Street 3 segment ******* ************************************ *** Vehicles to go straight/Right turn from Shahbaz Building to Naseem Nagar *** generate 1,0.5 arrive stret3 if grenS3=nu,waitS3

N. A. Shaikh

36

*time consumed by vehicle to cross the intersection (signal area) advance 0.5 goto joinS3 waitS3 waitif grenS3=nu *time consumed by vehicle to cross the intersection, if it waited for green signal advance 1,0.5 joinS3 depart stret3 terminate ************************************* ********* Street 4 segment ************* ************************************* *** Vehicles to go straight/Right turn from Shahbaz Building to Naseem Nagar *** generate 2,1 arrive stret4 if grenS4=nu,waitS4 6.

*time consumed by vehicle to cross intersection (signal area) advance 0.5 goto joinS4 waitS4 waitif grenS4=nu *time consumed by vehicle to cross intersection, if it waited for green signal advance 1,0.5 joinS4 depart stret4 terminate ***** stop segment ***** *simulation continues for 4 hours generate 14400 terminate 1 start 1 end

the

the

Results and Discussion Proposed time for this signal is given in the view of observations. First it should remain green for 16 seconds for vehicles to pass straight and right turn from Wahdat Colony to Qasim Chouk/Shahbaz Building round about, and then it remains green for 16 seconds for vehicles to pass straight/right turn from Qasim Chouk to Wahdat Colony/Naseem Nagar round about. After that it remain green for 12 seconds to allow vehicles to cross straight and right turn from Shahbaz Building to Naseem Nagar/Qasim Chouk round about, then it remain green for 12 seconds to allow vehicles straight and right turn from Naseem Nagar to Shahbaz Building/Wahdat Colony round about. Note that these timings are suggested specially for rush hours in the evening. First cross section of the road is situated at Thandi Sarak between Wahdat Colony round about and Gul Center round about. First it remain green for 18 seconds for vehicles to pass straight to/from Wahdat Colony and Gul Center, then it remain green for 12 seconds to allow right turn for both sides. After that it remain green for 12 seconds to allow vehicles to cross straight between Latifabad and Qasimabad Road, then it remain green for 10 seconds to allow right turn for vehicles at same sides. Finaly it remain green for 8 seconds to allow G.O.R side vehicles to go straight or right turn. Remember this information of green light is taken for rush hours (evening hours), the signal duration changes some seconds several times in a day for each side. 7. Acknowledgement The author expresses his thanks to Mr. Yasir Arafat Malkani, Lecturer, at Institute of Mathematics and Computer Science, University of Sindh, Jamshoro, for his help. Reference Brigham. G., (1955) "On a Congestion Problem in an Aircraft Factory" J. Oper. Res. Soc. Amer, (3): 412-428. Bhatt, U. V., and S.S. Rao, (1972) "A Statistical Technique for the Control of Traffic Intensity in the Queuing System M/G/I and Gt/M/I" Oper. Res. (20): 955-966. Hoover, S.V., and R.F. Perry, (1989) Simulation: a Problem-Solving approach, reading, M.A. Addison Wesley. Ingolf Stahl: Introduction to Simulation with GPSS Prentice Hall, U.S. Lo. H. K., and A.H.F Chow, (2004) Control Strategies for over Saturated Traffic, J. of Transportation Eng. 130 (4): pp 166.

Traffic Congestion and Reliability, trends and Advanced Strategies for Congestion Mitigation U.S. Federal Highway Administration, (2005).

Sindh Univ. Res. Jour. (Sci. Ser.) Vol: 40 (2) 37-40 (2008)

SURJ

Sindh Univ. Res. Jour. ECOLOGICAL STUDIES ON FRESHWATER GASTROPODS (SNAILS) OF INDUS RIVER AND ITS CANALS AT KOTRI BARRAGE SINDH, PAKISTAN G. H. Burdi, W. A. Baloch, F. Begum, A. N. Soomro and M. Y. Khuhawar* Department of Fresh Water Biology and Fisheries, University of Sindh, Jamshoro 76080,

(Received 16th October 2008 and Revised 21st November 2008)

Abstract

Occurrence of freshwater gastropods in Indus River and its canals at Kotri Barrage, near Jamshoro, Sindh were studied from (October 2005 to September 2006). Sampling area included five stations namely, Pinyari Canal, Phuleli Canal, K.B. Feeder, Chakumber (Ring Dam) and River Indus (downstream). A total of seven genera including 10 species of gastropods were identified in which Bellamya bengalensis being most dominant. Gastropod populations were fairly good and more or less same numbers of individuals were recorded at K.B. Feeder, Pinyari Canal Phuleli Canal and Chakumber. The Indus River (downstream) was very poor in live gastropods, where only dead shells were found. The lowest temperature was in January (16.7 °C) while highest being in August (32.5 °C). Lowest D.O was 5.0 mg/l at Chakumber in August and highest was 6.2 mg/l in January. TDS and conductivity were higher at Chakumber in January. Water transparency was very low (2.0 cm) throughout all stations during flood season (July­September) and higher (58.0 cm) during dry season (January-March). The Chakumber was more transparent than other stations because of its shallow and stagnant water. The pH ranged in between 7.4 to 8.2 whereas hardness ranged between 70 mg/l to 130 mg/l. The gastropod population showed a direct relationship with the temperature and strong correlation was evident. On the other hand pH, hardness and secchi transparency showed a negative correlation with the population density. Keywords: Gastropoda, occurrence, ecology, Indus River.

of the freshwater mollusks of Pakistan in her unpublished thesis. Khatoon and Ali (1978) also worked on Mollusca. A detailed work on taxonomy of bivalves was done by Begum and Nazneen (1991, 1992a, 1992b). These researchers have mostly concentrated on taxonomical studies and literature on ecological studies on gastropod fauna of Pakistan is not available. 2. Materials and Methods Sampling area Kotri Barrage is the last barrage on The distribution and diversity of Indus River to irrigate Phuleli, Pinyari and K.B. freshwater snails depend on their abilities of Feeder through lined channels and enhance colonization in a habitat and survival. That is agriculture in the lower Sindh region. regulated by the physico-chemical factors like Five stations were fixed for the sample temperature, hardness, pH, and pollution. collection. These included 1. K.B Feeder 2. Previous work on mollusks of East and Pinyari canal 3. Phuleli canal, 4. Indus River West Pakistan including marine mollusks was (downstream) and 5. Chakumber (Ring Dam) undertaken by Khan and Dastagir (1971) Later where water remains stagnant. Monthly samples on Tirmizi (1973) gave the taxonomic account *Dr. M.A. Kazi Institute of Chemistry, University of Sindh, Jamshoro, Pakistan (Oct. 2005 to Sep. 2006). were collected from 1. Introduction The gastropods play a vital role in the natural ecosystem. They purify water bodies because these are saprophytic animals. They eat algae, zooplankton and organic waste, and provide food for many types of fish, birds and also for human beings. They are passive indicators to environmental degradation. Some snails are of great importance for being intermediate hosts of infectious trematodes and other parasites of animals and human beings.

G. H. Burdi, et al.,

38

The specimens were collected by hand picking when or where water was shallow. During flood season however, fishermen were deployed for snail collection. The specimens were then taken to laboratory in wide-mouth plastic bottles. In the laboratory they were washed in freshwater and preserved in 70% alcohol. The identification of species was carried out using standard methods given by Preston (1915). Physico-chemical parameters were determined using digital meters were, temperature, E. conductivity and TDS were measured by using digital meter (WTW Cond 330i). Dissolved oxygen and pH were determined by (WTW Oxi 315i) and (Ecoscan pH5), respectively. The transparency was measured by secchi disk while hardness was determined by titration method in laboratory by EDTA method. 3. Results Water quality The temperature was lowest (16.5°C) in January that gradually increased and reached to highest (32.9°C) in August and September and decreased to the lower then after (Fig. 1a). An inverse proportion was evident in dissolved oxygen that was highest (6.2 mg/l) in January while lowest (5.0 mg/l) was noted in August (Fig. 1b). The TDS were more or less same in Main River Indus (down stream), Pinyari canal, Phuleli canal and K.B. Feeder, but in February highest TDS and conductivity were seen at Pinyari canal. In general among all stations Chakumber showed higher TDS and conductivity throughout whole year (Fig. 1c,d). The pH ranged from 7.4 to 8.2, where lowest being at Chakumber and Phuleli (September)

Name of species Bellamya bengalensis (Lamarck) Bellamya naticoides (Theobald) Thiara (Melanoides) tuberculata (Muller) Lymnaea acuminata chlamys Benson Lymnaea acuminata patula Troschel Lymnaea acuminata rufescens Gray Physa acuta Drapanaud Gabia arcula Benson Planorbis exustus Deshayes Gyraulus euphraticus (Mousson)

while highest at K.B. Feeder (August) and at Chakumber (June). The transparency was very high from January to April because there was no flood water. Highest transparency was 58.0 cm at Chakumber (Feb.) and it was lowest (2.0 cm) during July and August due to flooding at all stations except at Chakumber. The lowest hardness (70 mg /l) was noted in Jan. at Indus River (downstream) while it was highest in March and October (130 mg /l) at Pinyari canal. Gastropod populations A total of seven genera including 10 species of gastropods were identified (Table -1). The vivipaerids were abundant and very common throughout all stations. These included Bellamya bengalensis and Bellamya naticoides in which the former being most abundant. Genus Lymnaea was most diverse representing three species where Lymnaea acuminata chlamys was dominant among the three. On the population density basis however, it was secondly dominated genera (Table-1). Thiara tuberculata showed fair number of population density and ranked third in the community. The population of Physa acuta, Gabia arcula, Planorbis exustus and Gyraulus euphraticus were very scarce. Only dead shells of Gabia arcula and Gyraulus euphraticus were seen and no live specimen was collected. On the other hand live specimens of Physa acuta and Planorbis exustus were only collected from Chakumber station. The Indus River (downstream) was very poor in live snails and only dead shells were found. Whereas K.B. Feeder, Pinyari canal and Phuleli canal showed more or less equal number of individuals. Chakumber was also having low number of individuals.

Table-1. List of gastropod species and their average annual density.

St. 1 22.3 16.5 13.6 11.6 10.7 7.8 6.7 5.8 2.9 2.0 St. 2 20.2 12.8 12.8 10.6 9.6 9.6 7.4 8.6 5.4 3.2 Annual contribution (%) St. 3 St. 4 St. 5 25.9 16.9 23.4 17.3 13.6 17.8 13.6 13.6 14.9 12.3 11.8 13.1 9.9 8.6 10.3 8.6 16.9 8.4 6.2 6.7 6.5 3.7 8.6 3.7 1.2 1.6 0.9 1.2 1.6 0.9 Avg 21.7 15.6 13.7 11.9 9.8 10.3 6.7 6.1 2.4 1.8

St. 1 = K.B. Feeder, St. 2 = Pinyari, St. 3 = Phuleli, St. 4 = Indus, St. 5 = Chakumber, Avg = Average

Ecological studies on freshwater gastropods ...

39

Figure 1. Physico-chemical parameters.

Figure 2. Density versus water quality.

4.

Discussion The distribution of freshwater mollusks depends on their abilities to colonize a habitat and survive there. Their survival, in turn, is regulated by various physico- chemical factors that play major role to determine the ecological traits associated with a particular species. The factors like temperature, hardness, pH, altitude, size of water bodies and vegetation and pollution are among the significant aspects influencing the distribution and abundance of mollusks (Dillon, 2000). The population density was highest during June that showed a strong positive relationship (R = 0.777) with temperature (Fig. 2c).while a negative correlation with the hardness was evident (Fig. 2a). Chatterjee et al., (2008) found a same correlation of hardness and gastropods. The CaCO3 is generally considered to be utilized for shell construction and thus it has negative correlation with hardness. According to Russel-Hunter and Eversole, (1976) calcium salts in both food and water are important for growth of molluscan shell. The population density also showed a negative correlation with secchi transparency and pH. It indicated that in early monsoon the population was highest and it declined with highest transparency (Fig. 2b). The pH versus density showed (Fig. 2d) strong but negative correlation (R2= 0.623). Hardness and pH are considered important factors directly or indirectly influencing metabolic activities and thereby growth and abundance of freshwater mollusks (Eleutheriadis and Lazaridou-Dimitriadou, 1995). All the gastropods recorded are already documented from other water bodies of Pakistan (Begum and Nazneen, 1991, 1992a, 1992b). The vivipaerid (mystery-snail) females are equipped with a uterus in which they gestate the eggs until they become juvenile snails hence called viviparous. There were three species of Lymnaea altogether their number ranked them as secondly dominant species. Among three species Lymnaea acuminata chlymas was dominant

2

G. H. Burdi, et al.,

40

over other species. Lymnaeids are known to be distributed worldwide. They are host for larval stages of the helminthes parasites which cause diseases in man and domestic animals. In Pakistan however, the studies are restricted to taxonomical work only. Akhtar (1978) and Khatoon and Ali (1978) have described Lymnaea rufescens as separate species instead of a form (variety) of Lymnea acuminata. However, Preston (1915), Subha ­ rao and Mitra (1979), Subha ­ Rao et al., (1980), Goel and Sirivastava (1980), and Nazneen and Begum (1990) have described it as the form (variety) of Lymnaea acuminata. The living specimens of Physa acuta was first time recorded in 1990 from Layari River (Begum and Nazneen, 1990). The Physa snails are cosmopolitan and have been spread through human agency around the world (Dillon et al., 2002). Physa acuta is a "weedy" or R-selected in the sense of Dillon (2000). It is most common in lentic water bodies and was found live from the Chakumber station on algal mats. The dominance of viviparous (prosobranchs) and low number of pulmonates in these water bodies is justified because these water bodies are deeper, weed less and unpolluted. References Akhtar, S., (1978) On a collection of Freshwater Molluscs from Lahore. Biologia, (24): 437- 447. Begum, F., and S., Nazneen, (1991) Systematic study of Molluscan fauna of Layari River, Mesogastropoda (Suborder Taenioglossa). Bangladesh J. Zool., (19): 107-121. Begum, F., and S., Nazneen, (1992a) Systematic study of Molluscan fauna of Layari river Part-I. Archaeogastropoda (Aspidobranchia) from the Estuarine Region. Pak. J. Sci. Ind. Res. 35 10. Begum, F., and S., Nazneen, (1992b) Systemic study of Molluscan fauna of Layari river, part 7: Bivalves (Paleoheterodonta).The Philippine J. Sci. 121 (1): 121-133. Chatterjee, A., M., Jain, U. S., Roy and S. K., Mukhopadhayay. (2008) Limnochemical Factors influencing the Seasonal Population Density, Secondary Production, and Calcium-to-Tissue Ratio in the Freshwater Limpet Septaria lineate (Archaeogastropoda: Neritidae). Turk. J. Zool. (32): 245-252. Dillon, R.T. Jr., (2000) The Ecology of Freshwater Molluscs. Cambridge University Press, Cambridge, U.K. Dillon, R.T., Jr., A. R., Wethington, J. M., Rhett, and T. P., Smith, (2002) Populations of the European freshwater pulmonate Physa acuta are not reproductively isolated from American Physa heterostropha or Physa integra. Invert. Biol. 121(3): 226-234. Eleutheriadis, N., and M., Lazaridou-Dimitriadou. (1995a) Age related differential catabolism in the snail Bithynia graeca and its significance in the bioenergetics of sexual dimorphism. Malacologia (36): 139-146. Eleutheriadis, N., and M., Lazaridou-Dimitriadou. (1995b) Density and growth of freshwater snails (Bithynia graeca and Viviparous contectus) in relation to water chemistry in Serres, Northern Greece. J. Moll. Stud. (61): 347-352. Godan D.,(1983) Pest slugs and snails. Springer-Verlag, Berlin, Heidelberg, New York vi+445 Goel, H.C., and C.P., Srivastava. (1980) Freshwater snails of Gwaliar (M.P). J. Bombay Nat. Hist. Soc. (77): 215-222. Khan, M.D., and S.G., Dastagir. (1971) On the Mollusca, Gastropoda fauna of Pak. Rec. Zool. Surv. Pak. (1): 17-130.

[

Khatoon, S and S.R. Ali. (1978) Freshwater Mollusks of Pak. Bull. Hydrobiol. Res. Ser., (1): 518-525. Nazneen, S., and F., Begum. (1990) Systematic study of some freshwater gastropods of Sindh. Biologia, 36. Preston, B., (1915) A further report on Mollusca from Lake Chilka on the East coast of India. Res. Indian Mus., (11): 291-310.

Preston, H. B., (1915) The fauna of British India including Ceylon and Burma. Tailor and Francis Publisher, London. Russell-Hunter, W.D., and A.G., Eversole (1976) Evidence for tissue degrowth in starved freshwater pulmonate snails (Helisoms trivolvis) from carbon pollen nitrogen analys. Comp. Biocam. Phys. A. (54): 447-453. Tirmizi, F., (1973) Taxonomy of Freshwater Mollusca. M.Sc. Thesis, Karachi University.

Sindh Univ. Res. Jour. (Sci. Ser.) Vol: 40 (2) 41-50 (2008)

SURJ

Sindh Univ. Res. Jour. OPTIMIZATION STUDIES OF YEAST FEED FOR ENHANCED GROWTH FOR ETHANOL PRODUCTION BY SHAKE FLASK METHOD A. A. Noor and M. U. Dahot* Department of Microbiology, University of Sindh, Jamshoro

(Received 17th February. 2008 and Revised 13th April 2008) Abstract Yeasts are the industrially used microorganisms to provide the best possible combination of characteristics for the biological reactions with the given substrates. Yeast nutrition refers to how cells feed; translocate water and essential in-organic and organic nutrients from the ecosystem. Saccharomyces cerevisiae of bakery origin (strain S5) and soil origin wild strain (strain S6) were cultivated on nutrient agar and were activated on Saboraud's dextrose agar and Czepkdox agar, potato dextrose agar and malt extract agar by three subsequent inoculations for the maximum growth. Test strains S5 and S6 were optimized for their growth requirements in malt extract broth as initial medium; pH was maintained at 5.0 and incubated at 37°C for 72 hours, 110 rpm, 1.0 ml inocula size. Growth studies were under taken in three replicates after every 4 hours till 96 hours. Number of nutritional requirements were provided at the optimized concentrations in the form of yeast media 5 and 6 respectively and finally the growth was observed at 600 nm. The test strains grown on growth media (5 and 6) were also subjected for their tolerance to various chemical agents at the respective concentrations (ml v/v). Present study revealed the higher growth rate of S. cerevisiae strains S5 on Gm5 as compared to the strain S6 on Gm6 containing vitamins and amino acids as growth promoting factors at 110 rpm, pH 4.0, 0.5 and 1 ml. inocula size after 72 hours incubation by shake flask method which indicates that strain S5 is the best candidate for ethanol production. Keywords: Yeasts industrially, Yeast nutrition, Growth for ethanol production shake flask method

1.

* Institute of Biotechnology and Genetic Engineering, University of Sindh, Jamshoro

Introduction The industrial society developed by the accumulation of the scientific knowledge, the spread of technological innovations, and the exploitation of enormous natural resources (King and Clevedon, 1980). In the current epoch, the utilization of microorganisms in industrial processes or in process in which, their activities may become of industrial or technological significance that relates to the production of desired products including pharmaceuticals, organic acids, enzymes, organic solvents and synthetic fuels and also utilized to prevent their values (Presscott and Dunn, 1959, Zertuche and Zali, 1982). Yeasts are truly fascinating organisms, having 7000 species reported (Boekhout and Kurtzman, 1996; Hawksworth and Mouchacca, 1994). Yeasts, Saccharomyces cerevisiae are able to rapidly convert sugars to ethanol having a high

fermentation property, high show, high ethanol tolerance and osmotolerance, low pH, and high temperature optima, have high glycolytic, invertase activities and genetic stability (Codon and Bentez, 1985). 2. Methodology A-Isolation of cultures Saccharomyces cerevisiae cultures were received from local bakery (Strain-5) and the wild strain was isolated from 20 soil samples. Bakery strain was isolated by mixing bakery dough in the sterilized malt extract (ME) broth and incubated at 37°C for 12 hr. Later a loop full was inoculated on Sabouraud's dextrose agar (SDA) plate containing mycological peptone 10, dextrose 40, yeast extract 3 and agar-agar 20g / liter of distilled water, pH 5.6. Plates were incubated at 37°C for 24 hr to observe the

A. A. Noor and M. U. Dahot

42

Khoc cho nho thuong voi trong long, khoc cho noi sau nhe nhu khong. Bao nhieu yeu thuong nhung ngay qua da tan theo khoi may bay that xa... growth. Colonies were identified and maintained as pure culture in SDA slopes. Soil originated wild strain (S6) was isolated by pour plate method of Khan and Mahmood, (1992). 1ml of each suspension was poured over the surface of SDA agar plates containing 400mg of griseofulvin/liter antifungal agent (Hantscahke, 1968). B-Activation of cultures. All test strains were activated on Sabourad's dextrose agar (SDA) and Czapekdox agar (CZPA) composed according to Demain and Solomon, (1986) with yeast extract 4, sucrose 15, sodium nitrate 2, ferrous sulfate seven hydrate 0.01, di-potassium hydrogen phosphate 0.5, potassium chloride, 0.5, magnesium sulfate 0.5, agar-agar 15g / liter supplemented with streptomycin 30 mg / liter of distilled water. These plates were incubated at 37°C for 24 hours. The sub culturing of these strains were continued for five days in fresh media plates (Ghumro et al., 2001) and finally the master plates were maintained for further studies. C-Growth studies. Growth studies were under taken in three replicates after every 4 hr till 96 hr at room temperature, 37°C in static incubator and orbital shaker at 120 rpm and were examined at OD 600 (Choudhary et al., 1986). Further optimization of various sizes of inocula, pH, temperatures and agitation speeds in ME broth. D-Preparation of growth media (Gm) for the growth of test strains. After optimization studies of various nutritional factors, synthetic growth media (5 and 6) for the growth of test strains were prepared according to the optimized concentrations. E­Growth studies of test strains on growth media. Fresh cultures of test strains into 300 ml conical flask containing 100 ml of growth media, flasks were incubated at 30°C for 72 hr in orbital shaker at 110 rpm. Absorbance of all test strains was determined and compared after every 24 hr incubation with the absorbance of test strains grown in the malt extract broth and

later total viable count (Sherply, 1960; Noor and Hameed, 1996; Ghumro, 1996; Birch et. al., 2003) was obtained. F-Growth studies of test strains under stressed environments i - Effect of ultraviolet (UV) radiations Fresh inocula of test strains (S5 and S6) were separately inoculated in four sets of conical flasks containing 100 ml (v/v) of growth media and incubated at 30ºC for different time periods at 260 nm UV lamp. The irradiated cultures were sub cultured on fresh growth media and kept at 110 rpm, 120 rpm in orbital shaking incubator at 30ºC for 72 hrs at OD 600. ii- Effect of moist heat at various temperatures Fresh inocula of test strains were separately inoculated in seven sets of conical flasks containing 100 ml (v/v) of growth media and kept in water bath at various temperatures and various intervals. Heat treated cultures were sub cultured in fresh growth media (5 and 6) and kept at 110 rpm in orbital shaker incubator at 30°C and 36°C for 72 hr respectively and finally the absorbance was checked at OD 600. iii - Effect of mutagenic chemicals Fresh inocula of test strains (S5 and S6) were separately inoculated into seven sets of conical flasks containing 100 ml of growth media and mercuric chloride, ferric chloride, phenol at the concentration (v/v). All culture flasks were incubated for 72 hrs in shaking incubator at 30°C, 110 rpm, OD 600. 3. Results This research work has been performed at the Enzyme and Fermentation Biotechnology Laboratory, Institute of Biotechnology and Genetic Engineering, University of Sindh Jamshoro, Pakistan. Accordingly the test strains S5 and S6 grown well during 72 hr incubation at 37°C in orbital shaker incubator (at 120 rpm) as compared to the incubation at room temperature and in the static incubator (Fig. 1). Test strains S5 and S6 showed the maximum growth at 0.5 and 1.0 ml inoculum size, pH 4.0, temperature 30°C and 110 rpm agitation speed respectively (Table-1). During optimization studies for the yeast growth it was revealed that the mineral requirement of yeast growth ranges from 0.2 to 0.6% (w/v) in ME broth (Fig. 2). The optimized concentration of minerals supports and the growth of test strains increases

Optimization studies of yeast feed for enhanced growth...

43

at 30°C, pH 4.0, 0.5-1.0 ml inoculum size after incubation at 110rpm (Fig. 3-4). Saccharomyces cerevisiae strains S5 and S6 were tested for their tolerance to various stress conditions such as UV radiations, moist heat (in water bath) in various intervals and were also tested at various concentrations of mercuric chloride, ferric chloride and phenol, ethanol and methanol. This study has revealed that both S5 and S6 strains

survived for 15 minutes with little decrease in growth and thereafter a drastic decrease in growth occurred. Chemical stress revealed the tolerance at 0.2 ml v/v concentrations of mercuric chloride, ferric chloride and phenol. It was also observed that these test strains have a tolerance in ethanol at 11 and 06 ml and methanol at 6 and 04 ml v/v of the broth medium (Table- 2-3, Fig. 5).

Table -1. Growth of test strains (S5 and S6) of S. cerevisiae at 1.0 ml inoculum size with various sizes of inocula (ml v/v), pH values, temperatures °C and agitation speeds (rpm) in shaking incubator at 37ºC after 72 hr incubation Sizes of inoculum (ml v/v) 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 S5 0.229 0.228 0.225 0.220 0.215 0.207 0.198 0.191 0.184 0.177 S6 0.168 0.175 0.171 0.165 0.158 0.152 0.146 0.138 0.130 0.124 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 pH values S5 0.217 0.226 0.235 0.230 0.224 0.216 0.208 0.200 0.191 S6 0.158 0.163 0.170 0.177 0.171 0.164 0.157 0.152 0.146 0.139 Temperatures °C S5 0.221 0.226 0.231 0.238 0.244 0.251 0.247 0.242 0.236 0.230 S6 0.160 0.165 0.170 0.176 0.182 0.188 0.184 0.179 0.173 0.165 Agitation speeds (rpm) S5 0.238 0.241 0.250 0.259 0.255 0.249 0.242 0.233 S6 0.179 0.184 0.190 0.196 0.192 0.187 0.179 0.170

20 22 24 26 28 30 32 34 36 38

80 90 100 110 120 130 140 150

Table-2. Effect of U V radiations, heat (water bath temperature) at 20 - 50°C on the growth of test strains (S5 and S6 with initial O.D. 1.701, 1.062 respectively) in growth media at pH 4 and 0.5 - 1 ml inoculum size, 30°C 110 rpm after 72 hr incubation Time (minutes) 15 30 45 60 Various temperatures °C U.V. rays S5 1.679 1.563 1.534 1.500 S6 1.011 0.883 0.672 0.417 S5 1.679 1.563 1.534 1.500 20 S6 1.011 0.883 0.672 0.417 S5 1.700 1.700 1.696 1.693 30 S6 1.056 1.056 1.054 1.055 S5 1.669 1.637 1.595 1.568 40 S6 0.158 1.042 0.127 1.009 S5 1.657 1.618 1.559 1.503 50 S6 1.024 1.006 0.878 0.763

Mean values of three replicates Table-3. Effect of mercuric chloride, ferric chloride and phenol at various concentrations (0.2, 0.4 ­ 1.0 ml v/v) on the growth of test strains (S5 and S6 with initial O.D. 1.701, 1.062 respectively) in growth medium at pH 4 and 0.5 - 1 ml inoculum size, 30°C 110 rpm after 72 hr incubation Concentration (% v/v) 0.2 0.4 0.6 0.8 1.0 Mercuric chloride S5 S6 1.671 1.054 1.634 1.045 1.591 1.036 1.549 1.024 1.497 1.014 Ferric chloride S5 S6 1.674 1.058 1.645 1.051 1.611 1.045 1.576 1.036 1.531 1.025 Phenol S5 1.663 1.618 1.567 1.501 1.426 S6 1.055 1.042 1.030 1.018 1.001

Mean values of three replicates

A. A. Noor and M. U. Dahot

F ig . 1: D e t e rmina t io n o f g ro w t h o f S a c c ha ro my c e s c e re v is ia e s t ra ins S 5 a nd S 6 o n M E b ro t h a t ro o m t e mp e ra t ure , 3 7 C a t s t a t ic inc ub a t o r a nd a t 3 7 C in o rb it a l s ha king inc ub a t o r a t 110 rp m.

44

0 .2 2 0 .2 0 . 18 0 . 16 0 . 14 0 . 12 0 .1 0 .0 8 0 .0 6 0 .0 4 0 .0 2 0 R o o m t e mp e ra t ure

S5

S6

S t a t ic inc ub a t o r Te mp e ra t ure s a nd mo d e s o f inc ub a t io n

S ha king inc ub a t o r

Fig .2 : C o mp arat ive g ro w t h o f S .c e re v is iae s t rains S 5 and S 6 o n g ro w t h me d ia ( 5 and 6 ) re s p e c t ive ly at s t re s s c o nd it io ns af t e r 72 hr inc ub at io n. 1.6 1.4 1.2 1 0 .8 0 .6 0 .4 0 .2 0 S5 t e s t s t rains Init ial g ro w t h Final g ro w t h S6

Fig. 03: 0.25

Maximum growth of S. cerevisiae strain S5 at various minerals. Test strain S5

0.2

O 6 D 00

0.15

0.1

0.05

ZC n l2

K H O

N H aO

CS 4 uO

K 2P 4 H O

KH O 2 P 4

C (O )2 a H

N 4 l2 HC

(N 4 2S 4 H) O

NN 3 a O

NN 2 a O

K O 3P 4 0.2

MC n l2

M l2 gC

NC a l2

0.2

0.2

0.4

0.4

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0.2

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0.2

0.2

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Various chemicals (minerals) at respective contrations (% w/v)

N O aS 3

K l C

0

Optimization studies of yeast feed for enhanced growth...

Fig.4 Maximum growth of S. cerevisiae test strain S6 on various minerals 0.25 0.2 O 60 D0 0.15 0.1 0.05 ZC n l2 KH O NO a H N 4 l2 HC CS 4 uO MC n l2 NC a l2 K2 O HP 4 KH O 2 P4 C ( H2 aO ) KP 4 3O 0 Test strain S5

45

MC g l2

NN 3 a O

NN 2 a O

0.2 0.4 0.4 0.6 0.4 0.2 0.4 0.6 0.6 0.2 0.4 0.4 0.2 0.2 0.4 0.2 0.2 0.6 Various chemicals (minerals) at respective concentrations (% w/v)

Fig. 5 Comparasion of ethanol and methanol tolerance of S. cerevisiae strains S5 and S6 at various concentrations 1.8 1.6 1.4 1.2 O 60 D 0 1 0.8 0.6 0.4 0.2 0 S5 S. cerevisiae test strains S5 and S6 S6

MS 4 gO

Ethanol Methanol

4.

Discussions Globally, biofuel technology has emerged and being used as substitute of fuel oil by yeast from waste as a source of energy (Bawa and Yoshiyuki, 1992; Moritz and Sheldon, 1996; Noor and Hameed, 1998, 1999). The essence of the present study is to enhance the growth of Saccharomyces cerevisiae strains S5 and S6 using different growth requirements including the minerals (feed-I), sugars, amino acids and vitamins (feed-II) after treating under stress conditions for ethanol fermentation by shake flask method. Drying of baker's yeast causes rupture of cytoplasmic membrane and brings about changes in the composition of the nucleic acids, proteins, lipids, carbohydrates. The re-hydration provides the moisture level for the growth and metabolic activity (Alpas et al., 1996). Activation of yeast strains in malt medium supports the higher cellular mass and enzymatic activity. Sub culturing of lyophilized

cultures and synthetically prepared modified MYPGA in which peptone was replaced with Yeast Nitrogen Base i.e. Malt extract Yeast extract Yeast Nitrogen Base Agar (MYNGA). From these media, MYNGA was found a better medium because it contains yeast nitrogen base, which includes ammonium sulphate, asparagines, minerals, vitamins, trace elements and amino acids provides a good nitrogen sources as well as other growth factors for carbon assimilation and fermentation (Walker, 1997; Edgerton, 2001). The activated cultures were grown in basic medium e.g. ME broth Age and size of inocula were observed after four hour incubation, which revealed 72 hr age of test strains. This is due to the fact that the growth of the starter reaches its log phase, having faster metabolic activity and the optimized volume of inocula of active yeast culture competes more for available nutrients. Addition of various inorganic compounds in optimized concentration range could result the enhanced growth and have the stimulatory effects on ethanol production

NS 3 aO

Kl C

( H) N4

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during fermentation process. Growth rate and fermentation rate are not inversely proportional. This is due to the fact that the starter culture requires the specific growth rate, time, nutrients and other miscellaneous parameters for their enzymatic activity. Our observations pertaining to the size of inocula are in agreement with the results of Choudhary et al., (1986), Panday and Agarwal (1993), Noor and Hameed (1997). According to Stanford and Anslow (1996) the hydrogen ions such as protons are very significant for both intracellular and extracellular pH which has a dramatic influence on growth and metabolism of yeast cells. The effects of various pH ranges are due to the weak acids, which dissipate plasma membrane proton gradients and depressing cell pH when they dissociate into ions in the yeast cytoplasm. On the other hand, medium chain fatty acid causes rapid cell death by disruption of cell membrane integrity during growth. Oxygen is used for respiratory metabolism, temperature is important for inoculum development, maintenance of fermenter productivity in industrial ethanol production (Noor et al., 2003). An initial increase at 37°C showed increase in growth but the final cell mass concentration attained very low (Sedha et al., 1984, Williams and Munnecke, 1993). In present study, the maximum growth of yeast strains was observed at 30°C when incubated for 72 hours, which shows same pattern as reported by Tabera et al., (1985), Elahi et al., (1989), Enhanced growth of test strains S5 and S6 was observed when incubated in orbital shaking incubator adjusted at 110 rpm. This is because agitation speed provides oxygen supply, which activates the mitochondria for cyclic metabolism and also providing uniform distribution of nutrients. Agitations and temperatures beyond the optima results in cellular damage Choudhary et al., (1986). During the optimization studies of growth in various chemicals, it was observed that the maximum growth occurred in between 0.2 - 0.6 % (w/v) of ME broth. Concentration of minerals beyond the optima is toxic for the growth of yeast (Walker and Maynard, 1997; Gadd and Laurence, 1996; Avery et al., 1996). Phosphorus is essential for all yeasts which are present in nucleic acid and phospholipids.

The major part of sulfur is inorganic orthophosphate (Aiking and Tempest, 1976; Theobald et al., 1996a) which acts as a substrate and effectors for many enzymes, including those involved in energy transduction depending upon the levels that fluctuate according to the mode of sugar catabolism (Theobald et al., 1996b). It is reported that chloride transport occurs via a proton-chloride or sodium chloride symport mechanism which may be involved in yeast cell water content (Andre, 1995). Divalent cations in yeast cells act primarily as enzyme cofactors. Mg2+ ions transport mechanism is involved in regulating free Mg2+ ion in the yeast cytosol (Beeler et al., 1997). Manganese is essential for yeast growth and metabolism in trace concentrations, which may accumulate in vacuole and act as an intracellular regulator for key enzymes (Kihn et al., 1988; Auling, 1994). Ca++ ions have been linked to the cell cycle regulation and implicated in the transition from lag to exponential phase in batch cultures of S. cerevisiae (You att, 1993; Loukin and Kung, 1995). Potassium and magnesium are regarded as macro elements required to establish the main metallic cationic environment in the yeast cell. Copper and iron act as cofactors in several enzymes including the redox pigments of the respiratory chain (Joslyn, 1941; Walker, 1997). These undergo unique chemistry due to ability to adopt distinct redox states, either oxidized (Cu-II) or in the reduced (Cu-I) state (Maria et al., 1999). It is involved in a variety of biochemical processes, such as cytochrome oxidase, Cu, Zn, superoxide mutase, lysyl oxidase and dopamine-monooxygenase and also play a critical role in assimilation (David and Fred, 1999). They require traces of Zn2+ that is essential for structure-function of the enzymes during metabolism for example, the important terminal step enzymes in yeast alcoholic fermentation namely alcohol dehydrogenase is a zinc-metalloenzyme Zn2+ uptake is time, temperature and concentration dependent and saturable (Fuhrmann and Rothstein, 1968; Mowll and Gadd, 1983, White and Gadd, 1987). Sulphates are transported for assimilation into sulphur-containing amino acids like methionine and peptides like glutathione but no singular sulphate transporter gene has been identified in yeasts at present (Andre, 1995). Sulphur is required in the form of a variety of sulphur compounds e.g. for the biosynthesis of sulphur

Optimization studies of yeast feed for enhanced growth...

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contaning amino acids. In S. cerevisiae (in most of the microorganisms and in plants) the formation of sulfite occurs through reductive sulfate assimilation which is the route of methionine and cysteine biosynthesis (Walker, 1997; Thomass and Surdin-Kerjan, 1997). Sulphur prevents enzymatic and non-enzymatic browning reactions and in free form it prevents microbial growth of undesirable microorganisms, which allows the yeast growth in diverse form when provided in liquid form under optimal physical growth conditions for required time (Morris and Haugh, 1956; Walker and O'Neill, 1990; Gimeno et al., 1992; Tudor and Board, 1993; Thomas, 1993). Growth media (5 and 6) were prepared by addition of minerals in optimized concentrations along with sugars, amino acids and vitamins, which supported the growth of test strains S5 and S6 respectively. Growth under stressed conditions revealed that various physical and chemical agents, radiations are responsible to inhibit the yeast growth when they are exposed to various durations and concentrations beyond the optima respectively. During the observations of thermotolerance, it was reported that temperature beyond optima shows the greater cellular damage i.e. cell viability declines because the yeasts are unable to regulate their internal temperature and the high temperatures provided to them resulting the disruption of hydrogen bonding and hydrophobic interactions leading to general denaturation of protein and nucleic acids (Coote et al., 1991, Shah and Hameed, 2004). Yeast growing quickly in glucose rich medium is more sensitive to heat and other stresses compared to stationary phase cells. This may be due to the fact that glucose carriers act as the sensors of glucose availability, which rapidly adjust their growth and metabolism to changing environmental conditions (Lagunas, 1993). Sub-lethal heat shock treatment of yeast leads to the induction of synthesis of a specific set of highly conserved heat-shock proteins known (Ruis and Schuller, 1995; Walker, 1997). Mild water stress in yeasts occurs during osmostress caused either by hyperosmotic shock or by hypoosmotic shock (Piper, 1995; Halsworth 1998) producing dramatic changes in membrane structural arrangements and permeability properties (Walker, 1997).

Saccharomyces cerevisiae strain S5 and S6 showed insignificant decrease in the growth when examined under stressed growth conditions and a complete decline stage after the specific time period and concentration respectively. This is due to the activation of transcriptional control elements (STREs) in S. cerevisiae. The high osmolarity glycerol (Hog1) gene product encodes a mitogen-activated protein kinase and the pathway starts with the activation of plasma membrane bound receptor proteins which act as osmosensors. MAPK cascades transducer signals trigger by heat shock and UV radiations etc. Certain stress metabolites play role as stress-protectant molecule including osmotolerant, antidesicant, chemical detoxicant, cryoprotectant and thermoprotectant (Hino et al., 1990; De Virgillo et al., 1994). Radiations cause DNA damage in S. cerevisiae. UV causes dimerization, nicks and other lesions. These cause intrachromosomal recombination in cell cycle-dependent manner and can inhibit certain membrane functions by affecting membrane lipid phase transitions and by interacting with photosensitive chromogenic molecules within the cell (Galli and Shiestl, 1995; Walker, 1997). Chemical stresses in yeast cells may cause cellular damages during growth and metabolism due to acetaldehyde and ethanol stress produced during growth and fermentation. Low ethanol concentrations are inhibitory to yeast growth and cell division, but higher concentrations can be lethal beyond the concentration 20% v/v. Watson and Cavicchioli (1983) reported that, exposure to ethanol causes the increased membrane fluidity and decreased membrane integrity. Ethanol and methanol toxicity is reduced to the increase of fatty acyl chain length, unsaturated fatty acids and sterols in the cell membrane, membrane lipids, palmitic acid and cholesterol, phosphatidylserine, ergosterol or campesterol, linoleic acid, yeast hull which supports enhanced growth, viability and nutrient uptake. Higher concentration of ethanol destroys enzymes and kills the yeast cells in the medium due to the accumulation of dissolved toxic products, which slows down and stops the growth of test strains. Methanol on the other hand did not allow the test strains to grow beyond the concentration 4-6 % (v/v) in the fermentation medium. This is because the test strains are not the methanol utilizing yeasts and are unable to metabolize by an oxygen-

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dependent oxidase to formaldehyde, which is then converted into dihydroxyacetone by DAH synthase enzyme. Our results are well acquainted with Minier and Goma, (1982). Magnesium (Mg2+) is a known substance that protects yeast from ethanol stress by reducing its fermentative activity (Dombek and Ingram, 1986; Birch and Walker, 1996; Ciesarova et al., 1996) and increasing the proton and anion permeability caused by ethanol (Petrov and Okorokov, 1990). References Aiking, H., and D. W. Tempest, (1976) Growth and physiology of Candida utilis NCYC 321 in potassium limited chemostat culture. Archives of Microbiology, (108): 117-124. Alpas, H., M. Ozilgen, T.F. Bozoglu, and S. Katnas, (1996) Mathematical modeling of survival and weight loss of baker's yeast during drying. Enzyme and Microbial Technology. (19): 348-351. Andre, B., (1995) An over view of membrane transport proteins in S. cerevisiae. Yeast (11): 1575-1611. Auling, G., (1994) Manganese: function and transport in fungi. In metal ions in fungi (eds. G., Winkleman and d. Winge), Marcel Dekker, New York, 215-236. Avery, S. V., N.G. Howlett, and S. Radice, (1996) Copper toxicity towards Saccharomyces cerevisiae: dependence on plasma membrane fatty acid composition. Applied and Environmental Microbiology (62): 3960-3966. Bawa, D., and O. Yoshiyuki, (1992) Promotive action of ceramics on yeast ethanol production, and its relationship to pH, glycerol and alcohol dehydrogenase activity. Appl. Microbial Biotechnol., (36): 717-721. Beeler, T., K. Bruce, and T. Dunn, (1997) Regulation of cellular Mg 2+ by Saccharomyces cerevisiae. Biochimica Biophysica Acta1 (323): 310-318. Birch, R., and G. M. Walker, (1996) The role of magnesium in yeast stress responses. Abstracts of the 9th International Symposium on Yeasts (Sydney) 46-47. Birch, R.M., M. Ciani, and G.M. Walker, (2003) Magnesium, Calcium and fermentative metabolism wine yeasts. J.Wine Res.14(1): 3-15.

Boekhout, T., and C. P. Kurtzman, (1996) Principles and methods used in yeast classification, and an overview of currently accepted yeast genera. In: Nonconventional Yeast in Biotechnology. A hand book (ed. K. Wolf), Springer-Verlag, Berlin, Heidelberg,1-99. Ciesarova, Z., D. Smorrovicova, and Z. Domeny, (1996) Enhancement of yeast ethanol tolerance by calcium and magnesium. Folia Microbiologica (41): 485-488. Codon, A.C., and T. Benitz, (1985) Variability of physiological features of the nuclear and mitochondrial geneomes of Baker's yeasts. Syns. Appl. Microbial. (18): 343-352. Coote, P. J., M. B. Cole, and M.V. Jones, (1991) Induction of increased thermotolerance in Saccharomyces cerevisiae may be triggered by a mechanism involving intracellular pH. Journal of General Microbiology, 137: 1701-1708. David, A. P., and S. Fred, (1999) Toxicity of copper, cobalt and Nickel salts is dependent on histadine metabolism in the yeast Saccharomyces cerevisiae. J. Bacteriol. 181 (16): 4774-4779. Demain, A.L., and N.A. Solomon, (1986) Manual of microbiology and biotechnology. Am. Soc. For Microbiology, Washington, USA, 3-23. DeVirgillo, C., T. Hottiger, J. Dominguiz, T. Boller, and A. Wiemken, (1994) The role of trehalose synthesis for the acquisition of thermotolerance in yeast. I. Genetic evidence that trehalose is a thermoprotectant. Europeon Journal of Biochemistry (219): 179-186. Dombek, K.M., and L.O. Ingram, (1986) Nutrient limitation as a basis for the apparent toxicity of low levels of ethanol during fermentation. Journal of Industrial Microbiology (1): 219-225. Edgerton, J., (2001) A primer on yeast propagation technique and procedures. Technical Quarterly 38 (3): 167-175. Elahi, S., A. Hashmi, M. I. Rajoka, C. M. Akhter, and A. Ilahi, (1989) Production of ethanol from blackstrap molasses by Saccharomyces cerevisiae. Int. Symp. Biotech. for Energy, 16-21. Faisalabad 179-186.

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Fuhrmann, G. F., and A. Rothstein, (1968) The transport of Zn2+, Co2+, and Ni2+, into yeast cells. Biochem. Biophysics. Acta (163): 325-330. Gadd, G. M., K. Chalmers, and R. H. Reed, (1987) The role of trehalose in dehydration resistance of Saccharomyces cerevisiae. FEMS Microbiology Letters (48): 249-254. Gadd, G. M., and O. S. Laurence, (1996) Demonstration of high-affinity Mn2+ uptake in Saccharomyces cerevisiae ­ specificity and kinetics. Microbiology (142): 25-60. Galli, A., and R. H. Shiestl, (1995) On the mechanism of UV and -ray induced intrachromosomal recombination in yeast cells synchronized in different stages of cell cycle. Molecular and General Genetics (248): 301-310. Ghumro, P.B., A.A. Noor, and A. Hameed, (2001) Multidimensional study of maximum growth of protease producing Bacillus subtilis strains M1 and M2. Hamdard Medicus XLIX, (4): 97-102. Gimeno, C. J., P.O. Ljungdahl, C. A. Styles, and G. R., Fink, (1992) Unipolar cell divisions in the yeast S. cerevisiae lead to the filamentous growth: regulation by starvation and RAS. Cell (68): 1077-1090. Halsworth, J. E. (1998) Ethanol induced water stress in yeast. Journal of fermentation Bioengineering (85): 125-137. Hantschke, D., (1968) Eincolistin-NovobiocinActidion-Agar als Anzuchtmedium for humanpathogene Pilze. Mykosen (11): 769-778. Hawksworth, D. L., and J. Mouchacca, (1994) Ascomycetes systematics in the nineties. In: Ascomycetes systematics: Problems and Perspectives in the Nineties (ed. D. L. Hawksworth), Plenum Press, NewYork, 3-11. Hino, A., K., Mihara, K. Nakashima, and H., Takano, (1990) Trehalose levels and survival ratio of freeze tolerant versus freeze sensitive yeasts. Applied and Environmental Microbiology (56): 1386-1391. Josyln, M.A., (1941)The Mineral Metabolism of yeasts. Wallerstain labs. Communs 4(11): 49-65. Khan, A. M., and N. Mahmood, (1992) Laboratory Manual of Microbiology, 4th edn. Time Traders, Karachi, 83-128.

King, A., and H. Cleveland, (1980) The renewable way of life. In bioresources for development, Pergamon press, Oxford, 12-20. Kihn, J.C., C.M. Dassargues, and M. M. Mestdagh, (1988) Preliminary ESR study of Mn (I) retaintion by the yeast Saccharomyces cerevisiae. Canadian J. Microbiol. (34): 1230-1234. Lagunas, R., (1993) Sugar transport in Saccharomyces cerevisiae. FEMS Microbiology Reviews, (104): 229-242. Loukin, S., and C. Kung, (1995) Manganese effectively supports yeast cell cycle progression in place of calcium. Jour. Cell Biology (131): 1025-1037. Minier, M. K., and G., Goma, (1982) Ethanol production by extractive fermentation. Biotechnol. Bioeng. (24): 1565-1579. Moritz, J. W., and J.B.D. Sheldon, (1996) Simultaneous saccharification and extractive fermentation of cellulose substrates. Biotechnol. Bioeng. (49): 504-511. Morris, E.O., and J. S. Hough, (1956) Some aspects of the giant colony characteristics of S.cerevisiae. J. Inst. of Brewing (62): 446-469. Mowll, J. L., and G. M. Gadd, (1983) Zinc uptake and toxicity in the yeast Sporobolomyces roseus and Saccharomyces cerevisiae. J. Gen. Microbiol. (129): 3421-3425. Noor, A. A., and A. Hameed, (1996) Three dimensional screening of fermentation yeast Saccharomyces cerevisiae strain AAN-2. J. Sci. and Technol. (20): 1-7. Noor, A. A., and A. Hameed (1997) Yeast bioconversion of glucose from molasses to ethanol by Saccharomyces cerevisiae. Pak. J. Biochem. and Mol. Boil. (30): 97-103. Noor, A. A., and A. Hameed (1998) A multistep optimization study for maximum growth of saccharophilic yeast Saccharomyces cerevisiae strain ASN-3: A three-in-one approach. Sci. Sindh, Pak. (5): 108. Noor, A.A., and A. Hameed (1999) Optimization studies for bioconversion of corn steep liquor to ethanol by Saccharomyces cerevisiae strain KA-1. Pak. J. Biol. Sci. (2): 137-140.

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Noor, A. A., A. Hameed, K. P. Bhatti, and S. A. Tunio, (2003) Bio-ethanol fermentation by the bioconversion of sugar from dates by Saccharomyces cerevisiae strains ASN-3 and HA-4. Biotechnology 2(1): 8-17. Pandey, K., and P. K. Agrawal, (1993) Effect of EDTA, Potassium ferrocyanide, and Sodium potassium tartarate on the production of ethanol from molasses by Saccharomyces cerevisiae. Enzyme and Microbial Technol. (15): 887-898. Petrove, V. V., and L. A. Okorokov, (1990) Increase of the anion and proton permeability of Saccharomyces carlsbergensis plasmalemma by n-alcohols as a possible cause for de-energization. Yeast (6): 311-318. Piper, P. W., (1995) The heat shock and ethanol stress of yeast exhibit extensive similarity and functional overlap FEMS Microbiology Letters (134): 121-127. Prescott, S. C., and C. G. Dunn, (1959) Industrial Microbiology, 3rd. McGraw-Hill Book Co., Inc., New York, 1-62. Ruis, H., and C. Schuller, (1995) Stress signaling in yeast. Bioessays, (17): 959-965. Sedha, R. K., G., Verma, R. P., Gupta, and H. K., Tewari, (1984) Ethanol production from molasses using cell recycling of Saccharomyces cerevisiae. J. Ferm. Tech. 62 (5): 471-476. Shah, A. H., and A. Hameed, (2004) Nutritional and Mutational aspects of Indegenous lysine production by Corynebacterium glutamicum: Comprehensive studies in shake flask fermentation-I. J. Chem. Soc. Pak. 26 (4): 416-428. Sherply, J. M., (1960) Elementry Petroleum Microbiology, Wolf Publication Co. Houston, Texas. U.S.A. pp. 112. Stanford, M., and P. A. Anslow, (1996) Comparasion of the inhibitory action on Saccharomyces cerevisiae of weak acid preservatives, uncoupler and medium chain fatty acids. FEMS Microbiology Letters (142): 53-58. Tabera, J. I. M. A., I. Schnabel, and J. Garrido, (1985) Yeast strain screening method for a continuous process of alcoholic fermentation. Biotech. Letters. 7 (6): 437-442.

Theobold, U., J. Mohns, and M. Rizzi, (1996 b) Dynamics of orthophosphate in yeast cytoplasm. Biotechnology letters, (18): 461-466. Theobold, U., J. Mohns, and M. Rizzi, (1996b) Determination of in vivo cytoplasmic orthophosphate concentration in yeast. Biotechnology Techniques, (10): 1753-1790. Thomas, D.S., (1993) Yeast as spoilage organisms in beverages. In: The yeasts 2nd edn. Vol. 5, Yeast technol. (eds. A. H. Rose and J. S. Harrison), Academic Press, London,517-561. Thomass, D., and Y. Surdin-Kerjan, (1997) Metabolism of sulfur amino acids in Saccharomyceserevisiae. Microb. Mol. Biol. Rev. (61): 503-532. Tudor, E. A., and R. G. Board, (1993) Food spoilage yeasts. In The Yeasts 2nd edn. Vol. 5: Yeast Technology (eds. A.H. Rose and J.S. Harrison), Academic Press London, 435-516. White, C., and G. M. Gadd, (1987) The uptake and cellular distribution of zinc in Saccharomyces cerevisiae. J. Gen. Microbiol. (133): 727-737. Williams, D., and D. M., Munnecke, (1993) The production of ethanol by immobilized yeast cells, Biotechnol. Bioeng., (23): 1813-1815. Walker, G. M., and J. D. O'Neill, (1990) Morphological and metabolic changes in the yeast Kluyveromyces marxians var. marxians NRRLY 2415. J. Chemical Technology and Biotechnology (49): 75-89. Walker, G. M., and A. I. Maynard, (1997) Accumulation of magnesium ions during fermentative metabolism in Saccharomyces cerevisiae. J. of Industrial Microbiology and Biotechnology (18): 1-3. Watson, K., and R. Cavicchioli, (1983) Acquisition of ethanol tolerance in yeast cells by heat shock. Biotechnology Letters (4): 683-688. You att, J., (1993) Calcium and microorganisms. Critical Reviews in Microbiology (19): 83-97. Zertuche, L., and R. R. Zali. (1982) A study of producing ethanol from cellulose using Clostridium thermocellum. Biotechnology Bioeng. (24): 57-6.

Sindh Univ. Res. Jour. (Sci. Ser.) Vol: 40 (2) 41-50 (2008)

SURJ

Sindh Univ. Res. Jour. OPTIMIZATION STUDIES OF YEAST FEED FOR ENHANCED GROWTH FOR ETHANOL PRODUCTION BY SHAKE FLASK METHOD A. A. Noor and M. U. Dahot* Department of Microbiology, University of Sindh, Jamshoro

(Received 17th February. 2008 and Revised 13th April 2008) Abstract Yeasts are the industrially used microorganisms to provide the best possible combination of characteristics for the biological reactions with the given substrates. Yeast nutrition refers to how cells feed; translocate water and essential in-organic and organic nutrients from the ecosystem. Saccharomyces cerevisiae of bakery origin (strain S5) and soil origin wild strain (strain S6) were cultivated on nutrient agar and were activated on Saboraud's dextrose agar and Czepkdox agar, potato dextrose agar and malt extract agar by three subsequent inoculations for the maximum growth. Test strains S5 and S6 were optimized for their growth requirements in malt extract broth as initial medium; pH was maintained at 5.0 and incubated at 37°C for 72 hours, 110 rpm, 1.0 ml inocula size. Growth studies were under taken in three replicates after every 4 hours till 96 hours. Number of nutritional requirements were provided at the optimized concentrations in the form of yeast media 5 and 6 respectively and finally the growth was observed at 600 nm. The test strains grown on growth media (5 and 6) were also subjected for their tolerance to various chemical agents at the respective concentrations (ml v/v). Present study revealed the higher growth rate of S. cerevisiae strains S5 on Gm5 as compared to the strain S6 on Gm6 containing vitamins and amino acids as growth promoting factors at 110 rpm, pH 4.0, 0.5 and 1 ml. inocula size after 72 hours incubation by shake flask method which indicates that strain S5 is the best candidate for ethanol production. Keywords: Yeasts industrially, Yeast nutrition, Growth for ethanol production shake flask method

1.

* Institute of Biotechnology and Genetic Engineering, University of Sindh, Jamshoro

Introduction The industrial society developed by the accumulation of the scientific knowledge, the spread of technological innovations, and the exploitation of enormous natural resources (King and Clevedon, 1980). In the current epoch, the utilization of microorganisms in industrial processes or in process in which, their activities may become of industrial or technological significance that relates to the production of desired products including pharmaceuticals, organic acids, enzymes, organic solvents and synthetic fuels and also utilized to prevent their values (Presscott and Dunn, 1959, Zertuche and Zali, 1982). Yeasts are truly fascinating organisms, having 7000 species reported (Boekhout and Kurtzman, 1996; Hawksworth and Mouchacca, 1994). Yeasts, Saccharomyces cerevisiae are able to rapidly convert sugars to ethanol having a high

fermentation property, high show, high ethanol tolerance and osmotolerance, low pH, and high temperature optima, have high glycolytic, invertase activities and genetic stability (Codon and Bentez, 1985). 2. Methodology A-Isolation of cultures Saccharomyces cerevisiae cultures were received from local bakery (Strain-5) and the wild strain was isolated from 20 soil samples. Bakery strain was isolated by mixing bakery dough in the sterilized malt extract (ME) broth and incubated at 37°C for 12 hr. Later a loop full was inoculated on Sabouraud's dextrose agar (SDA) plate containing mycological peptone 10, dextrose 40, yeast extract 3 and agar-agar 20g / liter of distilled water, pH 5.6. Plates were incubated at 37°C for 24 hr to observe the

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Khoc cho nho thuong voi trong long, khoc cho noi sau nhe nhu khong. Bao nhieu yeu thuong nhung ngay qua da tan theo khoi may bay that xa... growth. Colonies were identified and maintained as pure culture in SDA slopes. Soil originated wild strain (S6) was isolated by pour plate method of Khan and Mahmood, (1992). 1ml of each suspension was poured over the surface of SDA agar plates containing 400mg of griseofulvin/liter antifungal agent (Hantscahke, 1968). B-Activation of cultures. All test strains were activated on Sabourad's dextrose agar (SDA) and Czapekdox agar (CZPA) composed according to Demain and Solomon, (1986) with yeast extract 4, sucrose 15, sodium nitrate 2, ferrous sulfate seven hydrate 0.01, di-potassium hydrogen phosphate 0.5, potassium chloride, 0.5, magnesium sulfate 0.5, agar-agar 15g / liter supplemented with streptomycin 30 mg / liter of distilled water. These plates were incubated at 37°C for 24 hours. The sub culturing of these strains were continued for five days in fresh media plates (Ghumro et al., 2001) and finally the master plates were maintained for further studies. C-Growth studies. Growth studies were under taken in three replicates after every 4 hr till 96 hr at room temperature, 37°C in static incubator and orbital shaker at 120 rpm and were examined at OD 600 (Choudhary et al., 1986). Further optimization of various sizes of inocula, pH, temperatures and agitation speeds in ME broth. D-Preparation of growth media (Gm) for the growth of test strains. After optimization studies of various nutritional factors, synthetic growth media (5 and 6) for the growth of test strains were prepared according to the optimized concentrations. E­Growth studies of test strains on growth media. Fresh cultures of test strains into 300 ml conical flask containing 100 ml of growth media, flasks were incubated at 30°C for 72 hr in orbital shaker at 110 rpm. Absorbance of all test strains was determined and compared after every 24 hr incubation with the absorbance of test strains grown in the malt extract broth and

later total viable count (Sherply, 1960; Noor and Hameed, 1996; Ghumro, 1996; Birch et. al., 2003) was obtained. F-Growth studies of test strains under stressed environments i - Effect of ultraviolet (UV) radiations Fresh inocula of test strains (S5 and S6) were separately inoculated in four sets of conical flasks containing 100 ml (v/v) of growth media and incubated at 30ºC for different time periods at 260 nm UV lamp. The irradiated cultures were sub cultured on fresh growth media and kept at 110 rpm, 120 rpm in orbital shaking incubator at 30ºC for 72 hrs at OD 600. ii- Effect of moist heat at various temperatures Fresh inocula of test strains were separately inoculated in seven sets of conical flasks containing 100 ml (v/v) of growth media and kept in water bath at various temperatures and various intervals. Heat treated cultures were sub cultured in fresh growth media (5 and 6) and kept at 110 rpm in orbital shaker incubator at 30°C and 36°C for 72 hr respectively and finally the absorbance was checked at OD 600. iii - Effect of mutagenic chemicals Fresh inocula of test strains (S5 and S6) were separately inoculated into seven sets of conical flasks containing 100 ml of growth media and mercuric chloride, ferric chloride, phenol at the concentration (v/v). All culture flasks were incubated for 72 hrs in shaking incubator at 30°C, 110 rpm, OD 600. 3. Results This research work has been performed at the Enzyme and Fermentation Biotechnology Laboratory, Institute of Biotechnology and Genetic Engineering, University of Sindh Jamshoro, Pakistan. Accordingly the test strains S5 and S6 grown well during 72 hr incubation at 37°C in orbital shaker incubator (at 120 rpm) as compared to the incubation at room temperature and in the static incubator (Fig. 1). Test strains S5 and S6 showed the maximum growth at 0.5 and 1.0 ml inoculum size, pH 4.0, temperature 30°C and 110 rpm agitation speed respectively (Table-1). During optimization studies for the yeast growth it was revealed that the mineral requirement of yeast growth ranges from 0.2 to 0.6% (w/v) in ME broth (Fig. 2). The optimized concentration of minerals supports and the growth of test strains increases

Optimization studies of yeast feed for enhanced growth...

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at 30°C, pH 4.0, 0.5-1.0 ml inoculum size after incubation at 110rpm (Fig. 3-4). Saccharomyces cerevisiae strains S5 and S6 were tested for their tolerance to various stress conditions such as UV radiations, moist heat (in water bath) in various intervals and were also tested at various concentrations of mercuric chloride, ferric chloride and phenol, ethanol and methanol. This study has revealed that both S5 and S6 strains

survived for 15 minutes with little decrease in growth and thereafter a drastic decrease in growth occurred. Chemical stress revealed the tolerance at 0.2 ml v/v concentrations of mercuric chloride, ferric chloride and phenol. It was also observed that these test strains have a tolerance in ethanol at 11 and 06 ml and methanol at 6 and 04 ml v/v of the broth medium (Table- 2-3, Fig. 5).

Table -1. Growth of test strains (S5 and S6) of S. cerevisiae at 1.0 ml inoculum size with various sizes of inocula (ml v/v), pH values, temperatures °C and agitation speeds (rpm) in shaking incubator at 37ºC after 72 hr incubation Sizes of inoculum (ml v/v) 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 S5 0.229 0.228 0.225 0.220 0.215 0.207 0.198 0.191 0.184 0.177 S6 0.168 0.175 0.171 0.165 0.158 0.152 0.146 0.138 0.130 0.124 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 pH values S5 0.217 0.226 0.235 0.230 0.224 0.216 0.208 0.200 0.191 S6 0.158 0.163 0.170 0.177 0.171 0.164 0.157 0.152 0.146 0.139 Temperatures °C S5 0.221 0.226 0.231 0.238 0.244 0.251 0.247 0.242 0.236 0.230 S6 0.160 0.165 0.170 0.176 0.182 0.188 0.184 0.179 0.173 0.165 Agitation speeds (rpm) S5 0.238 0.241 0.250 0.259 0.255 0.249 0.242 0.233 S6 0.179 0.184 0.190 0.196 0.192 0.187 0.179 0.170

20 22 24 26 28 30 32 34 36 38

80 90 100 110 120 130 140 150

Table-2. Effect of U V radiations, heat (water bath temperature) at 20 - 50°C on the growth of test strains (S5 and S6 with initial O.D. 1.701, 1.062 respectively) in growth media at pH 4 and 0.5 - 1 ml inoculum size, 30°C 110 rpm after 72 hr incubation Time (minutes) 15 30 45 60 Various temperatures °C U.V. rays S5 1.679 1.563 1.534 1.500 S6 1.011 0.883 0.672 0.417 S5 1.679 1.563 1.534 1.500 20 S6 1.011 0.883 0.672 0.417 S5 1.700 1.700 1.696 1.693 30 S6 1.056 1.056 1.054 1.055 S5 1.669 1.637 1.595 1.568 40 S6 0.158 1.042 0.127 1.009 S5 1.657 1.618 1.559 1.503 50 S6 1.024 1.006 0.878 0.763

Mean values of three replicates Table-3. Effect of mercuric chloride, ferric chloride and phenol at various concentrations (0.2, 0.4 ­ 1.0 ml v/v) on the growth of test strains (S5 and S6 with initial O.D. 1.701, 1.062 respectively) in growth medium at pH 4 and 0.5 - 1 ml inoculum size, 30°C 110 rpm after 72 hr incubation Concentration (% v/v) 0.2 0.4 0.6 0.8 1.0 Mercuric chloride S5 S6 1.671 1.054 1.634 1.045 1.591 1.036 1.549 1.024 1.497 1.014 Ferric chloride S5 S6 1.674 1.058 1.645 1.051 1.611 1.045 1.576 1.036 1.531 1.025 Phenol S5 1.663 1.618 1.567 1.501 1.426 S6 1.055 1.042 1.030 1.018 1.001

Mean values of three replicates

A. A. Noor and M. U. Dahot

F ig . 1: D e t e rmina t io n o f g ro w t h o f S a c c ha ro my c e s c e re v is ia e s t ra ins S 5 a nd S 6 o n M E b ro t h a t ro o m t e mp e ra t ure , 3 7 C a t s t a t ic inc ub a t o r a nd a t 3 7 C in o rb it a l s ha king inc ub a t o r a t 110 rp m.

44

0 .2 2 0 .2 0 . 18 0 . 16 0 . 14 0 . 12 0 .1 0 .0 8 0 .0 6 0 .0 4 0 .0 2 0 R o o m t e mp e ra t ure

S5

S6

S t a t ic inc ub a t o r Te mp e ra t ure s a nd mo d e s o f inc ub a t io n

S ha king inc ub a t o r

Fig .2 : C o mp arat ive g ro w t h o f S .c e re v is iae s t rains S 5 and S 6 o n g ro w t h me d ia ( 5 and 6 ) re s p e c t ive ly at s t re s s c o nd it io ns af t e r 72 hr inc ub at io n. 1.6 1.4 1.2 1 0 .8 0 .6 0 .4 0 .2 0 S5 t e s t s t rains Init ial g ro w t h Final g ro w t h S6

Fig. 03: 0.25

Maximum growth of S. cerevisiae strain S5 at various minerals. Test strain S5

0.2

O 6 D 00

0.15

0.1

0.05

ZC n l2

K H O

N H aO

CS 4 uO

K 2P 4 H O

KH O 2 P 4

C (O )2 a H

N 4 l2 HC

(N 4 2S 4 H) O

NN 3 a O

NN 2 a O

K O 3P 4 0.2

MC n l2

M l2 gC

NC a l2

0.2

0.2

0.4

0.4

0.6

0.2

0.2

0.4

0.2

0.2

0.2

0.2

MS 4 gO

0.4

0.2

0.2

0.2

0.6

Various chemicals (minerals) at respective contrations (% w/v)

N O aS 3

K l C

0

Optimization studies of yeast feed for enhanced growth...

Fig.4 Maximum growth of S. cerevisiae test strain S6 on various minerals 0.25 0.2 O 60 D0 0.15 0.1 0.05 ZC n l2 KH O NO a H N 4 l2 HC CS 4 uO MC n l2 NC a l2 K2 O HP 4 KH O 2 P4 C ( H2 aO ) KP 4 3O 0 Test strain S5

45

MC g l2

NN 3 a O

NN 2 a O

0.2 0.4 0.4 0.6 0.4 0.2 0.4 0.6 0.6 0.2 0.4 0.4 0.2 0.2 0.4 0.2 0.2 0.6 Various chemicals (minerals) at respective concentrations (% w/v)

Fig. 5 Comparasion of ethanol and methanol tolerance of S. cerevisiae strains S5 and S6 at various concentrations 1.8 1.6 1.4 1.2 O 60 D 0 1 0.8 0.6 0.4 0.2 0 S5 S. cerevisiae test strains S5 and S6 S6

MS 4 gO

Ethanol Methanol

4.

Discussions Globally, biofuel technology has emerged and being used as substitute of fuel oil by yeast from waste as a source of energy (Bawa and Yoshiyuki, 1992; Moritz and Sheldon, 1996; Noor and Hameed, 1998, 1999). The essence of the present study is to enhance the growth of Saccharomyces cerevisiae strains S5 and S6 using different growth requirements including the minerals (feed-I), sugars, amino acids and vitamins (feed-II) after treating under stress conditions for ethanol fermentation by shake flask method. Drying of baker's yeast causes rupture of cytoplasmic membrane and brings about changes in the composition of the nucleic acids, proteins, lipids, carbohydrates. The re-hydration provides the moisture level for the growth and metabolic activity (Alpas et al., 1996). Activation of yeast strains in malt medium supports the higher cellular mass and enzymatic activity. Sub culturing of lyophilized

cultures and synthetically prepared modified MYPGA in which peptone was replaced with Yeast Nitrogen Base i.e. Malt extract Yeast extract Yeast Nitrogen Base Agar (MYNGA). From these media, MYNGA was found a better medium because it contains yeast nitrogen base, which includes ammonium sulphate, asparagines, minerals, vitamins, trace elements and amino acids provides a good nitrogen sources as well as other growth factors for carbon assimilation and fermentation (Walker, 1997; Edgerton, 2001). The activated cultures were grown in basic medium e.g. ME broth Age and size of inocula were observed after four hour incubation, which revealed 72 hr age of test strains. This is due to the fact that the growth of the starter reaches its log phase, having faster metabolic activity and the optimized volume of inocula of active yeast culture competes more for available nutrients. Addition of various inorganic compounds in optimized concentration range could result the enhanced growth and have the stimulatory effects on ethanol production

NS 3 aO

Kl C

( H) N4

A. A. Noor and M. U. Dahot

46

during fermentation process. Growth rate and fermentation rate are not inversely proportional. This is due to the fact that the starter culture requires the specific growth rate, time, nutrients and other miscellaneous parameters for their enzymatic activity. Our observations pertaining to the size of inocula are in agreement with the results of Choudhary et al., (1986), Panday and Agarwal (1993), Noor and Hameed (1997). According to Stanford and Anslow (1996) the hydrogen ions such as protons are very significant for both intracellular and extracellular pH which has a dramatic influence on growth and metabolism of yeast cells. The effects of various pH ranges are due to the weak acids, which dissipate plasma membrane proton gradients and depressing cell pH when they dissociate into ions in the yeast cytoplasm. On the other hand, medium chain fatty acid causes rapid cell death by disruption of cell membrane integrity during growth. Oxygen is used for respiratory metabolism, temperature is important for inoculum development, maintenance of fermenter productivity in industrial ethanol production (Noor et al., 2003). An initial increase at 37°C showed increase in growth but the final cell mass concentration attained very low (Sedha et al., 1984, Williams and Munnecke, 1993). In present study, the maximum growth of yeast strains was observed at 30°C when incubated for 72 hours, which shows same pattern as reported by Tabera et al., (1985), Elahi et al., (1989), Enhanced growth of test strains S5 and S6 was observed when incubated in orbital shaking incubator adjusted at 110 rpm. This is because agitation speed provides oxygen supply, which activates the mitochondria for cyclic metabolism and also providing uniform distribution of nutrients. Agitations and temperatures beyond the optima results in cellular damage Choudhary et al., (1986). During the optimization studies of growth in various chemicals, it was observed that the maximum growth occurred in between 0.2 - 0.6 % (w/v) of ME broth. Concentration of minerals beyond the optima is toxic for the growth of yeast (Walker and Maynard, 1997; Gadd and Laurence, 1996; Avery et al., 1996). Phosphorus is essential for all yeasts which are present in nucleic acid and phospholipids.

The major part of sulfur is inorganic orthophosphate (Aiking and Tempest, 1976; Theobald et al., 1996a) which acts as a substrate and effectors for many enzymes, including those involved in energy transduction depending upon the levels that fluctuate according to the mode of sugar catabolism (Theobald et al., 1996b). It is reported that chloride transport occurs via a proton-chloride or sodium chloride symport mechanism which may be involved in yeast cell water content (Andre, 1995). Divalent cations in yeast cells act primarily as enzyme cofactors. Mg2+ ions transport mechanism is involved in regulating free Mg2+ ion in the yeast cytosol (Beeler et al., 1997). Manganese is essential for yeast growth and metabolism in trace concentrations, which may accumulate in vacuole and act as an intracellular regulator for key enzymes (Kihn et al., 1988; Auling, 1994). Ca++ ions have been linked to the cell cycle regulation and implicated in the transition from lag to exponential phase in batch cultures of S. cerevisiae (You att, 1993; Loukin and Kung, 1995). Potassium and magnesium are regarded as macro elements required to establish the main metallic cationic environment in the yeast cell. Copper and iron act as cofactors in several enzymes including the redox pigments of the respiratory chain (Joslyn, 1941; Walker, 1997). These undergo unique chemistry due to ability to adopt distinct redox states, either oxidized (Cu-II) or in the reduced (Cu-I) state (Maria et al., 1999). It is involved in a variety of biochemical processes, such as cytochrome oxidase, Cu, Zn, superoxide mutase, lysyl oxidase and dopamine-monooxygenase and also play a critical role in assimilation (David and Fred, 1999). They require traces of Zn2+ that is essential for structure-function of the enzymes during metabolism for example, the important terminal step enzymes in yeast alcoholic fermentation namely alcohol dehydrogenase is a zinc-metalloenzyme Zn2+ uptake is time, temperature and concentration dependent and saturable (Fuhrmann and Rothstein, 1968; Mowll and Gadd, 1983, White and Gadd, 1987). Sulphates are transported for assimilation into sulphur-containing amino acids like methionine and peptides like glutathione but no singular sulphate transporter gene has been identified in yeasts at present (Andre, 1995). Sulphur is required in the form of a variety of sulphur compounds e.g. for the biosynthesis of sulphur

Optimization studies of yeast feed for enhanced growth...

47

contaning amino acids. In S. cerevisiae (in most of the microorganisms and in plants) the formation of sulfite occurs through reductive sulfate assimilation which is the route of methionine and cysteine biosynthesis (Walker, 1997; Thomass and Surdin-Kerjan, 1997). Sulphur prevents enzymatic and non-enzymatic browning reactions and in free form it prevents microbial growth of undesirable microorganisms, which allows the yeast growth in diverse form when provided in liquid form under optimal physical growth conditions for required time (Morris and Haugh, 1956; Walker and O'Neill, 1990; Gimeno et al., 1992; Tudor and Board, 1993; Thomas, 1993). Growth media (5 and 6) were prepared by addition of minerals in optimized concentrations along with sugars, amino acids and vitamins, which supported the growth of test strains S5 and S6 respectively. Growth under stressed conditions revealed that various physical and chemical agents, radiations are responsible to inhibit the yeast growth when they are exposed to various durations and concentrations beyond the optima respectively. During the observations of thermotolerance, it was reported that temperature beyond optima shows the greater cellular damage i.e. cell viability declines because the yeasts are unable to regulate their internal temperature and the high temperatures provided to them resulting the disruption of hydrogen bonding and hydrophobic interactions leading to general denaturation of protein and nucleic acids (Coote et al., 1991, Shah and Hameed, 2004). Yeast growing quickly in glucose rich medium is more sensitive to heat and other stresses compared to stationary phase cells. This may be due to the fact that glucose carriers act as the sensors of glucose availability, which rapidly adjust their growth and metabolism to changing environmental conditions (Lagunas, 1993). Sub-lethal heat shock treatment of yeast leads to the induction of synthesis of a specific set of highly conserved heat-shock proteins known (Ruis and Schuller, 1995; Walker, 1997). Mild water stress in yeasts occurs during osmostress caused either by hyperosmotic shock or by hypoosmotic shock (Piper, 1995; Halsworth 1998) producing dramatic changes in membrane structural arrangements and permeability properties (Walker, 1997).

Saccharomyces cerevisiae strain S5 and S6 showed insignificant decrease in the growth when examined under stressed growth conditions and a complete decline stage after the specific time period and concentration respectively. This is due to the activation of transcriptional control elements (STREs) in S. cerevisiae. The high osmolarity glycerol (Hog1) gene product encodes a mitogen-activated protein kinase and the pathway starts with the activation of plasma membrane bound receptor proteins which act as osmosensors. MAPK cascades transducer signals trigger by heat shock and UV radiations etc. Certain stress metabolites play role as stress-protectant molecule including osmotolerant, antidesicant, chemical detoxicant, cryoprotectant and thermoprotectant (Hino et al., 1990; De Virgillo et al., 1994). Radiations cause DNA damage in S. cerevisiae. UV causes dimerization, nicks and other lesions. These cause intrachromosomal recombination in cell cycle-dependent manner and can inhibit certain membrane functions by affecting membrane lipid phase transitions and by interacting with photosensitive chromogenic molecules within the cell (Galli and Shiestl, 1995; Walker, 1997). Chemical stresses in yeast cells may cause cellular damages during growth and metabolism due to acetaldehyde and ethanol stress produced during growth and fermentation. Low ethanol concentrations are inhibitory to yeast growth and cell division, but higher concentrations can be lethal beyond the concentration 20% v/v. Watson and Cavicchioli (1983) reported that, exposure to ethanol causes the increased membrane fluidity and decreased membrane integrity. Ethanol and methanol toxicity is reduced to the increase of fatty acyl chain length, unsaturated fatty acids and sterols in the cell membrane, membrane lipids, palmitic acid and cholesterol, phosphatidylserine, ergosterol or campesterol, linoleic acid, yeast hull which supports enhanced growth, viability and nutrient uptake. Higher concentration of ethanol destroys enzymes and kills the yeast cells in the medium due to the accumulation of dissolved toxic products, which slows down and stops the growth of test strains. Methanol on the other hand did not allow the test strains to grow beyond the concentration 4-6 % (v/v) in the fermentation medium. This is because the test strains are not the methanol utilizing yeasts and are unable to metabolize by an oxygen-

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dependent oxidase to formaldehyde, which is then converted into dihydroxyacetone by DAH synthase enzyme. Our results are well acquainted with Minier and Goma, (1982). Magnesium (Mg2+) is a known substance that protects yeast from ethanol stress by reducing its fermentative activity (Dombek and Ingram, 1986; Birch and Walker, 1996; Ciesarova et al., 1996) and increasing the proton and anion permeability caused by ethanol (Petrov and Okorokov, 1990). References Aiking, H., and D. W. Tempest, (1976) Growth and physiology of Candida utilis NCYC 321 in potassium limited chemostat culture. Archives of Microbiology, (108): 117-124. Alpas, H., M. Ozilgen, T.F. Bozoglu, and S. Katnas, (1996) Mathematical modeling of survival and weight loss of baker's yeast during drying. Enzyme and Microbial Technology. (19): 348-351. Andre, B., (1995) An over view of membrane transport proteins in S. cerevisiae. Yeast (11): 1575-1611. Auling, G., (1994) Manganese: function and transport in fungi. In metal ions in fungi (eds. G., Winkleman and d. Winge), Marcel Dekker, New York, 215-236. Avery, S. V., N.G. Howlett, and S. Radice, (1996) Copper toxicity towards Saccharomyces cerevisiae: dependence on plasma membrane fatty acid composition. Applied and Environmental Microbiology (62): 3960-3966. Bawa, D., and O. Yoshiyuki, (1992) Promotive action of ceramics on yeast ethanol production, and its relationship to pH, glycerol and alcohol dehydrogenase activity. Appl. Microbial Biotechnol., (36): 717-721. Beeler, T., K. Bruce, and T. Dunn, (1997) Regulation of cellular Mg 2+ by Saccharomyces cerevisiae. Biochimica Biophysica Acta1 (323): 310-318. Birch, R., and G. M. Walker, (1996) The role of magnesium in yeast stress responses. Abstracts of the 9th International Symposium on Yeasts (Sydney) 46-47. Birch, R.M., M. Ciani, and G.M. Walker, (2003) Magnesium, Calcium and fermentative metabolism wine yeasts. J.Wine Res.14(1): 3-15.

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Sindh Univ. Res. Jour. (Sci. Ser.) Vol: 40 (2) 51-54 (2008)

SURJ

Sindh Univ. Res. Jour. INFLUENCE OF TRICHODERMA HARZIANUM AND AZOTOBACTER ON DECOMPOSITION OF ORGANIC SUBSTANCES OF STRAW AND FLAX OF WHEAT IN TATARISTAN, RUSSIA G. A. Maka and F. Alimova* Departmentof Pharmaceutics, Faculty of Pharmacy, University of Sindh Jamshoro.

(Received 10th October 2008 and Revised 22nd November 2008) Abstract The study was carried to observe the effect of Trichoderma harzianum and Azotobacter on decomposition of organic substances of straw and flax in Tataristan Russia. The results indicated that the additional inoculation of cellulose decomposing microorganisms with straw and flax of wheat had increased the process of decomposition of organic substances. The sterilized and non sterilized straw and flax were used. Trichoderma harzianum in comparision to Azotobacter has shown high activity in non sterilized straw and flax. Keywords Trichoderma harzianum and Azotobacter, in Tataristan Russia, in comparision to Azotobacter

1.

Introduction The cereal straw is the crop residues. It Adebowale et al., (1989) from Microscopic *Department of Microbiology, Kazan State University Tataristan, Russia. examination is the feed source of ruminant live stock in many of cell wall of Graminaceae during the microbial countries. To improve the quality of straw for effect showed that some wall types and parts of the good production of live stock is yet an individual walls are more easy to attack than important issue (Preston and being 1987). other (Chesson A. et al., 1986) At the farm level the use of ammonia treatment has not became In soil microorganisms get the carbon economically successful (Devendra 1991). and energy from plant residues and the nitrogen fixation also take place with the decomposition Azotobacter in soil is a cellulolytic of plant residues (Rice et al., 1972). Straw was diazotroph producing organism and with treated physically and chemically to improve its association of Trichoderma resulted in the quality. (Sundstol 1974). Straw fractions are utilization of plant residues as source of carbon nodes, internodes and leaves. The ratio of these and energy for nitrogen fixation. (Halsall et al., fractions depend upon the kind of species and 1986). environmental factors. Such as maturity at Therefore the biological method of harvest, soil and climate conditions etc. The straw treatment is an alternative to the chemical wheat straw has the different percentage of (Jackson 1978). The cellulose decomposing fraction as node 4-8, internode 54-73 and microorganism are widely distributed in nature leaves 20-41(Shand et al., 1988). Therefore the which play important role in the process chemical composition of straw changes of conversion of organic substances and according to consequence (Theander 1984). mineralization of soil components. Some of the important representative are verticillium, The chemical composition of leaves and Trichoderma, fusarium, Alternaria and nodes is similar that the hemicelluloses is more Aspergillus.

in both but internode has more cellulose. The silica is highest in leaves (DEs. Thiago et al., 1982). Trichoderma harzianum play role for

G. A. Maka and F. Alimova

100ml of medium. Method 2gm of sterilized and non sterilized straw and flax were placed in Petri plates

52

nitrogen fixation by utilizing cellulose as a carbon and energy source. Trichoderma harzianum produce extra cellular enzyme called as cellulose and makes able the diazotroph to use cellulose as a carbon and energy source (veal et al., 1984). The cell wall of straw contain amount of carbohydrates, small quantity of protein and minerals (walls et al., 1988). 2. Material and Method Organism Trichoderma harzianum, a cellulolytic fungus was isolated from green house of Tataristan Russia. Grown on Potato dextrose agar. The Azotobacter was obtained from the laboratory of the department of microbiology, Kazan state University of Russia. It is nitrogen fixing bacteria which promotes the soil fertility. Media Stock cultures of Trichoderma harzianum were maintained on slopes of potato dextrose agar media. Azotobacter grown on Azotobacter basal media (K2 HPO4 1.0g, MgSO4 0.2g NaCl 0.2g, FeSO4 0.005g Agar 15.0g tap water 900ml). Inocula Preparation Plates of Trichoderma harzianum which showed good spore production after 12-18 days were used as inoculum. To each plate 10 ml of sterile distilled water containing 08-10 glass beeds were added and spore dislodged by gentle agitation. Than spore suspension was centrifuged (2000 rpm) for ten minutes. After that resuspended in potassium phosphate buffer pH 7. Suspension (100µl) containing 1.5x107 spores (Helber chamber count) was used to inoculate 100 ml of medium. The colonies of Azotobacter were transferred from stock slopes to liquid medium (10ml) and incubated at 27°C for 24 hours. After that culture was centrifuged and re-suspended in Potassium phosphate buffer pH 7. The culture (100L) containing approximately 3x107 cells (Helber chamber count) was used to inoculate

separately, moistened with 2ml of nitrogen maintained solution and experiment was repeated nine times and then inoculated with two drops of fourteen days old suspension of fungal culture in every Petri plate and incubated at 22 °C up to seven days. The Azotobacter was also inoculated together, individually and repeated in same method. After incubation, dried and finally the decomposed mass was determined quantitatively. Result and Discussion. It was observed that inoculation in sterilized straw the Trichoderma harzianum increased the decomposition of straw organic substances. The Trichoderma harzianum showed 4% difference of decomposed in relationship of control. The quantity of decomposed substances was 72mg. In combination the decomposed effect was more that was 95 mg and percent was 5.2 in relation with control (Tab-1).The non sterilized straw result was 152 mg of decomposed organic substances by Trichoderma. And its percentage was 8.2 with the relationship of control. In combination of Azotobacter and Trichoderma result was 115 mg of the decomposed organic substances the percentage was 6.2 with the relationship of control (Table-2). In the sterilized flax the Trichoderma harzianum showed result of decomposed organic substances 70mg. It was the 37 percentage in the relationship of the control but in combination with Azotobacter the result was 123 mg. Which was the 6.8% in relationship of control (Table-3). In non-sterilized flax the result by Trichoderma harzianum was 95mg of the decomposed organic substance. It was 6.2 percent in the relationship of the control. In combination with the Azotobacter the result was 135 mg of decomposed organic substances. It was 9.1 Percent. 1 in the relationship of control (Table-4). The results have the difference between sterilized and non sterilized straw and flax. In non sterilized straw, the combined 3.

decomposition result was more and also flax non sterilized result was more. The result had the difference between sterilized , non sterilized, combine and individual use of Trichodrema

Influence of trichoderma harzianum...

combination with Azotobacter. The aim to calculate the degradation of straw and flax by both organism, the method showed a new insight in to the mode of action. Finally our results are

53

harzianum. It is due to the presence of own flora of straw and flax.In this work Trichoderma harzianum inoculated individually and in

coin size with the results of (Weimar et al., 1985).

Table-1. Decomposition of organic substances of straw and flax after inoculation of Trichoderma harzianum and Azotobacter. Inoculation of straw and flax decomposing microorganism STERILSED STRAW Trichoderma harzianum Trachoderma harzianum and Azotobacteria Loss of mass after decomposition mg 81 99 Contents of organic substance in decomposed straw. mg 1780 1757 Table-2. Inoculation of straw and flax decomposing microorganism Loss of mass after decomposition mg NON STERILSED STRAW Trichoderma harzianum Trichoderma Azotobacter Control Inoculation of straw and flax decomposing microorganism STERILSED FLAX Trichoderma harzianum Trichoderma harzianum and Azotobacter Inoculation of straw and flax decomposing microorganism Contents of organic substance via decomposed straw. mg Quantity of decomposed organic substance. mg % 174 128 90 1700 1737 1852 Table-3 Contents of organic substance in decomposed straw. mg 1853 1800 Table-4. Loss of mass after decomposition mg NON STERILSED FLAX Contents of organic substance via decomposed straw. mg Quantity of decomposed organic substance. mg Difference of decomposed substances in relation of control % 152 115 88 Quantity of decomposed organic substance. mg 70 123 8.2 6.2 100 Difference of decomposed substances in relation of control % 3.7 6.8 Difference of decomposed substances in relation of control Quantity of decomposed organic substance. mg 72 95 Difference of decomposed substances in relation of control % 4.0 5.2

Loss of mass after decomposition mg 81 130

Trichoderma harzianum Trichoderma harzianumand Azotobacter G. A. Maka and F. Alimova Control

88 150

1800 1740

95 135

6.2 9.1

54 44 1923 42 100

References Adebowale., E.A., E.R. Orskov, and P.M. Hotten, (1989) rumen degradaion of straw 8; effect of alkaline hydrogen peroxide on degradation of straw using either sodium hydroxide or gaseous ammonia as source of alkali Amin. Prod., (48): 553-559. Chesson, A., C.S. K. Stewart, K. Dalgaro, King (1986) degradation of isolated grass mesophyll, epidermis and fiber cell wall in the rumen and by cellulolytic rumen bacteria in axemic culture, J. Appl. Bacteriol. (60): 327-336. Devendra I.C. (1991) technology currently used for the improvement of straw utilization in ruminant feeding system in Asia paper presented at the workshop on the utilization of straw in ruminant production system. Kuala Lumpur, Malaysian agricultural research and development institute, 7-11. Hals all, D.M.S., A.H. Gibson, (1986) comparison of two cellulomonas strains and their interaction with azospirillum brasilense in degradation of wheat straw and associated nitrogen fixation. Applied and environmental microbiology (51): 855-861. Haerper, S.H.T., J.M. lych, (1984) nitrogen fixation by cellulytic communities at arobicanaeobric interfaces in straw. Journal of applied microbiology (57): 131-137. Jackson M.G. (1978) treated straw for animal feeding an assessment of its technical economic feasibility world animal review (28): 38-43. Preston T.R and R.A leng (1987) matching ruminant production system with available resources in the tropics and substropics. Penambul books, armidale Australia. Rice, W.A., S E.A. Paul, (1972) the organism and biological process involved in a symbiotic nitrogen fixation in water logged soil amended with straw. Canadian journal of Microbiology (18): 715-723. Sundstol, F. (1974) improvement of poor quality forages and roughage. In feed Sci. vol. no (13): 257-276. Shand, W.J., E.R Orskov, A.FL. Morrice, (1988) rumen degradation of straw 5. Botanical fraction and degradability of different varieties of oat and wheat straws Anim. Prod., (47): 387392. Theader, O., P. Aman (1984) in straw and other fibrous by products as feed. Ed. Sundstal and Owen. Elseevier. Amsterdam. 45-78. Thiago, DE.S., R.C. Kellaway (1982) botanical composition and extent of lignification affecting

digestibility of wheat and oat straw and paspalum hay. Amin. Feed Sci. Technol., (7):

71-81.

Veal, D. A., J. M lynch, (1984) associate cellutolysis and dinitrogen fixation by cocultures of trichoderma harzyium and clostridium butyricum. Nature (310): 695-696. Walli, T.K., E.R. Orskov, P.K., Bhargava, (1988) rumen degradation of straw 3. Botanical fraction of two rice straw varieties and effect of ammonia treatment. Amin. Prod, (46): 347-352. Weimer, P.J., W.M. Weston (1985) relatioship between the fine structure of native cellulose and cellulose degradability of cellulose comptexas of Trichoderma reesi and Clostridium the rmocellum. Biotechnol. Bioeng. (27): 1540-1547.

Sindh Univ. Res. Jour. (Sci. Ser.) Vol: 40 (2) 55- 78 (2008)

Sindh Univ. Res. Jour.

SURJ

M. Sadiq Malkani

MARISAURUS (BALOCHISAURIDAE, TITANOSAURIA) REMAINS FROM THE LATEST CRETACEOUS OF PAKISTAN Paleontology and Stratigraphy Branch, Geological Survey of Pakistan, Sariab Road, Quetta, Pakistan.

E. mail, [email protected] (Received 14th June 2008 and Revised 17th Sep. 2008) Abstract Marisaurus associated remains has been found from the latest Cretaceous Vitakri member of Pab Formation in Mari Bohri, Darwaza, Bor, Kinwa, Top Kinwa, Alam Kali Kakor and some other localitie of Sulaiman Basin, Pakistan. Marisaurus from Alam Kali Kakor locality provides the opportunity to view the most complete (more than one third) skeleton of titanosaur yet discovered. It is also hoped other remaining preserved assemblages can be found on further excavations at many sites. The Marisaurus (Balochisaurus and Pakisaurus) bear the associated cranial, vertebral and limb elements found from Pakistan, because previously no such associations are found except Rapetosaurus from Madagascar and a few recent discoveries from Argentina. It also helps to resolve the position of Mongolian genera like Nemegtosaurus and Quaesitosaurus. It provides new insights in to skull and postcranial morphology. Multipurpose tail special with trispinous distalmost caudal centrum of Marisaurus (and Balochisaurus) of Balochisauridae and some or all genera of Pakisauridae found from Pakistan provide a new look of titanosaurs. The skull discoveries of Marisaurus (and Balochisaurus) adds the general morphs of sauropod skulls like moderate inclined, elongate and camellate (with large open internal cells), in the previously reported two morphs like high and short, and low and elongate. Due to dearth of cranial data and lack of common associated elements in titanosaurs has left even the most basic skeletal morphology of the clade controversial and has precluded detailed study of its higher and lower level phylogeny, but now Pakistan has this advantage which produced associated cranial, axial and appendicular elements of Marisaurus (and also Balochisaurus and Pakisaurus). The Rapetosaurus, Marisaurus, Balochisaurus, Pakisaurus, some Argentinean and Mongolian forms have great potential to resolve paleobiogeography, lower and higher level phylogeny of titanosaurs. Keywords: Marisaurus, Balochisauridae, Titanosauria, Vitakri member, Pab Formation, Late Cretaceous, Pakistan, Malkani. Introduction From 1864 to 1999, India was the only source of Mesozoic vertebrates (Hislop 1864, Falconer 1868, Lydekker 1877, 79; Huene and Matley, 1933, Jain and Bandyopadhyay, 1997). Since 2000, about 3000 fossils collected by author from latest Cretaceous strata of Sulaiman Basin, Pakistan (Fig. 1A), which are the basis of Khetranisaurus, Sulaimanisaurus and Pakisaurus of Pakisauridae, and Marisaurus and Balochisaurus of Balochisauridae (Titanosauria, Sauropoda), Vitakridrinda abelisaurid theropod, and Pabwehshi baurusuchid mesoeucrocodile (Malkani & Anwar, 2000; Malkani et al., 2001; Malkani, 2003a,b,c; 2004a,b; 2006a,b,c,d,e; 2007a,b,c,d,e; 2008a,b,c,d,e; Wilson et al., 2001, 2005). Most of these materials 1. were found as fragmentary but seems to be associated due to clusters or heap forming occurrences. The Brohisaurus kirthari (Titanosauria) from the J/K boundary of lower Indus (Kirthar Range) Basin has also established (Malkani 2003c). The trackways of wide gauge titanosaurian sauropods (Malakhelisaurus mianwali) confronted by a running narrow gauge abelisaurian theropod (Samanadrinda surghari) found from the Middle Jurassic limestone of upper Indus (Kohat and Pothwar) Basin (Malkani 2007a). Marisaurus jeffi was reported first time on the basis of articulated partial skull (Malkani 2003a). The five genera and its two family of latest Cretaceous Titanosauria from Pakistan, which are

based on associated caudal vertebrae (especially mid-caudal) due to their most common and most diverse nature, presented at the occasion of 5th Pakistan Geological Congress (Malkani 2004a) and this classification is formally published in (Malkani M. Sadiq Malkani 2006b). Here associated and fragmentary remains of Marisaurus collected from Pakistan are being figured and described. 2. Systematic Paleontology Dinosauria (Owen, 1842) Order Saurischia (Seeley, 1888) Infraorder Sauropoda (Marsh, 1878) Titanosauria (Bonaparte & Coria, 1993)

Family Balochisauridae (Malkani, 2004a, 2006b) Marisaurus (Malkani, 2003a, 2004a, 2006b) Holotypic specimens and locality; The holotype of A Marisaurus MSM-7-15 biconvex first caudal, MSM56 29-15, MSM-30-15, MSM-31-15, MSM-32-15 and MSM-33-15 five fragmentary but associated caudal vertebrae were collected from Mari Bohri locality (Malkani, 2006b; fig.9-12). These remains seem to be associated with referred material of this locality. These specimens are housed at the Museum of Geological Survey of Pakistan, Quetta.

B

C

D

E

F

Marisaurus (balochisauridae, titanosauria)...

57

G

H

I

Fig. 1. A, Grey oval represents the study area in Pakistan. B, MSM-256-16 cervical/anterior dorsal vertebra, MSM-774-16, MSM-51116 ventral ridge forming tall dorsal vertebrae. C, MSM-153-16 caudal with prezygapophyses and postzygapophyses ? and neural spine. D, MSM-815-15, MSM-506-15 caudal vertebrae. E, MSM-359-2 cervical vertebra, MSM-441-2 dorsal vertebra. F, Generalized 5 types of caudals from Pakistan. G, Upper row, MSM-295-2, MSM-279-2, MSM-685-2, MSM-566-2, MSM-278-2, MSM-686-2, MSM-1029-2, MSM-688-2 proximal metacarpals; Lower row, MSM-277-2,MSM-1028-2, MSM-285-2, MSM-370-2, MSM-684-2, MSM-687-2, MSM361-2, MSM-683-2 distal metacarpals. H, upper MSM-643-2, and lower MSM-1031-2, MSM-1030-2 metatarsals. I, MSM-1036-16 distal metacarpal. Scale, Each black digit is 1 cm.

Referred specimens and localities; The referred specimen were collected from Mari Bohri (PDL-15), Darwaza Gumbrak (PDL-8), Kinwa (PDL-4), Top Kinwa (PDL-16), Bor (PDL-2), Alam Kali Kakor (PDL-19) and other localities. The partial skull (MSM-79-19, MSM-80-19) of Marisaurus was described Malkani (2003a; Figs. 2,3,4,5; here reconstructed Fig. 2A). The braincase (GSP/MSM-216 or GSP-UM 7000) was collected by the present author and described by Wilson et al., (2005; Figs. 2,3,4) and here referred to Marisaurus due to associated axial and appendicular remains of Marisaurus. The presacral vertebrae MSM-120-2 cervicodorsal, MSM-121-2, MSM-122-2, MSM-123-2, MSM-124-2, MSM-125-2 and a pair of sacral MSM135-2 found from the south-central site just on the base of resistant elongated ridge of Bor locality, and precaudal vertebrae MSM-131-16, MSM-13216cervicodorsal, MSM-131-16, and MSM-137-16 sacral pair, found from the Top Kinwa locality, and one presacral vertebra MSM-134-8 from Darwaz Gumbrak locality were described by Malkani (2006c; figs.2-5) and here being referred to Marisaurus due to associated caudal vertebrae and other elements. The association is mostly interpreted on the basis of heap or cluster occurrences of specimens. Five fragmentary but seems to be associated caudal vertebrae MSM-364, MSM-37-4, MSM-38-4, MSM- 39a-4 and MSM-394 from Kinwa locality and two fragmentary but associated MSM-34-16 and MSM-35-16 were described by Malkani (2006b:figs. 9-12). An articulated atlas-axis complex (GSP/MSM-82-4) of Marisaurus was described by Malkani (2008a; figs. 2-6). Two fragmentary but seems to be associated caudal vertebrae MSM-34-16 and MSM-35-16 from Mari Bohri locality, one fragmentary caudal vertebra MSM-40-8 from Darwaza Gumbrak, and two fragmentary but associated caudal vertebrae MSM-412 and MSM-42-2 from Bor locality were described by Malkani (2006b;figs 9-12). The following remains of Marisaurus are being referred and described here. The Alam Kali Kakor locality has yielded associated remains of Marisaurus (adult animal) including the partial skull

(articulated) (Malkani, 2003a), vertebral and limb elements. Cervical vertebrae (MSM-107-19, MSM108-19, MSM-109-19; Fig. 2B,C), (MSM-437-19, MSM-220-19, MSM-502-19; Fig. 4B); dorsal vertebrae (MSM-110-19, MSM-111-19, MSM-112-19; Fig. 2B,C), (MSM-617-19; Fig. 4B); caudal vertebrae (MSM-113-19, MSM-114-19, MSM-115-19, MSM116-19, MSM-117; Fig. 2B,C), (MSM-219-19, MSM218-19, MSM-696-19, MSM-777-19, MSM-221-19; Fig. 3B,C), left femur (MSM-213-19, MSM-118-19; Fig. 4A), proximal radius (MSM-215-19; Fig. 4A), Left proximal tibia (MSM-119-19; Fig. 4A), a pair of left and right distal tibiae (MSM-710-19, MSM-56919; Fig. 4A); a pair of left and right distal scapulae (MSM-1100-19, MSM-217-19; Fig. 3C), sternal plate partial ilia (MSM-216-19; Fig. 4B) and diapophysis (MSM-638-19c; Fig. 7A). A large number of fossils/pieces, which are covered by brown muddy matrix, are also collected from this site. The whole picture of this animal can be clear after cleaning of matrix from collected materials, and the possible further exploration and excavation at this site. Bor Kali Kakor locality yielded cervical (MSM-359-2; Fig. 1E), and dorsal (MSM-441-2; Fig. 1E) vertebrae, metacarpals (MSM-295-2, MSM279-2, MSM-685-2, MSM-566-2, MSM-278-2, MSM686-2, MSM-1029-2, MSM-688-2, MSM-277-2, MSM- 1028-2, MSM-285-2, MSM-370-2, MSM684-2, MSM-687-2, MSM-361-2, MSM-683-2; Fig. 1G), metatarsals (MSM-643-2, MSM-1031-2, MSM1030-2; Fig. 1H), proximal and distal femur (MSM178-2, MSM-182-2; Fig. 5A), distal scapula (MSM-

573-2; Fig. 5A), proximal ulna (MSM-271-2; Fig. 5A), proximal ischium (MSM-184-2; Fig. 5A ), humerus parts (MSM-850-2, MSM559-2, MSM-287-2, MSM-852-2, MSM-181-2, MSM-362-2, MSM-362-2,MSM-343-2; Fig. 5A),

sternal (MSM-565-2; Fig. 7A ), (MSM-1004-2; Fig. 7A ), coracoids (MSM-560-2; Fig. 7A), distal portion of dorsal rib (MSM-187-2; Fig. 7A), neural spines (MSM-301-2, MSM-792-2, MSM-784-2; Fig. 7A) and trispinous distalmost caudal centrum (MSM- 302-2; Fig. 6A,B,C).

M. Sadiq Malkani

58

Kinwa locality yielded mid-scapula with ventral ridge (MSM-838-4; Fig. 7A). Top Kinwa yielded cervical or anterior dorsal (MSM-256-16; Fig. 1B), dorsal vertebrae (MSM-774-16, MSM-511-16; Fig. 1B), caudal with prezygapophyses and neural spine (MSM-153-16; Fig. 1C), distal metacarpal (MSM-1036-16; Fig. 1,i), proximal ulna (MSM-17516, MSM-240-16; Fig. 5E), distal ulna (MSM-74-16; Fig. 5E), a pair of distal scapulae (MSM-250-16, MSM-176-16; Fig. 5G), part of ilia (MSM-557-16; Fig. 5H), (MSM-150-16; Fig. 7A), acetabulum (MSM147-16, 148-16; Fig. 7A), sternal (MSM-1014-16, MSM-604-16; Fig. 7A), metatarsal/phalanges (MSM149-16; Fig. 7A), cervical rib (MSM-328-16, MSM767-16, MSM-329-16; Fig. 7A), sacral (MSM-776-16; Fig. 7A), chevron (MSM-330-16; Fig. 7A), prezygapophyses (MSM-327-16; Fig. 7A) and armour plate (MSM-1035-16; Fig. 7A). Mari Bohri locality yielded caudals vertebrae (MSM-815-15, MSM506-15; Fig. 1D), a femur (MSM-169-15, MSM-7015; Fig. 5B), a distal scapula (MSM-163-15; Fig. 5D), proximal and distal pubis (MSM-165-15, MSM164-15; Fig. 5C). These specimens are housed at the Museum of Geological Survey of Pakistan, Quetta. Horizons; These remains are collected from the latest Pab Formation of Sulaiman Basin, Pakistan. The Pab Formation (20-400m) is divided in to Dhoala, Kali and Vitakri members. Dhaola member (white quartzose sandstone with minor to moderate black weathering; Fort Munro and Dhaola are best reference sections) represents the environments of proximal delta, near the coastline and consistent in the central and eastern Sulaiman Basin. Kali member (shale and black weathering sandstone; Dhaola and Tor Thana are reference sections) represent middle and distal deltaic environments and exposed in the western part of Sulaiman Basin. Vitakri member (15-35m, exposed in central system of middle Indus Basin; Dhaola and vitakri are best reference sections) consist of alternated two units of red mud/clay (2-15m each unit) of over bank flood plain deposits and two quartzose sandstone units (2-15meach unit) with black weathering of meandering river system. Lower red mud horizon is based on Kali member or Dhaola member and capped by middle sandstone horizon of Vitakri member. The upper red mud horizon is based on middle sandstone horizon and capped by a resistant sandstone horizon. Its coeval strata (coal, carbonaceous shale and sandstone) represent the lacustrine and deltaic environment, and laterite represent the erosional disconformity. Topography is semiplain to rugged. The relief of dinosaur host area varies from 800 to 2000m. Low hills capped by Vitakri Member with gentle dips produced many associated bones. The lateral extensions of dinosaur beds (30km Northwestnorth direction and 100km eastnortheast direction; gray oval in Fig. 1A) have been observed in

the Vitakri-Mari Bohri, Dhaola, Phulali (Pikal-Siah Koh), and Fort Munro anticlinoriums (Malkani, 2006d). The sauropods from the Vitakri member demonstrate extreme morphological variation in caudal vertebrae. Age; The age of the dinosaur beds (Vitakri) Member of upper Pab Formation is considered as the latest Cretaceous (Maastrichtian) (Malkani 2006d). 3. Partial skull of Marisaurus (Balochisauridae) from Pakistan Titanosaurian sauropods have one of the most extensive geographic and temporal ranges of any dinosaurian clade. However due to the persistent lack of associated cranial and post cranial material, a clear understanding of even their most basic morphological attributes remains elusive (Curry Roger & Forster, 1999; 2001). Traditionally, titanosaurids were considered as bearing a Diplodocus-type skull (Salgado et al., 1997). In contrast with the wealth of data derived from the study of the postcranial skeleton, the information on titanosaurid skull morphology is scarce (Salgado et al., 1997). No complete titanosaurid skull has been found to date. A few well-preserved braincases and some fragments constitute all the available material attributable to titanosaurids (Salgado et al., 1997). Cranial evidence links titanosaurids with other sauropods is poorly understood. In this regards, an important question should be answered: do titanosaurids have diplodocus type skull, as previously stated? (Salgado et al., 1997). But here the associated materials such as partial skull (articulated), and post crania of Marisaurus have a vantage point to solve most of the questions and problems. The partial skull is reconstructed (Fig. 2A) by the specimens MSM-79-19 and MSM-80-19 (Malkani, 2003a). These specimen were collected 50 meters apart towards east from its postcranial material, which may belongs to same animal. It may be dragons away by the Vitakridrinda theropod abelisaurs because the skull of Vitakridrinda abelisaurian theropod (Malkani, 2006e) is found near the skull of Marisaurus. The braincase (GSP/MSM-2-16; Wilson et al, 2005) of Kinwa Top locality, which is found about half km toward north from Marisaurus skull locality. An atlasaxis complex (Malkani, 2008a), which is found 1 km apart towards west from Marisaurus skull locality. On the basis of the matrix, horizon/age, and shape, this partial skull, braincase and atlas-axis complex may belong to Marisaurus. The size of skull matches with the appendicular and axial fossils from Alam locality but matrix is slightly different. These skull specimens represent large internal open cells like Kinwa atlasaxis complex and Marisaurus cervical of Alam locality, which is also a basis of its assignment to Marisaurus.

Marisaurus (balochisauridae, titanosauria)...

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4. Vertebral materials of Marisaurus (Balochisauridae) from Pakistan Marisaurus jeffi based on first biconvex caudal centrum; moderate short, heavy and slightly tall with broad ventral groove (u shape) on posterior parts of anterior caudal centra (except some anteriormost caudals) due to well developed posterior chevron facets; moderate short, heavy and slightly tall with broad ventral groove (u shape) of mid-caudals due to well developed posterior and faint anterior chevron facets; the ratio of dorsal view width to mid-ventral width of anterior caudal centra (except some anteriormost caudal centra), and mid-caudal centra is around 1.5; lateral surfaces on ventral view are clearly observed in the anterior and mid caudals due to middorsal width greater than ventral width; robust femora; thick transversely proximal tibiae; narrow lateral width (mediolaterally elongated oval shape) of distal tibiae; and mid-mediolateral width is greater than midanteroposterior width of distal tibiae (Malkani, 2006b). First time, the caudal vertebrae of 3 genera of Pakisauridae and 2 genera of Balochisauridae Titanosauria from Pakistan were described and figured by Malkani, (2006b). Second time, the sacral and presacral vertebrae of Pakistani Titanosauria are reported and figured by Malkani, (2006c). Third time, an articulated atlas-axis complex of Marisaurus are figured and described (Malkani, 2008a). Fourth time it is being descried here. The precaudal vertebrae are recovered from Alam, Bor, Top Kinwa and Darwaza localities. The caudal vertebrae are recovered from Mari Bohri, Kinwa, Alam, Bor, Top Kinwa, Darwaza, and some other localities. The Alam locality has produced the associated materials of Marisaurus including about 6 cervical, 4 dorsal, and 10 caudal vertebrae along with partial left femur, partial left and right tibiae, and partial radius, a pair of partial distal scapulae, partial sternal plate/ilia, etc. Although fragmentary and disarticulated but it is associated because all of these material is found within one site of same locality. The field study show that these materials are associated due to following reason. Some vertebrae are found aligned according to strike direction in about 10 m length and appendicular elements were some 3 m down slope. However, some vertebrae were little transported down slope. There is no any duplication from this site. The field evidence indicate all these materials are found in situ host formation and excavation can reveals remaining preserved assemblages. The nature of matrix, relative size and collection from one locality represents association and belongs to same adult animal. However a partial articulated skull (Malkani, 2003a) is found 50 meter apart from the axial and appendicular elements and relative size show one animal but their matrix is slightly different. Top Kinwa and Bor localities have produced associated vertebral and appendicular elements. Mari Bohri locality has also

yielded holotypic and some referred caudal vertebrae, a femur, a pubis and a distal scapula, which may belong to one animal. Cervical vertebrae; Axis parapophysis is robust and rectangular (posteriormost part is subrounded) in shape. Axis parapophyses are located in the central and posterior half of the ventral surface of centrum. The parapophysis is rectangular, and positioned on the ventral surface of axis centrum. In remaining cervicals parapophysis is becoming anteroposteriorly elliptical, and position is shifting to the mid and then anterior of centra as the centra proceeding posterior. The parapophyses seems to directed outward and downward. It begins to travel anteriorly as the series going backward (toward posterior). The rectangular nature of parapophysis is becoming oval as the series going backward of cervical series. The centra have almost flat and slightly concave ventral surface because anterior cone and posterior articular surfaces are trending to lower level than central surface of centra. Centra are broad, long and opisthocoelous. Pleurocoel is small and elongated. The centra are constricted in the middle and successively increase in length. The posterior concavity seems to be larger than anterior convexity. The broadness and length is increasing much backward in all discontinuous series. The transverse processes are directed laterally. The well developed diapophysis have small diameter in the anterior most cervical but become some what relatively with larger diameter and stouter, projecting outward and little upwards as proceeding backward in the series. The prezygapophyses are becoming wide divergent backward depending on the broadness of centrum. The neural canal is uniformly rounded to sub rounded in all the cervicals. The fragmentary neural spine is single and seems to be low. The postzygapophyses are high and divergent and may change position according to prezygapophyses. The cervical vertebrae have camellae/pneumatic texture. The fragmentary pieces of cervical ribs are collected having boat shape. These ribs are pneumatic (large open internal cells). Measurements of some cervical centra are shown in (Table 1).

Table 1. Measurement of cervical centra in centimeters. Sample no MSM-107-19 MSM-108-19 MSM-109-19 Length 18 28 30 Width 18 18 Height 13 13 -

Dorsal vertebrae; There are three opisthotic morphs. First morph is short and broad centra with smooth ventral surface, open wide pleurocoel and small ventrolateral surface below the pleurocoel, as characterised by the first cervicodorsal vertebrae and may be found in other anterior dorsals. Second morph is short and circular centra, which may be in the

transition between the anterior and posterior dorsals as hemisperical to cylinderical centra. Third morph is tall and relatively long and has sagital ventral ridge/keel, and narrow and long pleurocoels may found in posterior dorsals. Posterior concavity seems to be larger than anterior convexity. From morphology I can guess that the transeversely hemisphericity decreases up to mid dorsal. In posterior dorsal the transeversely hemisperity shifted to the ventrodorsally hemisperity. The broadness is going to be less i.e., transeverse ellipticity is going to be low as going backward. Early middle centra may be circular. The remaining middle and all posterior centra seems to be tall and relatively

long and having ventral saggital ridge and long pleurocoel. Keel/ridge may be transition from dorsal to sacral. The broken dorsal vertebrae represent the camellae/spongy/ pneumatic texture. The neural canal is subrounded. Parapophyses come close to diapophyses in mid dorsals. There are interprezygapophyses lamina, centro diapophyseal laminae, post zygapophyses and spine laminae. The postzygapophseal lamina is directed backward to the postzygapophysis and the supradiapophseal lamina is directed upward to the spine. There are two-separated prespinal laminas.

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A

B

C

Fig. 2. A, reconstructed partial skull. B, Upper (top) row, MSM-107-19 cervical vertebra, left posterolateral view, MSM-108-19, MSM-109-19 cervical vertebrae, left lateral view; Middle row, MSM110-19, MSM-111-19, MSM-112-19 dorsal vertebrae, left lateral view; Lower row, MSM-113-19, MSM114-19, MSM-115-19, MSM-116-19, MSM-117-19 caudal vertebrae, right lateral view; C, Upper row, MSM-107-19, MSM-108-19, MSM-109-19 cervical vertebrae, anterior view; Middle row, MSM-110-19, MSM-111-19, MSM-112-19 dorsal vertebrae, anterior view; Lower row, MSM-113-19, MSM-114-19, MSM-115-19, MSM-116-19, MSM-117-19 caudal vertebrae, posterior view. Scale, Each black digit is 1 cm.Most of the specimens in this research are partial. Specimen numbers started in each row from left to right. The specimen number like GSP/MSM-107-19 represents GSP Geological Survey of Pakistan; MSM collector name (M. Sadiq Malkani); 107 represents sample number; 19 represents Pakistani Dinosaurs locality number or PDL-19.

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A

B

C

Fig. 3. A, MSM-219-19, MSM-218-19, MSM-696-19, MSM-777-19, MSM-221-19 caudal vertebrae, posterior view. B, Upper row, MSM-219-19, MSM-218-19, MSM-221-19 caudal vertebrae, right lateral view. Lower row, MSM-217-19 partial distal right scapula; MSM-696-19, MSM-777-19 caudal vertebrae, posteroventral view. C, MSM-1100-19, MSM-217-19 a pair of left and right partial distal scapula. Scale, Each black digit is 1 cm

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A

B

anterior. The prezygapophyses and postzygapophyses are placed quite wide in anterior dorsals while placed close in posterior dorsals. There is no hyposphene-hypantrum in any of the collected dorsals. The neural spine is

Fig. 4. A, Upper row, MSM-215-19 proximal radius, posterior view; MSM-119-19 proximal left tibia, lateral view; MSM-213-19 proximal left femur, ventroposterior view; Lower row, MSM-569-19 distal right tibia, ventral view; MSM-710-19 left distal tibia, ventral view; MSM-118-19 left distal femur, posteroventral view. B, Upper row, The transverse MSM-220-19, MSM-502-19 cervical vertebrae left lateral upward. Therow, MSM-617-19 are quite MSM-437-19, processes are robust and directed laterally and little view; lower articular facets dorsal prominent in all lateroventral view, MSM-216-19 sternal plate/ partial ilia. Scale, the parapophyseal 1facets from the vertebra, right the dorsals. There is a gradual change in the position of Each black digit is cm.

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undivided. The spine is directed perpendicular to the neural canal and may slightly inclined backward. Some rib pieces are also collected. The tuberculum and capitulum are in sub equal size. Capitulum is subrounded while tuberculum is oval to suboval. Both are marked with a notch in between. There is a marked ridge running along the length of the rib shaft bifurcating just before the proximal notch and terminating toward the end of the capitulum and tuberculum. Medially there is a marked depression running anteriorly. The proximal part of the rib is spongy and concavo-convex in cross section and distally massive and plano-convex. Measurements of dorsal centra are shown in Table 2.

Table 2. Measurement of dorsal centra in centimeters. Sample no Length MSM-110-19 20 MSM-111-19 23 MSM-112-19 18 Width Height 16 15 17 15 16 14

5.A new look of titanosaurs: multipurpose tail special with trispinous distalmost caudal centrum found from Pakistan. The first and new tail special with trispinous caudal centrum which provide a new look of Titanosauria, has been found from the latest Cretaceous Vitakri member of Upper Pab Formation, Sulaiman Basin of Pakistan. Tail of titanosaurs from Pakistan is unique among World. It seems to be robust and relatively short which may act for multipurpose like good defending tool for its foe, balancing body as third support during foraging from tall tree, and mating. This tail resemble with Shunosaurus of China (Laurasia). This discovery has an advantage for evolutionary history from Middle Jurassic Shunosaurus to latest Cretaceous Pakisauridae and Balochisauridae (Titanosauria) from Pakistan (Gondwana). Five terminal caudal centra (Fig.6 A,B,C) of different genera are found from Barkhan District of Balochistan Province, Pakistan. These caudals show anterior surface with concave ring (Fig.1 A), like other caudal centra (except anteriormost caudal centrum in some genera) but their posterior, ventral and dorsal surfaces are specialized trispinous (Fig.6 B,C) forming unique character. Distalmost caudal centrum represents three enlarged and thick triradial spines (Fig. 6 B,C). Two spines are found in the ventroposterior region of posterior centrum which are diverging with low angle, while one spine is found on the mid of dorsal surface of centrum. The bases of spines are only preserved and remaining portions damaged. It may be broken in defending with its foe or taking support during foraging from tall tree or by erosion and weathering. The cross section of these spines at the base shows oval nature with maximum width varies from 3.5 to 6.5 cm and depth varies from 1.5 to 3.5 cm. The oval axis of dorsal spine shows two different directions (Fig.6 C). Three distalmost caudal centra show its oval axis trend posterolateral towards right ventral spine, while the other two trend posterolateral towards left ventral spine. It shows sexual dimorphism (male and female) or two genera or two rows of centra at terminal tail. The right and left ventral spines are symmetrical and directed lateroposteroventrally (Fig.6 C). The dorsal spine directed upward. Tail of titanosaurs from Pakistan seems to be robust (and relatively

Caudal Vertebrae; All the caudal vertebrae are strongly procoelous (except anteriormost and distalmost caudals). Both the height and width of caudal centra reduces much backward with relative to their length, which reduces less. Anterior caudals are broad while the mid caudals are slightly tall and posterior caudals are long and cylindrical with anterior circular articular and posterior squarish to circular posterior articular region. The chevron facets are not found on the anterior most caudals and posterior caudals, while located in the remaining anterior and middle caudals. The neural arch is situated on the anterior half of the caudal centra. The prezygapophyses are rod like while post zygapophyses also have lateral small surface for attachment. The middle and posterior caudal spines are laterally compressed and seems to be directed posteriorly. Prominent ridge facets occur which seems like blunted in anterior caudals. Measurements of some caudal centra are shown in (Table) 3.

Table 3. Measurements of caudal centra in centimeters. Sample no Length Width Height MSM-113-19 17 15 14 MSM-114-19 16 13 12 MSM-115-19 14 13 13 MSM-116-19 13 12 11 MSM-117-19 13 11 10

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short), due to robust distalmost caudal centrum. These tails may act for multipurpose like good defending tool for its foe like Vitakridrinda and Pabwehshi etc, balancing body as third support during foraging from tall tree, and mating. Since 2000 (first discovery to until) about 3000 (cranial and postcranial) fossils have been collected by present author from the latest Cretaceous Vitakri member (Dinosaur beds) of Pab Formation which are the basis for Pakisaurus, Khetranisaurus and Sulaimanisaurus of Pakisauridae, and Marisaurus and Balochisaurus of Balochisauridae Titanosauria, and Vitakridrinda of abelisaurian theropod dinosaurs, and Pabwehshi of baurusuchid mesoeucrocodylian fauna (Malkani, 2006b). A short tail of about 35 stout caudal vertebrae characterizes titanosaurs while primitive sauropods have about 50 caudal vertebrae and diplodocids have about 80 caudal vertebrae (Wilson, 2002). This shortened tail might have function as a third support when derived titanosaurs reared during feeding or mating (Borsuk-Bialynicka, 1977, Wilson, 2006). This idea is also strengthened by the present discovery. Sauropod is characterized by various tail specializations, including the fusion of the distalmost three or four caudal vertebrae in to a bony tail club in Shunosaurus (Dong, et al., 1989), and the short series of mobile, biconvex caudals in unnamed titanosaurs (neosauropod) from Argentina, which is modified in to a `whiplash' tail in diplodocids6. Terminal tail with trispines of Titanosauria from Pakistan is a unique record among world. The tail club of Shunosaurus, which appears to fuse late in growth, composed of atleast three enlarged, coossified caudal vertebrae with atleast two dermal spine (Dong et al., 1989; Zhang, 1988), while Pakistani Titanosauria has three spines on a single centrum. Present discovery shows clues of resemblance with Chinese form of Laurasia. Its temporal and spatial variation from Middle Jurassic (China; Shunosaurus lii) to latest Cretaceous (Pakistan; Balochisauridae and Pakisauridae) has an advantage for the hypothesis of evolutionary history and paleobiogeography.

6. Appendicular elements of Marisaurus (Balochisauridae) from Pakistan. Appendicular proportion is dominant with reference to cranial and axial elements. Here the most representative of limb elements are being briefly described. Sternal Plate; Partial fragments of sternal plates are collected. One piece which is expanded and represents the anteroventral crest. Pectoral girdle; Pectoral girdle is represented by fragmentary partial mid scapula and distal scapula, and coracoids. The thickness and width of middle part (and also probably proximal part) is remarkably less than distal ends. The outer surface represents slight convexing curvature and internal surface is concave, forming D shape cross section. One section of right scapula having ventral crest. Distal blade is expanded and represents broadening and thickening at the distal end forming a deep and spoon shape glenoid for humerus head, and nearby rugosity for the attachment of corocoid and developed into an area for the attachment of the muscles over almost the entire width.. Distal scapula and coracoid are separate. The acromian size seems to be narrow (and acromial edge is not expanded). The distal scapula of Marisaurus (and also Balochisaurus) is not deflected laterodorsally i.e., it is straight or slightly deflected medially and also not expanded in the lateroventral side of mid scapular blade, while the scapula of Pakisaurridae (Pakisaurus) is deflected laterodorsally and expanded in the lateroventral side of mid scapular blade. The coracoid showing glenoid for humerus and rugose area for attachment of distal scapula. The remaining area seems to be plate like. The corocoid glenoid area is at the same level as plate but elevated or raised in other side of plate Humerus; The anterior view of the distal humerus have prominent ridge, and posterior part is divided well in to two condyles. Humerus having a continuous ridge (convexing part) on the posterior view of humerus starting from head to downward may be running up to constricted middle part of humerus. Proximal humerus has head and represents deltopectoral crest which may be slightly twisted. The proximal humerus is slightly rugose while distal humerus has intense

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rugosities. Humeral midshaft cross section shape seems to be elliptical, with long axis oriented transversely. The supracondylar ridges are prominent forming well developed depression for the adjustment of prominent olecranon process. Ulna; The proximal ulna is rugose and bears a prominent olecranon process. The proximal ulna represents a triradiate structure. The ulna is gracile. There is a marked depression on the proximal lateral side for the adjustment of radius. It has also depression on the medial side also. The posterior side have slight depression and almost smooth. The ulna gradually tapers toward distal end which is subovate in outline and smaller than the proximal end. Distally the shaft has marked depression for the reception of distal radius. Ulna has well developed olecranon process. Relative length of Ulnar proximal condylar processes are unequal. Radius; Both the ends of radius are expanding well. Proximal end is elongated sub oval type, while the distal end is sub rectangle type. The proximal part show slight concavity or depression on proximal view. The distal end is expanded and is directed downward medially or makes medial inclination on the axis of length of radius bone. It also shows some smooth slight concavity on the distal view. Distal condyle flattened posteriorly and articulatind in front of ulna. Radius distal breadth is approximately twice the midshaft breadth. Radial distal condyle shape is beveled approximately 20-degree proximolaterally relative to long axis of shaft. Metacarpals; Metacarpals are elongated and triangular and have rugose articular proximal surfaces. Distal condyle shape is undivided and has no articular rugosities. A proximal and distal end is expanded with triangle shape. Pelvic girdle; Pelvic girdle is represented by partial ilium, pubis and ischium. Ilium; Partial ilium represented by acetabulum along with large pubic peduncle condyle (broken) and short and laminar ischium peduncle. Ventrally the acetabulum is long and oval and bears parallel ridges indicating calcified tendons. Up of the pubis peduncle a robust subrounded broken bone is located which may join to the

preacetabular process. The pubic peduncle is large and subovate or flat and D shape. In contrast the ischium peduncle is feeble. The acetabulum forms the embayment for the reception and movement of femur heads. The broken part of ilia represents the parallel spongy/pneumatic texture with large open internal cells. The preacetabular process of ilia is semicircular on one side and nearly straight in other side. Pubis; The proximal pubis bears a large glenoid surface for femur head. The anterior side of pubis is broad, cylindrical to sub oval, and posterior side is platy and possibly sigmoid. Proximal pubis has a ventral ridge. The distalmost end of pubis is slightly expanded and has rugose junction to its fellow. Ischium; Partial ischium is also found. The proximal part is platy having iliac condyle and long, thin acetabular glenoid and then articular surface for pubis. The proximal surface is thin as comparable to proximal pubis. Femur; Marisaurus (and Balochisaurus) has stocky femur. The slender femur is assigned to Pakisauridae (Pakisaurus) (Malkani, 2006b; 17a, 17b). The slender type has posterior elongated dominant rugosities starting from proximal part to downward constricted part, while the stocky type has no dominant rugosities. Proximal femora are beveled one third medially. Distal end is bifurcated in two condyles as tibial and fibular condyles. The tibial condyle is relatively more deep than fibular condyle while the fibular condyle is relatively more wide than tibial condyle. The tibial condyle is wheel like. The fibular condyle is posteriorly divided in two sub condyles. The distal femora may be divided atleast into two types like robust and slender. The cross section of shaft is elliptical eccentric and have constricted portion in the lower part. The constricted part of slender type seems to be relatively high. The parallel ridge on side seems to be running from proximal part/greater trochanter to downward. Femoral shaft is elliptical. It may become subcircular to circular in the minimum transverse diameter, relatively close to distal condyle than proximal condyle. In one type, the articular face shape is expanded onto anterior portion of femoral shaft. Femora show a posterior ridge on proximal shaft. The femora

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represent proximal one third medially deflected, laterally beveled knee condyles and transverse elliptical eccentricity, which may help for wide gauge tracks and trackways. Tibia; Two types of tibia are collected (Malkani, 2006b; fig, 18a,b, 19a). First type is elongate and thin/narrow proximal surface observed on proximal view and lunate distal surface observing on distal view, while the other has a broad and thick proximal surface and oval shape distal surfaces. Tibia cnemial crest orientation represent also two type of orientation, one is projecting laterally and others projecting anteriorly. Tibial distal posteroventral process is also two types, the size of first type is broad transversely and size of second type is reduced. The stocky tibia is assigned to Marisaurus and Balochisaurus. Fibula; A pair (left and right) of proximal fibula has been collected (Malkani, 2006b; fig. 19b). Proximal fibula have medial scar for attachments of tibia. The shape of fibular proximal tibial scar is well marked and deepening anteriorly. The distal fibula is oval shape. Fibular distal condyle size is not equal to shaft but expanded transeversely more than twice midshaft breadth. . Fibular distal condyle is broad transversely like also Barapasaurus. Metatarsals; Metatarsals represent irregular concave proximal and distal expanded surfaces. Unguals; The ungulas are sickle-shaped, much deeper dorsoventrally than broad transversely. Their assignments are in process. 7. Osteoderms of Marisaurus (and other genera) from Pakistan. Four types of dermal armour bones, which are lacking distinct pit patterns as in crocodilians, are referred to different genera from Titanosauria found from Pakistan. The armour plate found from Top Kinwa locality 16 is about 9 cm in diameter are made up of polygon like ossicles of irregularly shaped varying diameter ranging from half centimeter to 4 centimeters, separated by internal ducts of 1-3mm wide. This plate is 1-1.5 centimeters thick and partially preserved (Malkani, 2003b). This armour plate may belong to Marisaurus-a member of Balochisauridae due to bone association in the

field. Now another armour plate found from the same site (Fig. 7A) has above mentioned characters is also referred to Marisaurus. An oval plate (MSM-84-7) is sub circular plate which has slight concavity and rugosities on smooth ventral surface, and rugosities on lateral and dorsal surfaces. Dorsal surface has an asymmetrical low ridge/cone. The ossicles on the ridge area are tightly packed and fibrous like directing toward the tip of ridge making radial pattern, having centre at tip; and remaining dorsal and lateral area has irregularly shaped ossicles and internal ducts. The ridge seems to be directed dorsoposterior. The maximum length and width of ventral sub circular surface are 19 and 16 cm respectively; and dorsoventral depth of dorsal ridge from ventral surface to preserved apex is 12 cm. Apex/tip of cone/ridge is not preserved. This armour plate is being likely attributed to Balochisaurus due to field association with its caudal vertebrae. Another different oval plate (MSM-85-4) is also found. Its ventral surface is semi plain slightly convexing downward, with irregularly rugose ossicles and internal ducts pattern. The dorsal surface has well developed asymmetrical, low and sharp cone/ridge having possibly dorsoposterior directed tip/apex. The ossicles on the ridge area are tightly packed and course fibrous like directing toward the tip of ridge making radial pattern, having centre at tip. In the centre of dorsal surface of plate has a transverse median concavity belt just in front of ridge. The remaining dorsal and lateral surfaces have irregular rugosities of ossicles and internal ducts. The length and width of ventral oval surface are about 21 and 13 cm respectively, and dorsoventral depth of dorsal ridge/cone is 13 cm i.e., from ventral surface to apex or tip of ridge/cone. The third and fourth varieties have resemblance in shape but differ in nature of smooth, concave, convex, circular and oval ventral surfaces, and transverse median concavity belt on dorsal surface. The ventral surface of third type is slightly concave and smooth having corroded rugosities on circular ventral surface. The fourth type shows slight convexity and well developed, irregular rugosities on oval ventral surface, and it bears a transverse median concave belt on dorsal surface. This locality has produced diverse fauna of Pakisauridae and Balochisauridae. This type of armour plate probably belongs to Pakisaurus (Pakisauridae)

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because of resemblance to armour bone of basal Malawisaurus. The discovery of larger and smaller osteoderms from Pakistan provides a further opportunity for evolutionary history and correlation with the armor bones already discovered from Madagascar, Argentina, Brazil, France, Malawi, Romania and Spain. 8. Wide gauge skeletal morphology of Titanosauria found from Pakistan. Wide-gauge trackways have been defined as those in which manus and pes impressions are well away from the trackway midline, whereas those of narrow gauge tracks are close to or even intersect the trackway midline (Farlow 1992). Sauropod trackways are notable in that they document distinct gauges. Variation in track and trackway morphology of other dinosaur clades appears to be circumstantial rather than systematic, serving to differentiate speeds, sizes, or individual ichnotaxa. (Wilson and Carrano, 1999). Pakistani Titanosaurian Sauropod dinosaurs represent also the obligatory quadrupedal posture. The forelimb proximal segment like humerus, middle segment like the tibia and fibula, and the distal segment like metacarpals seems to be vertical or subvertical orientations. So the forelimb of Pakistani titanosauria represents columnar digitigrade posture. The hindlimb proximal segment like the femur (proximal part deflected medially), middle segment like tibia and fibula seems to be vertical or subvertical orientation, while the last segment (metapodials and unguals) may be semi plantigrade or plantigrade. But it is also known from morphology of appendicular elements that the Pakistani form is wide gauge quadrupedal. The theropod belongs to bipedal movements while Sauropod belongs to quadrupedal movements. Further among sauropod it is narrow gauge and wide gauge. The Pakistani titanosauria is a wide gauge, which is confirmed by the collected fossils of dinosaurs from Pakistan. The distal scapula in some Pakistani taxa is deflected laterodorsally creating more wide gauge movements while in some other Pakistani taxa the distal scapula is straight or inclined medially with less wide gauge movements. Further acetabulum orientation and horizontal preacetabular process can help for wide gauge. The femur of titanosaurian Pakistani forms have proximal part medially deflected, the

shaft have eccentricity represented by transversely greater width than anteroposterior width and the distal condyles are more inclined outward. These features like femur proximal deflected medially, femoral eccentricity, laterally beveled fibular condyle of femur, broad sacrum and ilia, laterodorsally deflected distal scapula and large sternal plates are basic theme going to wide gauge trackways. 9. Trackways; interaction among a theropod and a group of wide gauge titanosaurian sauropod from Middle Jurassic limestone of Pakistan. The trackways of wide gauge titanosaurian sauropods (Malakhelisaurus mianwali, renamed here due to previously engaged name Malasaurus) confronted by a running narrow gauge abelisaurian theropod (Samanadrinda surghari) found from the Middle Jurassic Samanasuk Limestone of upper Indus (Kohat and Pothwar) Basin. I report here the reanalysis of trackways representing interaction among a theropod and a group of wide gauge titanosaurian sauropod which were interpreted narrow gauge movements of sauropods along with overlaps of manus by large pes print (Malkani, 2007a.). The host limestone dip 520 west and strike north 50 east. These trackways were formed on coastal plain. These ichnofossils are deep footprints probably due to good granolometric sorting and the high plasticity of the limy substrate. In the vicinity, some footprints and tracks of possibly avian or nonavian small bodied theropods are also found. The footprint bearing strata are found in the upper successions of Samanasuk marine limestone, which show the slight regression of sea and area, was exposed as beach. This ichnocoenosis consists of exposed more than 15 footprints, and 4 short trackways. These trackways of titanosaurian sauropods trend in a southeast direction and are parallel to each other, which are confronted at about 1170 angle by a theropod trackway with north-northeast trend (Fig. 7 B). These are indicative of a wide gauge titanosaurian sauropods herd composed of 3 or more individuals defending the attack of a solitary predatory theropod. The footprints are well preserved and only show overprinting some in the back area. The exposed area is small like 60m2. The excavation along dipward can clear

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further these trackways and interaction among herbivores and carnivores. The large prints belong to titanosaurs are diagnosed on the basis of the D shape manus impression represents that metacarpals were arranged in D shape, and there is no indication of manus claw on digit I (Fig. 7 B). The pollex claw and all manual phalanges appear to absent in titanosaurs, and distal end of the metacarpals have reduced or lost the well developed articular surface present in most basal sauropods (Salgado, et al., 1997), thus the absence of the pollex claw impressions and wide gauge category (external trackway width, 340cm; internal trackway width, 60cm) in this site are the indication of titanosaur affinities. This site characterized the largest known pes trackmakers (pes length 150cm, width 140cm: manus length 60cm, width 70cm). The manus/pes heteropody area ratio is about 1/5 as compared with ratios ½ of middle Jurassic sauropods (Galinha, Portugal), 1/3 of Cretaceous Brontopodus birdi (Texas), and ¼ to 1/5 of Late Jurassic sauropods (Colorado) tracks (Santos et al., 1994). Manus without claw and pes with toes impressions are well preserved. The apparent absence of pes prints from many known sauropods trackways may be due to overlapping (Day, et al., 2004) but here the pes along with toes are best preserved (Fig. 7 B).The sagittal axis of the manus (passing through digit III) and pes prints show no rotation. However the right manus print of right side titanosaur is rotated inwards which may be due to danger of interaction. Theropod trackway is represented by large tridactyl prints with median symmetry. The pes print consists of three large weight bearing toes (digit II-IV) that are moderately divergent (Fig. 1). The other toed are not marked on footprints. The pes prints having maximum length about 60cm and width about 45cm are diagnosed only for a running large bodied probably abelisaurian theropod. Its assignment to lower level is difficult, because until now, no theropod fossils from these strata are collected. The common vertebrate fossils belong to titanosaurian sauropods (Pakisaurus, Khetranisaurus and Sulaimanisaurus of Pakisauridae; Marisaurus and Balochisaurus of Balochisauridae) and abelisaurian theropod (Vitakridrinda) dinosaurs, and mesoeucrocodylian (Pabwehshi) fauna from

the latest Cretaceous of Pakistan (Malkani, 2006b). Sauropod trackways are by far the most abundant and are representative of taxa that exhibit both narrow and wide gauge styles of locomotion (Day, et al., 2004). Narrow gauge limb posture, acquired early in sauropod evolution, was retained by all basal sauropods, diplodocoids, and basal macronarians (Day, et al., 2002). "The Titanosauria, in contrast, developed a number of derived skeletal features indicating that their limbs were held further from the midline of the animal, suggesting that this advanced clade of largely Cretaceous sauropods was responsible for wide gauge trackways (Wilson and Carrano, 1999). Both the narrow gauge sauropod tracks and those belonging to the theropods (Day, et al., 2004) include claw impressions. The large manus claw on digit I is found in diplodocoids and basal macronarians. In brachiosaurs [the sister group to titanosaurs (Upchurch, 1998)], the claw is somewhat reduced, and in titanosaurs, it is believed to have been entirely lost (Upchurch, 1998). The loss of manus claw is a derived state that supports the view that wide gauge trackways were made by titanosaurs" (Day, et al., 2002). "Several Middle Jurassic localities are reported as putative wide gauge tracks (Ishigaki, 1989, Santos et al., 1994); however, there is no evidence that these trackways were formed by titanosaurs, because they are associated with manus claw marks and in some cases are not truly wide gauge. Therefore the previous earliest substantiated "titanosaur" tracks are from the Late Jurassic (Wilson, and Carrano, 1999)" (Day et al, 2002). The oldest known remains of titanosaurs belong to Janenschia (Wild, 1991) and Brohisaurus (Malkani, 2003c) from the Late Jurassic of Tanzania and Pakistan respectively. Thus after the wide gauge sauropod tracks from Ardley (U.K), the Baroch site (Pakistan) is the second locality which provide the direct evidence that titanosaurs had diverged from other sauropods by the Bathonian. Baroch site is very significant due to following reasons. Middle Jurassic tracks and the vertebrate record in general are very sparse globally, therefore, any new information is valuable. These tracks of wide gauge Titanosauria and a running Abelisauria help for its early evolution. The manus impressions of Titanosauria are well preserved (even the manus has no claw)

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that help resolve a long standing discussion about the character of claw impressions associated with digit first (Beaumont and Dematheu, 1980, Farlow, et al., 1989). Heteropody ratios are hard to determine for many trackways due to absence of pes impressions (Day et al., 2004), while the present site has heteropody ratio 1/5 of titanosaur. Baroch site has much potential for picnic and academic point of view. It is the largest known pes trackmakers. It is among the largest known sauropod and theropod footprints. It is the first oldest locality which reveals the interaction among a titanosaurian sauropod herd and an abelisaurian theropod. It is another example of marine platform carbonates and fall in to Brontopodus ichnofacies. It adds considerably to the taxonomic composition of Middle Jurassic ecosystem. Many dinosaur groups radiated during the Middle Jurassic, replacing Late Triassic and Early Jurassic faunas (Weishampel, et al., 1990; Sereno, 1997). It represents the titanosaurian locomotion as herds while the theropod attacking solitary confirming the idea that the nonavian theropods were likely more solitary in nature, engaging in agonistic, ora like feeding aggregations (Roachand Brinkman 2007). It is the first oldest locality, which has highest depth of theropod footprint more than 10cm, and sauropod about 7 cm or more. Deeper footprints are formed due to increasing water content in the substrate. This tracksite of wide gauge titanosaurians from middle Jurassic limestone may be the second locality (after the Ardley site of UK) in the world and first locality in Asia. Morphological details such as digital pads or skin textures have not yet been recorded from any footprint (Day, et al., 2004), but this site has preserved pes digits especially ungual pads. As with extant mammals, titanosaurs may have formed a herd as part of defense against predation. The Baroch site adds significantly to our understanding of locomotor styles, potential trackmaker, evolutionary history, faunal diversity, echnology, behavior and social interaction of saurischian dinosaurs. 10. Discussions and conclusions Before 2000, India was the only source of Mesozoic vertebrates but since 2000 to so far about 3000 fossils are collected by author which include the Khetranisaurus barkhani (slightly broad midcaudals), Sulaimanisaurus gingerichi (squarish long midcaudals), and Pakisaurus

balochistani (tall midcaudals) of Pakisauridae, and Marisaurus jeffi (heavy, slightly tall midcaudals), and Balochisaurus malkani (heavy, short, slightly to moderate broad midcaudals) of Balochisauridae established, with the ratio of mid transverse width above and below of midcaudal centra which are less than 1, about 1, 1, 1.5 and 2 respectively (Fig. 1F). Further the stocky limbs (especially stocky tibia) and straight or slight medial deflection deflection of distal scapula is attributed to Balochisauridae, while slender limbs (especially slender tibia) and laterodorsal deflection of distal scapula may belong to one or more genera of Pakisauridae. The Saltasauridae (Titanosauria) may be considered for the extreme broad or dorsoventral compression of caudal centra. The Andesauridae include basal characters of titanosaurs like hyposphenehapantrum articulation and some caudals with mild procoelic nature. Many titanosaurs are named from India like Titanosaurus indicus, Titanosaurus blanfordi, and Titanosaurus (Isisaurus) colberti, Jainosaurus, etc however some fossils are also referred to Titanosaurus madagascariensis. Wilson and Upchurch (2003) have considered the genus Titanosaurus as invalid. Consequently the genus Titanosaurus and its species are considered nomen dubium and its family Titanosauridae was abandoned by them. The remains from Pakistan are diagnostic and can not be referred to Titanosaurus which is invalid, and so these remains have received the new family, generic and specific names like Khetranisaurus, Sulaimanisaurus and Pakisaurus, of Pakisauridae, and Balochisaurus and Marisaurus of Balochisauridae. The Saltasauridae are clearly differentiated from Balochisauridae on the basis of strongly broad anterior, mid and posterior caudal centra. The Pakisauridae and Balochisauridae are different from other families of Titanosauria. As a result Sulaimanisaurus or Isisaurus, Pakisaurus, and Khetranisaurus of Pakisauridae, and Balochisaurus and Marisaurus of Balochisauridae are distict titanosaur taxa that coexisted in Indo-Pakistan subcontinent just on or below the Cretaceous Tertiary Boundary. Sulaimanisaurus is different from Isisaurus on having trispinous distalmost caudal centrum.

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A

B F E

C

D

G H

Fig. 5. A, First column, MSM-178-2, MSM-182-2 proximal and distal femur; Second column, MSM-573-2 distal scapula, MSM-271-2 proximal ulna, MSM-184-2 proximal ischium; Third column, MSM-850-2, MSM-559-2, MSM-287-2, MSM-852-2 humerus parts; Fourth Column, MSM-181-2, MSM-362-2, MSM-343-2 humeus parts. B, MSM-169-15, MSM-70-15 proximal and distal femur. C, MSM-165-15 proximal pubis, MSM-164-15 distal pubis/ischium; D, MSM-163-15 distal right scapula; E, MSM-175-16, MSM-240-16 Proximal ulnae MSM-74-16 distal ulna. F, MSM-164-15 distal pubis/ischium; G, MSM-250-16, MSM-176-16 a pair of left and right distal scapulae. H, MSM-557-16 spongy may be part of ilia. Scale, Each black digit is 1 cm.

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A B

C

Fig. 6. Distalmost caudal centra with trispinous structure found from Sulaiman Basin of Pakistan. A, MSM-5213 (Shallghara Locality; Sulaimanisaurus), MSM-302-2 (Bor Kali Kakor Locality; Marisaurus), MSM-325-15 (Mari Bohri Locality; Balochisaurus), MSM-507-15 (Mari Bohri Locality; Marisaurus), and MSM-519-4 (Kinwa Kali Kakor Locality; Pakisaurus), anterior view. B, posterior view. C. MSM-325-15, MSM-507-15, right lateral view, MSM-507-15, posterior view. Scale, Each black digit is 1 cm.

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Fig. 7. A, First row, MSM565-2 anterolateral part of sternal, MSM-1004-2 sternal part?, MSM-147-16, 148-16 acetabulum part, MSM-150-16 part of ilia. Second row, MSM1014-16 convex part of sternal, MSM-604-16 sternal part, MSM-560-2 corocoid, MSM149-16 metatarsal/phalange, MSM-838-4 mid scapula with ridge. Third row, MSM-328-16 cervical rib, MSM-767-16 cervical rib, MSM-329-16 cervical rib, MSM-187-2 distal part of dorsal rib, MSM-63819c diapophysis, MSM-776-16 sacral. Fourth row, MSM-301-2 neural spine, MSM-792-2 neural spine, MSM-330-16 chevron, MSM327-16 prezygapophyses, MSM-784-2 neural spine, MSM-391-16 neural spine, MSM-1035-16 osteoderm. Scale, Each black digit is 1 cm. B. Trackways; Interaction among a running narrow gauge abelisaurid theropod, and a group of wide gauge titanosaurian sauropods, found from Middle Jurassic Limestone of Pakistan. Longer arrow shows the advancing movement of an abelisaur, while the smaller arrow shows the advancing movements of titanosaurian herd. Scale, hammer in photo or black digit which is 30 cm.

A

B

The Jainosaurus from India was named on the basis of braincase. The braincase from Pakistan (GSP/MSM-2-16 or GSP-UM-2007) assigned to Marisaurus, has many differences from Jainosaurus. The braincases from Bara Simla (ISI R 467) and Dongargaon (ISI R 199) in which the occipital condyle is ventrally inclined from skull roof are referable to Balochisauridae (Marisaurus and Balochisaurus), and from Bara Simla (GSI K27/497) and Rahioli (ISI R 162) in which occipital condyle is parallel to skull roof are referable to Pakisauridae (Pakisaurus, Khetranisaurus, and Isisaurus or Sulaimanisaurus) (Malkani, in process). According to Wilson (2002), the braincases with transversely broad basal tubera resemble with the Nemegtosaurus. Further it provides a clue that the Mongolian forms like Nemegtosaurus and Quaesitosaurus may belong to a slender limbed family Pakisauridae (Titanosauria). It also strengthens the above-mentioned assignment of braincases with narrow transversely basal tubera to Marisaurus (a stocky limbed family Balochisauridae of Titanosauria). With the few exceptions of new discoveries in Argentina, Madagascar, and Mongolia, most titanosaurs are known only from fragmentary postcranial skeletons and rare isolated skull elements.

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Titanosaurian postcrania are found worldwide, but their skulls are exceptionally rare. This nagging lack of association has left even the most basic skeletal morphology of the clade controversial and has precluded detailed study of the higher and lowers level phylogeny of the group (Curry Rogers and Forster, 2004). Titanosaurs with cranial materials are Ampelosaurus (teeth, dentary, 3 braincases), Antarctosaurus (braincase, dentary), Baja Santa Rosa (2 partial skulls), Jainosaurus (3 partial braincase, maxilla, squamosal), Jabalpur indet. (1 partial braincase), Lirainosaurus (partial basicranium, teeth), Magyarosaurus (partial braincase), Malawisaurus (partial skull, 2 dentary, teeth), Nemegtosaurus (partial skull, dentaries), Quaesitosaurus (partial skull, dentaries), Rapetosaurus (partial juvenile and adult skulls), Saltasaurus (1 juvenile braincase), and Titanosaurus indet. (Partial skull) (Curry Rogers and Forster, 2004). Recently Marisaurus (Partial skull), and Balochisaurus (Partial skull, in review) are also included. In fact, no titanosaur skull found in association with postcrania have ever been fully described or illustrated, and very instances of direct association of skull and postcrania have been documented. This nagging lack of association has left even the most basic skeletal morphology of the clade controversial and has precluded detailed study of the higher and lower level phylogeny of the group (e.g., Salgado et al., 1997; Upchurch, 1998; Wilson and Sereno, 2006, Curry Rogers and Forster, 2004). Titanosaurs comprised nearly half of all known sauropod genera, and were the only sauropod lineage to survive to the end of the Cretaceous. Their post crania are found worldwide, but their skulls are exceptionally rare. Cranial material is known only from ten titanosaurs and is limited to isolated elements and fragmentary braincases (Curry Rogers and Forster, 2004). The Marisaurus (and Balochisaurus, in review; and Pakisaurus, in review) bear the associated cranial, vertebral and limb elements, because previously no such associations are found in most derived titanosaurs except Rapetosaurus from Madagascar (Curry Rogers & Forster, 2004). It provides new insights in to skull and postcranial morphology. Due to dearth of cranial data for titanosaurs, the Rapetosaurus, Marisaurus, Balochisaurus, some Argentinean and Mongolian forms have great potential to solve paleobiogeography, lower and higher level phylogeny of titanosaurs. Marisaurus provides the opportunity to view the most complete (associated cranial, axial and limb elements) titanosaurs yet discovered. The skull of Marisaurus is articulated while the Rapetosaurus is disarticulated. The skull of Marisaurus is associated with vertebral and limb elements while Nemegtosaurus and Quaesitosaurus lack the postcranial articulation and association. Nemegtosaurus and Quaesitosaurus are known from skulls, and have been alternately placed within the Titanosauria or the Diplodocoidea. With regards to these Mongolian genera, Rapetosaurus is particularly illuminating because its elongated skull with retracted nares exhibits a general similarity to diplodocoids, whereas it's skeleton striking commonalities with brachiosaurids and other titanosaurs (Curry Rogers and Forster, 2004). Marisaurus offers an opportunity to test the monophyly of Titanosauriformes with abundant associated skull, axial and limb elements. It also offers the best opportunity yet to resolve the phylogenetic positions of two controversial Mongolian sauropods, Nemegtosaurus and Quaesitosaurus. Malawisaurus has short and high macronarian skull and amphicoelous caudals (except some anteriormost) vertebrae, which is differentiated from Marisaurus with moderate inclined and elongated Balochisaurids skulls and procoelous caudal (except biconvex anteriormost caudal) vertebrae. Karongasaurus has low and elongate macronarian skull, which is differentiated from Marisaurus with moderate, inclined and elongated Balochisaurids skulls. Marisaurus differs from the broad centra of Adamantisaurus, heart shape caudals and first procoelous caudal of Gondwanatitan, squared caudals of Isisaurus, squared caudals and first biconvex caudal of Baurutitan (Series C), slightly procoelous mid caudals of basal titanosaur Mendazasaurus. The ventral view of Trigonosaurus pricei (series B) from Peiropolis are not described and not figured (Campos et al. 2005). Marisaurus differs from the Austrosaurus which is more derived than Malawisaurus.

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Marisaurus differs from the broad caudal centra of Saltasaurus and Neuquensaurus, squared jaws and perpendicular dentary symphyses of Bonitasaura, perpendicular dentary symphyses of Mongolian taxa Nemegtosaurus and Quaesitosaurus, ventral crest of caudal centra of Bonatitan, and very long cervical vertebrae of Futalognkosauria. Marisaurus is most derived titanosaurs and differs from the basal titanosaurs like Haplocanthosaurus, Chubutisaurus, Amargatitanis, and Gobititan. Marisaurus also differs from the limb elements of Argyrosaurus. Marisaurus show a procoelous caudal vertebrae differentiated from basal forms like Andesaurus and Malawisaurus. Marisaurus also lack hyposphene-hypantrum articulation observed in basal titanosaurids like Andesauridae (Argentinosaurus, Andesaurus, Epachthosaurus). In general there are two types of skull among sauropods like Camarasaurus-type and Diplodocus-type. Camarasaurus-type skull can be characterized as a short, high skull with broad nares separated from each other by an arch formed by the premaxillae and nasals, quaderates nearly vertical oriented, basioccipital condyle posteroventrally directed, basipterygoid processes ventrally projecting and broad spoon shaped teeth inserted on the entire margins of the maxilla and dentary. On the other hand, the Diplodocus type is a long skull with confluent external nares located posterodorsally between the orbits, quaderate anteriorly inclined, basioccipital condyle ventrally directed, basipterygoid processes anteroventrally oriented and slender peg like teeth confined to the anterior part of the snout (Salgado et al., 1997). Traditionally, titanosaurids were considered as bearing a Diplodocus-type skull (Salgado et al., 1997). Calvo (1994a, 1994b) recognized the existence of at least four different tooth shapes: peg like teeth (in diplodocids), spoon like teeth (in camarasaurids), compressed cone chisel like teeth (in Brachiosaurus and Astrodon) and Chisel like teeth (in titanosaurids, Nemegtosaurus Nowinski and Quaesitosaurus Kurzanov and Banikov) (Salgado and Calvo, 1997). The wear facets are useful in investigating mechanisms of mandibular occlusion and feeding habits (Calvo, 1994a, 1994b). Prosauropods are characterized by the absence of tooth-to-tooth contact. In Camarasaurus each tooth contacts two teeth of the opposite jaw. Brachiosaurus and titanosaurids are similar in that one upper tooth can meet either one or two lower ones and vice versa (Calvo , 1994a, 1994b). Diplodocus is characterized by precise tooth to tooth contact (Calvo, 1994a, 1994b). Calvo (1994a, 1994b) pointed out titanosaurids resemble diplodocids by the slenderness of their crowns but they differ in the pattern of wear facets and tooth to tooth contact. However there are some indications of the probable Camarasaurus like skull. Powell, (2003) mentioned a premaxilla bearing slender peg teeth from Argentina is too high to correspond to a Diplodocus type skull. Further Huene and Matley (1933) mentioned a premaxilla which is also high. In this way, Jacobs et al. (1993) mentioned the complete premaxilla of the basal titanosaurids Malawisaurus dixeyii with doubled external nares which are placed anteriorly. Seeing the above mentioned discussion, the skulls of Balochisaurus (Malkani, in process), and Marisaurus (Malkani, 2003a, in process) can solve many cranial problems among world. The skull of Marisaurus is more incline than diplodocoids. However tooth distribution throughout the upper and lower jaws is more similar to those of macronarians like Camarasaurus and Brachiosaurus but skull inclination is less than these genera. The skull discoveries of Marisaurus and Balochisaurus adds the general morphs of sauropod skulls like moderate inclined, elongate and spongy (with large open internal cell) in the previously reported two morphs like high and short, and low and elongate. All analyses of titanosaur interrelationships have lacked a full body view for a diverse taxonomic sample due to the sheer lack of skeletal data for almost all titanosaur genera. Rapetosaurus is a keystone taxon that is most significant because it preserves cranial and postcranial materials (Curry Rogers 2005). Recently Pakistan has produced two articulated partial skulls and associated postcrania of Titanosauria which will also clear the morphology of titanosaurs. Pakisauridae (Pakisaurus) teeth on jaws are widely spaced than Marisaurus which are again more widely spaced than more derived Balochisaurus (Balochisauridae). This character adds help for evolution.

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References Beaumont, G.D. and G. Dematheu, (1980) Remarques sur les extremites anterieures sauropods (Reptiles, saurischians), Compt. Rend. Sci., (15): 191-198. Bonaparte, J.F., and Coria, R.A. (1993) Un neuo y gigantesco sauropodo titanosaurio de la formation Rio Limay (Albiano-Cenomanio) de la provincial del Neuquen, Argentina, Ameghiniana, (30): 271282. Borsuk-Bialynicka, M. A., (1977) new camarasaurid Opisthocoelicaudia gen. n. sp. n. from the Upper Cretaceous of Springer-Verlag, New York, 562 pp. Calvo, J.O. (1994a) Feeding mechanism in sauropod dinosaurs. MS. Thesis. University of Illinois at Chicago (Unpublished). Calvo, J.O. (1994b) Jaw mechanics in sauropod dinosaurs. Gaia (10): 183-193. Campos, D.D,A., A.W.A., Kellner, R.J., Bertini, R.M. Santucci, (2005) On a titanosaurid (Dinosauria, Sauropoda) vertebral column from the Bauru Group, Late Cretaceous of Brazil. Arquivos do Museu Nacional, Rio de Janeiro, 63 (3): 565-593. Curry Rogers, K. (2005) Titanosauria, A phylogenetic overview. In K. A. Curry Rogers, J.A. Wilson (eds), Sauropod Paleobiology. University of California Press, Berkeley, 50-77. Curry Rogers, K., and C.A., Forster, (2001) The last of the dinosaur titans: a new sauropod from Madagascar. Nature (412): 530-534. Curry Rogers, K., and C.A., Forster, (2004) The Skull of Rapetosaurus krausei (Sauropoda: Titanosauria) from the Late Cretaceous of Madagascar. Journal of Vertebrate Paleontology, 24 (1): 121-144. Day, J.J., D.B., Normann, A.S., Gale, P., Upchurch, H.P. Powell, (2004) A Middle Jurassic dinosaur trackway site from Oxfordshire, U.K. Paleontology, 47(2): 319-348. Day, J.J., P.,Upchurch, D.B., Normann, A.S., Gale, H.P. evolution, and behavior. Science, (296): 1659pp. Powell, (2002) Sauropod trackways,

Dong, Z., G. Peng, and D. Huang (1989) The discovery of the bony tail club of sauropods. Vertebrata PalAsiatica (27): 219-224. Falconer, H. (1868) Memorandum of two remarkable vertebrae, sent by Dr. Oldham from JubbalpurSpilsbury's bed. Paleontological memoirs and Notes of the late Hugh Falconer (1): 418-419. Farlow, J.O., J.G. Pittman, and J.M. Hawthorne, (1989) Brontopodus birdi Lower Cretaceous sauropods footprints from the U. S. Gulf coastal plain. In, D.D. Gillette, M.G. Lockley, (eds). Dinosaur tracks and traces, Cambridge Univ. Press, Cambridge, 371-394. Hislop S. (1864) Extracts from letters relating to the further discovery of fossil teeth and bones of reptiles in Central India. Quaterly journal of the Geological Society of London, (20): 280-282. Huene, F.V., and C.A. Matley, (1933) Cretaceous Saurischia and Ornithischia of the central provinces of India, Paleontologia Indica, (21): 1-74. Ishigaki, S. (1989) Footprints of swimming sauropods from Morocco. In D.D. Gillette and M.G. Lockley, (eds). Dinosaur tracks and traces, Cambridge Univ. Press, Cambridge, 83-86.

Jacobs, L.L., D.A., Winkler, W.R., Downs, E.M. Gomani, (1993) New material of an Early Cretaceous titanosaurid sauropod dinosaur from Malawi. Paleobiology (36): 523-534. Jain, S.L. and S., Bandyopadhyay, (1997) New titanosaurid (Dinosauria: Sauropoda) from the Late Cretaceous of Central India. Jour. Vert. Paleontol., (17): 114-136.

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Lydekker, R. (1877) Notes of new and other vertebrata from Indian Tyertiary and Secondary rocks. Record Geological Survey of India (10): 30-43. Lydekker, R., (1879) Indian pre-tertiary vertebrata. Part 3. Fossil reptilia and batrachia. PaleontologiaIndica, Series 4 (1): 325-326. Malkani, M. S. (2003a) Discovery of Partial Skull and Dentary of Titanosauria (Sauropod dinosaur) from the Late Cretaceous Pab Formation of Vitakri area, Barkhan district, Balochistan, Pakistan. Geol. Bull. Univ. Peshawar, (36):65-71. Malkani, M. S. (2003b) Pakistani Titanosauria; are armoured dinosaurs?. Geol. Bull. Univ. Peshawar, (36): 85-91. Malkani, M. S. (2003c). First Jurassic dinosaur fossils found from Kirthar range, Khuzdar District, Balochistan, Pakistan. Geol. Bull. Univ. Peshawar, 36, 73-83. Malkani, M. S. (2004a) Saurischian dinosaurs from Late Cretaceous of Pakistan. In: Abstract volume of Fifth Pakistan Geological Congress (Hussain, S.S., and Akbar, H. D., eds.), National Geological Society of Pakistan, Pakistan Museum of Natural History, Islamabad, 71-73. Malkani, M. S. (2004b) First diagnostic fossils of Late Cretaceous Crocodyliform (Mesueucrocoreptilia) from Pakistan. In: Abstract volume of Fifth Pakistan Geological Congress (Hussain, S.S., and Akbar, H. D., eds.), National Geological Society of Pakistan, Pakistan Museum of Natural History, Islamabad, 68-70. Malkani, M. S. (2006a) Diversity of Saurischian dinosaurs from Pakistan. In: Ist International Conference on Biotechnology and Informatics, 10th -12th April,2006, Quetta, Pakistan, Additional abstracts volume, Faculty of Biotechnology and Informatics, Balochistan University of Information Technology and Management Sciences, Quetta, Pakistan, 103. Malkani, M. S. (2006b) Biodiversity of saurischian dinosaurs from the latest Cretaceous Park of Pakistan. Journal of Applied and Emerging Sciences, 1(3): 108-140. Malkani, M. S. (2006c) Cervicodorsal, Dorsal and Sacral vertebrae of Titanosauria (Sauropod Dinosaurs) discovered from the Latest Cretaceous Dinosaur beds/Vitakri Member of Pab Formation, Sulaiman Foldbelt, Pakistan. Journal of Applied and Emerging Sciences, 1(3): 188-196. Malkani, M. S. (2006d) Lithofacies and Lateral extension of Latest Cretaceous Dinosaur beds from Sulaiman foldbelt, Pakistan. Sindh University Research J.(Sci. Ser.), 38 (1): 1-32. Malkani, M. S. (2006e) First Rostrum of Carnivorous Vitakridrinda (Abelisaurids Theropod dinosaur) found from the Latest Cretaceous Dinosaur beds (Vitakri) Member of Pab Formation, Alam Kali Kakor Locality of Vitakri area, Barkhan District, Balochistan, Pakistan. Sindh University Research Journal (Sci. Ser.), 38 (2): 5-24.

Malkani, M. S. (2007a) Trackways evidence of sauropod dinosaurs confronted by a theropod found from Middle Jurassic Samana Suk Limestone of Pakistan. Sindh University Research Journal (Sci. Ser.), 39 (1): 1-14. Malkani, M. S. (2007b) Cretaceous Geology and dinosaurs from terrestrial strata of Pakistan. In: Lee, Y.I., Paik, I.S., Cheong. D.K., Huh, M., Lee, Y.U. (eds.), Paleoclimates in Asia during the Cretaceous: theirvariations, causes, and biotic and environmental responses. Proceedings of the 2nd International Symposium of the IGCP Project No. 507, August, 20-21, 2007, Seoul, Korea, 57-63. Malkani, M.S., (2007c) Lateral and vertical rapid variable Cretaceous depositional environments and Terrestrial dinosaurs from Pakistan. In; Y., Huang, P., Wang, J., Gu, and S.Jing, eds. Abstracts volume, Joint Workshop on Rapid Environmental/Climate Change in Cretaceous Greenhouse World: OceanLand Interaction and Deep Terrestrial Scientific Drilling Project of the Cretaceous Songliao Basin, August 28-30, (2007), Daqing, China, 44-47 Malkani, M.S., (2007d) First diagnostic fossils of Late Cretaceous Crocodyliform (Mesoeucrocodylia, Reptilia) from Vitakri area, Barkhan District, Balochistan, Pakistan. In; M., Ashraf, S. S., Hussain,

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and H. D. Akbar, eds. Contribution to Geology of Pakistan (2007), Proceedings of 5 Pakistan Geological Congress (2004), A Publication of the National Geological Society of Pakistan, Pakistan Museum of Natural History, Islamabad, Pakistan, 241-259. Malkani, M. S. (2007e) Paleobiogeographic implications of titanosaurian sauropod and abelisaurian theropod dinosaurs from Pakistan. Sindh University Research Journal (Science Series), 39 (2): 33-54. Malkani, M.S. (2008a) First articulated Atlas-axis complex of Titanosauria (Sauropoda, Dinosauria) uncovered from the latest Cretaceous Vitakri member (Dinosaur beds) of upper Pab Formation, Kinwa locality of Sulaiman Basin, Pakistan. Sindh University Research Journal (Sci. Ser.), 40 (1): 55-70. Malkani, M.S. (2008b) Mesozoic terrestrial ecosystem from Pakistan. In, Sundquist, B. (Science Programme Coordinator), Abstracts of the 33rd International Geological Congress, Oslo, 1p. (Abstract: CD-ROM). Malkani, M.S. (2008c) Mesozoic terrestrial ecosystem from Pakistan. In: Lee, Y.I., Khand, Yo, & Ichinnorov, N. eds., Paleoclimates in Asia during the Cretaceous: their variations, causes, and biotic and environmental responses. Proceedings of the 3rd International Symposium of the IGCP Project No. 507, August, 16-17, Ulaanbaatar, Mongolia, 51-55. Malkani, M.S. (2008d) Titanosaur (Dinosauria, Sauropoda) osteoderms from Pakistan. In: Lee, Y.I., Khand, Yo, and Ichinnorov, N. eds., Paleoclimates in Asia during the Cretaceous: their variations, causes, and biotic and environmental responses. Proceedings of the 3rd International Symposium of the IGCP Project No. 507, August, 16-17, Ulaanbaatar, Mongolia, 56-60. Malkani, M. S. (2008e) Mesozoic Continental Vertebrate Community from Pakistan-An overview. Journal of Vertebrate Paleontology Vol. (28): Supplement to Number (3): 111A. Malkani, M.S., and C.M. Anwar, (2000) Discovery of first dinosaur fossil in Pakistan, Barkhan District, Balochistan. Geological Survey of Pakistan Information Release, 732, 1-16. Malkani, M.S., J.A. Wilson, and P.D. Gingerich, (2001) First Dinosaurs from Pakistan. Journal of Vertebrate Paleontology Volume 21, Supplement to No. 3, 77A. Marsh, O.C., (1878) Principal Characters of American Jurassic dinosaurs. Part I. Am. J. Sci., (16): 411416.

Owen, R., (1842) Report on British fossil reptiles,pt.II. Reptiles. Report of the British Association for the Advancement of Science, 1841, 60-204. Powell, J.E. (2003) Revision of South American titanosaurid dinosaurs: paleobiological, paleobiogeographical and phylogenetic aspects. Records of the Queen Victoria Museum (111): 194. Roach, B.T. and D.L. Brinkman, (2007) A reevaluation of cooperative pack hunting and gregariousness in Deinonychus antirrhopus and other nonavian theropods. Bulletin of the Peabody Museum of Natural history 48 (1): 103-138. Salgado, L., R.A. Coria, and J. O. Calvo, (1997) Evolution of titanosaurid sauropods. 1: Phylogenitic analysis based on the postcranial evidence. Ameghiniana (34): 3-32. Santos, V.F.D., M.G. Lockley, C.A. Meyer, J. de Carvalho, A.M.G. Carvalho, J.J. Moratalla, (1994) A new sauropod tracksite from the Middle Jurassic of Portugal. Gaia 10, 5-13. Seeley, H.G., (1888) The classification of the Dinosauria. British Association for the advancement of Science, Report, 1887, 698-699. Upchurch, P., (1998) The phylogenetic relationships of sauropod dinosaurs. Zoological Journal of the Linnean Society, (124) 43-103. Weishampel, D.B., et al., (1990) The Dinosauria, University of California Press, Berkley 733pp. Wild, R. (1991) Janenschia n.g. robusta (E. Rraas 1908) pro Tornieria robusta (E. Fraas 1908 ) (Reptilia, Saurischia, Sauropodomorpha). Sttuttgarter eitr. Naturk. B 173, 1-4.

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Wilson, J.A. (2002) Sauropod phylogeny, a critique and cladistic analysis. Zoological Journal of the Linnean Society, (136): 217-276. Wilson, J.A., and M.T. Carrano, (1999) Titanosaur locomotion and the origion of "wide gauge" trackways: a biomechanical and systematic perspective on sauropod locomotion. Paleobiology, (25): 252-263. Wilson, J. A, M. S. Malkani, and P. D., Gingerich, (2001) New Crocodyliform (Reptilia, Mesoeucrocodylia) form the upper Cretaceous Pab Formation of Vitakri, Balochistan (Pakistan), Contributions form the Museum of Paleontology, The University of Michigan, 30 (12): 321-336. Wilson, J.A, M.S. Malkani, and P.D., Gingerich, (2005) A sauropod braincase from the Pab Formation (Upper Cretaceous, Maastrichtian) of Balochistan, Pakistan. Gondwana Geol. Mag., Spec. Vol. (8): 101-109. Wilson, J.A. and P. Upchurch, (2003) A revision of Titanosaurus Lydekker (Dinosauria-Sauropoda), the first dinosaur genus with a `Gondwanan' distribution. Journal of Systematic Paleontology (1): 125160.

Wilson, J.A. (2006) An overview of titanosaur evolution and phylogeny. En (Colectivo ArqueologicoPaleontologico Salense, Ed.), Actas de las III Jornadas sobre Dinosaurios y su Entorno 169-190. Sala de los Infrantes, Burgos Espana. Zhang, Y. (1988) The Middle Jurassic dinosaur fauna from Dashanpu, Zigong, Sichuan: sauropod dinosaurs (1) Shunosaurus. Sichuan Publishing House of Science and Technology, Chengdu, 89pp. 15 pls. (in Chinese, English summary).

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Sindh Univ. Res. Jour. ELEMENTAL INVESTIGATION AND ANTI MICROBIAL ACTIVITY OF MEDICINAL PLANT NERIUM INDICUM MILL. AGAINST DERMATOPHYTIC FUNGI A. J. Pirzada, W. Shaikh, A. M. Jakhar, S. I. Shah* and M. P. Pirzada** Institute of Botany, University of Sindh. Jamshoro, Pakistan

(Received 12th February 2008 and Revised 25th August 2008)

Abstract

Five extracts such as ethanol, methanol, ethylacetate, chloroform and aqueous were obtained from the medicinal plant Nerium indicum. These extracts were tested against the Dermatophytic fungi, Aspergillus niger, Aspergillus flavus, Paecilomyces varioti, Microsporum gypseum, and Trichophyton rubrum. All crude extracts were found to be effective against test organism. Methanol, ethanol and aqueous extracts appeared to be most effective antifungal agents as compared to chloroform and ethylacetate extract. More over in present study nine elements, Al, Ca, Cu, Fe, Mg, Mn, P,S and Zn have been determined from the medicinal plant, Nerium indicum. by using atomic absorption spectrophotometry and U.V spectrophotometry. The medicinal plant Nerium indicum. contains considerable amount of elements which have therapeutic effects in skin diseases. Keywords: The medicinal plant Nerium indicum the Dermatophytic fungi, Microsporum gypseum, and Trichophyton rubrum

1.

Introduction Nerium indicum. commonly known as Kanur is a traditional medicine which is utilized in many part of Pakistan for the treatment of various fungal skin diseases like Tinea capitis, Tinea pedis, Tinea manum, and Tinea corporis etc. The docection is recommended to reduce swellings and oil prepared from the root bark is cure for skin diseases for scaly nature and leprosy. The flower has a bitter sharp taste, good for inflammation, chronic pain in the muscles and the joints, in lumbago, headache and scabies. (Baquar 1989, Kirtikar and Basu 1935).

lotion of zinc solution have a soothing and cooling effects. Calcium, Manganese, Magnesium are used in the formation of collagen, connective tissue and maintains electrical potential of nerve and muscles membrane for the transmission of impulses across junction. Phosphorus and sulpher are used for the treatment of scabies and leprosy. All living cells require iron for growth, replication, respiration and DNA synthesis. It can promote resistance against many diseases included skin disease. (Sahito et al., 2003, soderberg et al., 1982 underwood 1981). Skin disease is one of the main problem of Sindh province which is usually caused by fungi. The present report describe the antifungal potential of different solvent extract of N. indicum against fungi causing skin diseases. 2. Materials and Methods Plant Material:Plant material the N. indicum. leaves and shoots were collected from different area of Kohistan region District Dadu and reference

Metals and its elements as well as their compounds have been used since ancient times for therapeutic as well as cosmetic effects on skin. Aluminum acetate solution is used as skin disinfectant and cleansing agents. Copper sulphate is probably still used in some countries as mild astringent and antiseptic preparation to treat vitiligenous skin. In leprosy seven elements are found to have therapeutic effect such as the use of (Zn) tape for the treatment of leprosy wounds and

*Seed Certification Officer, Federal Seed Certification and Registration, Department Govt. of Pakistan, Bahawalpur **Govt. Zubaida Girls College, Hyderabad, Sindh

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sample were identified through literature Flora of Pakistan (Nasir and Ali 1990). The collected plant material were washed with distilled water and placed in shade at room temperature for two weeks. One kg of dried plant material was dip in five litter (L) of ethanol solvent in bottle for 20 days for cold percolation. The extract was filtered and concentrated under reduced pressure below 400C using rotary evaporator. The residue completely was dried as the syrupy liquid form. From the residue five different extracts such as ethanol, ethylacetate, chloroform, methanol and aqueous extract were prepared using separating funnel. The extracts were left at room temperature. The solvent were completely evaporated so that organic compounds remain in the dry form. These extracts so obtained were mixed with the sterilize water (1g:5ml) and each extracts sample was applied for antifungal activity. Collection of Dermatophytes The dermatophytic fungi namely: Aspergillus niger, Aspergillus flavus, Paecilomyces varioti, Microsporeum gypseum, Tricophytron rubrum were scraped from the different body parts at skin out patient departments of Liaquat University Hospital Jamshoro and Hyderabad. Preparation of fungal culture Sabourad glucose-agar media. Following composition were used for this purpose. Pepton 10g, glucose 20g, Agar 20g, distil water 1000ml with 5.4 pH. All the contents were mixed and dissolved in distilled water. The solution were autoclaved at 1200 C, 15 lb/sq inch pressure for 20 minutes. Treatment of different solvent extract layers:The human skin pathogen were treated with different extracts and result were taken after 72 hours at 300C. The percentage of mycelial inhibition was calculated as follows: Usmanghani and Shameel (1986), Ali Shtayeh and Suheil (1999) % Mycillial inhibition = [(dc-d1)/dc]x100 dc=colony diameter in control, d1= colony diameter in treatment Methodology for Elements determination. 4. 3.

A suitable dissolution method for biological sample to yield homogenous solution is a crucial first step to determine in atomic absorption spectrophotometric and U.V. techniques. The decomposition of organic matter must be completed to avoid interference by organic residue. Sample was digested with nitric acid: 30% hydrogen peroxide for determination of mineral elements. Appropriate working standard solution of Aluminum (Al), Calcium (Ca), copper (Cu), iron (Fe), Magnesium (Mg), Manganese (Mn), Phosphorus (P), Sulpher (S), Zinc (Zn) were prepared from stock standard solution (1000 ppm), in 2N nitric acid. Calibration curves were drawn for each elements using atomic absorption spectrophotometer Hitachi model 180-50 and UV-Spectrophotometer. The calibration curves obtained for concentration VS. absorbance data were statistically analyzed using fitting of state line by least square method. A blank reading was also taken and necessary correction was made during the calculation of percentage concentration of various elements. The efficiency of extraction method was checked by standard addition method. The sample was spiked with known standards and digested with nitric acid and hydrogen peroxide mixture. The matrix of standard and sample solution was same. The percentage recovery test for different elements by digestion method adopted was 98.5-99% in range. 5. Results All the crude extracts has significant anti fungal activity against most of the fungi, but the activity of inhibition varied for the fungi with respect to the type of plant extract. (Table-1). Ethanol extract: The maximum inhibition activity was observed against A. Flavus and T.rubrum 100 % each while moderate inhibition activity against P. varioti, A. niger and M. Gypseum was 96.37%, 90% and 80% respectively. (Table-1). Methanol extract: The maximum inhibition activity was observed against P. varioti, T. rubrum and A.niger 89.1%, 84% and 75% respectively while moderate inhibition activity against A. flavus, 71.41% and minimum inhibition activity against M.gypseum was noticed 50.%. (Table-1).

Elemental investigation and anti microbial activity ...

81

Chloroform extract: The maximum inhibition activity was found against M. gypseum, and P.varioti 60% and 54.55% each. While moderate inhibition activity against, A. niger 50.%, and minimum inhibition activity against A. flavus and T.rubrum 42.85% , 40% was recorded. (Table-1). Ethylacetate extract: The maximum inhibition activity was noticed against, P. varioti and M. gypseum 54.55% and 50 % respectively. While moderate inhibition activity against A. flavus and A.niger 34.29% and 30.% and minimum inhibition activity against T. rubrum 28.% was measured. (Table-1).

Aqueous extract: The maximum inhibition activity was observed against A.niger T.rubrum, and P. varioti 87.5%, 87.28% and 80% respectively. While moderate inhibition activity against M.gypseum 50% and minimum inhibition activity against A.flavus 48.58 % was determined. (Table-1). Elements:-The considerable amount of various elements such as: Aluminum (Al), calcium (Ca), copper (Cu), iron (Fe), Magnesium (Mg), Manganese (Mn), Phosphorus (P), Sulpher (S), Zinc (Zn) have been determined from the medicinal plant Nerium indicum (Table-2) These elements are biologically very much important for the treatment of different skin diseases.

Table-1. Antifungal activity of different solvent extract of N. indicum Mill against test organisms. Test extract

Ethanol Controlled reading at 300C after 72 hours (mm) Inhibited reading at 300C after 72 hours (mm) Inhibited (%). Methanol Controlled reading at 300C after 72 hours (mm) Inhibited reading at 300C after 72 hours (mm) Inhibited (%). Chloroform Controlled reading at 300C after 72 hours (mm) Inhibited reading at 300C after 72 hours (mm) Inhibited (%). Ethyl acetate Controlled reading at 300C after 72 hours (mm) Inhibited reading at 300C after 72 hours (mm) Inhibited (%). Aqueous Controlled reading at 300C after 72 hours (mm) Inhibited reading at 300C after 72 hours (mm) Inhibited (%).

A . niger

40 04 90

A .flavus 35 00 100

P. varioti 55 02 96.37

M. gypseum 30 06 80

T. rubrum 25 00 100

40 06 75

35 10 71.41

55 06 89.10

30 15 50

25 04 84

40 20 50

35 20 42.86

55 25 54.54

30 12 60

25 15 40

40 28 30

35 23 34.29

55 25 54.55

30 15 50

25 18 28

40 05 87.50

35 18 48.58

55 07 87.28

30 15 50

25 05 80

A. J. Pirzada, et al., Table-2. Quantity of different elements in N . indicum Mill Name of Elements Aluminum Calcium Copper Iron Magnesium Manganese Phosphorus Sulphur Zinc Formula Al Ca Cu Fe Mg Mn P S Zn Amount mg/ kg 5.91-6.42 20956.97-22811.57 8.26 ­ 9.69 73.49-127.84 6601.89 ­ 6863.49 81.00 ­ 94.3 65.24 ­ 76.59 314.59 ­ 326.12 31.26 ­ 44.52

82

6.

Discussion In the present study crude extracts of the plant material obtained in polar and less polar organic solvent were tested against fungi causing skin diseases. All the crude extracts had significance antifungal activity on most of the fungi, Ethanol, Methanol and Aqueous extract had maximum inhibition activity 48.58% - 100% against test pethogens as compared to Chloroform and Ethylacetate extract. The extracts exhibited a very active inhibition activity 40­60% against test dermatophytes. While Ethylacetate extract had weak inhibition activity (28 % -54.55% against test organism. Similar work have been done by Anjum and Khan (2003), where chloroform extract of Cuscuta reflexa Roxb. displayed minimum inhibition zone against A.niger. Adekunel et al., (2002), reported that water extract of Chasmanthera dependens Hochest did not inhibit the growth of A.niger and A.flavus. Sakharkar et al., (1999), cited that crude extract of Cassia alata linn had shown significant anti fungal activity against A.niger. However Ficker et al., (2003) claimed that the crude extract of Juglans cinerea L. were found to be very active inhibitory agent against M.gypseum. Thebo and Abro (2000) described leaves extract of Azadirachta indica A.Juss were very active against M.gypseum and T.rubrum. While Natarjan et al., (2003) provided evidence that ethonal extract of Azadirachta indica A. Juss exerted highly inhibitory activity against T.rubrum. Usmanghani and Shameel (1986) communicated that methanolic extract of some marine algal species were found very active to inhibit A.niger. While (Shaikh et al., 1990), observed that crude extract of S. marginatum were very active against T-rubrum, but activity was devoid of against A.niger and A. flavus. Pirzada et al., (2007) reported that crude extract of Rhazya stricta have strong anti fungal activity against A. flavus, A. niger, M. gypseum, P. varioti and T. rubrum. In present study this was the first attempt to study the anti fungal activity of the medicinal plant N. indicum against the dermatophytic Fungi such as A.niger, A.flavus, P.varioti, M. gypseum, T.rubrum causing different skin diseases. In this study all the solvent extract were very active to inhibit test organism except Ethylacetate which was weak to inhibit T.rubrum. In the continuation of this study nine elements (Al), Calcium (Ca),Copper (Cu), Iron (Fe), Magnesium (Mg), Manganese (Mn), Phosphorous (P), Sulphar (S) and Zinc (Z) have been analized in variable range from the medicinal plant N. indicum. However, calcium (Ca), Manganese (Mn) and Zinc (Zn) were found in sufficient amount in the range of (20956.97 ­ 22811,57) mg/kg, (81.00 ­ 94.31) mg/kg and (31.26 ­ 44.52) mg/kg respectively. All these elements play essential role for the treatment of skin disease. (Janjua 1990, Saily et al., 1994). Copper, Aluminum, Zinc and their salts are used in skin infection as disinfectant and cleansing agents. Zinc is co-factor for many enzymes required for the healing damaged skin. Sahito et al., (2003). This current experiment provides information about mineral contents present in N. indicum and scientific justification for the utilization of crude extract of N.indicum for the treatment of different skin disease such as Tinea capitis, Tinea carporis, Tinea manum, Tinea pedis etc to researchers and to the person dealing with herbal extracts.

References Ali Shtayeh, M.S., and I. S. A. Ghdeib (1999) Antifungal activity of plants extracts against dermatophytes, Mycoses (42): 665-672. Adekunle, A. A., and S.O. Okoli, (2002) Antifungal activity of crude extracts of Alfia barteri Oliver (Apocynaceae) and Chasmanthera dependens Hochst (Menispermaceae). Hamd. Med. XLV, (3):5256.

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Anjum N., and Z. Khan, (2003) Antimicrobial activity of the crude extract of Cuscuta reflexa Rox b. Pak. J. Bot. 35 (5): 999-1007. Baquar S.R., (1989) Medicinal and Poisonous Plants of Pak. Printas, Karachi. . Ficker C.E., J.T. Arnason, P.S. Vindas, (2003) Inhibition of human pathogenic fungi by ethnobotanically selected plant extracts. Mycoses (46): 29-37. Janjua. K.M., (1990) Trace elements in Allium cepa (Onion) and their therapeutic importance. Hamd. Med. XXXIII, (2): 87-90. Kirtikar. K. R., and B.D. Basu, (1935) Indian Medicinal Plants 1-IV Lalit Mohan Basu, Allahabad, India. 426- 428. Nasir, E., and S. I. Ali, (1990) Flora of Pakistan, Department of Botany, University of Karachi. Natarjan. V., P.V. Venugopal, and T. Memon, (2003) effect of Azadirachta indica (neem) on the growth pattern of darmatophytes. Ind. J. Med. Microbiol (21): 2,1-4. Pirzada, A.J., W. Shaikh, and T.G. Kazi, (2007) Isolation of essential elements and inhibition activity of medicinal plant Rhazya stricta Dcne. Against dermatophytic fungi. Pak. J. Agri., Agril. Engg., Vet. Sci., 23 (1): 34-38. Sahito, S.R., M.A. Memon, T.G. Kazi, and G. H. Kazi, (2003) Evaluation of mineral contents in medicinal plant Azadirachta indica (neem). J. Chem. Soc.Pak. (25): (2), 139-143. Shaikh, W., M. Shameel, A. Hayee-Memon, K. Usmanghani, S. Bano and V.U. Ahmed (1990) Isolation and characterization of chemical constituents of Stoechospermum marginatum (Dictyotales, Phaeophyta) and their antimicrobial activity. Pak. J. Pharm. Sci. 3 (2): 1-9. Saily, A., R. Sahu, B. Gupta and S.M. Sondhi. (1994) Analysis of mineral elements of medicinal plants used for the treatment of asthma, syphilis, diarrhea, skin diseases and rheumatism, Hamd. Med. XXXVII (4): 18-22. Sakharkar, P.R., and A.T. Patil, (1999) Antifungal activity of Cassia alata. Hamd. Med. XLI, (3): 2022. Soderberg, T., and G. Halimans, (1982) Treatment of leprosy with adhesive Zinc tapes. Lep. Rev., (53): (4) 271. Thebo, N. K., and H. Abro, (2000) Antifungal activity of Azadirachta indica against human pathogenic fungi Sindh Univ. Res. J. (Sci. Ser.) 32 (2): 35-42. Underwood. E., (1981) Trace metal in human and animal health J. Hum.nutr. 53, 37.

Usmanghani, K., and M. Shameel, (1986) Studies on the antimicrobial activity of certain sea weads from Karachi coast. In: R. Ahmad and A. San Pietro, (eds.): Prospects for Biosaline Res. Proc. USPak Biosal. Res. Workshop; Karachi, 519- 526.

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Sindh Univ. Res. Jour. SEARCHING OF OPTIMAL VERTICES IN A HAPHAZARD ROUTES N. Khan, F. H. Khan*and S. Inayat Ullah Department of Mathematics, University of Karachi, Karachi *Corresponding Author: [email protected]

(Received 12th March 2008 and Revised 09th May 2008)

Abstract

In this paper we are going to develop a new and efficient algorithm for finding a shortest cyclic tour of the network with the repetition of each vertex at least once. Problem under consideration is a traveling salesman problem, for repeating vertex. Penalizing the distance and choosing the shortest tour has been achieved from the weighted matrix of the graph. Keywords: Traveling salesman problem, Optimal salesman problem

1.

Introduction Finding the shortest tour within the city or area is a very useful requirement. The optimality is defined in terms of the cost it takes to complete the traversal. This cost depends on the number of times of each edge traversed through out the tour. In this paper we proposed a new algorithm to find the optimal tour in a directed network, called Asymmetric optimal sales man problem. Our algorithm is based on traveling sales man problem for repeated vertex, first we shall find all possible tours that each starts and ends at fixed point, then we combine all these tours, in the end we apply the removal and insertion to find the optimal tour. Solving TSPs is an important part of application in many areas including vehicle routing, computer wiring, machine sequencing and scheduling, frequency assignment in communication network and structuring of matrices (Lenstra and Kan, 1975; Punnen, 2002). Given a graph network, finding the tour of minimum cost (e.g. time or distance) that visits each node of the graph at least once is called optimal sales man problem. The literature on the graph theory includes many variants of the problem of graph traversal with respect to different constraints. One of the classic problems in graph theory is the problem of Chinese postman tour (CPT) in which a post man needs to travel along every road, with the least possible cost to deliver mails. Another problem is the Traveling sales man problem (TSP) Given a collection for cities and the cost of travel between each city pair, TSP is to find the cheapest way of visiting of all the cities and retuning to the starting point (i.e, finding a Hamiltonian cycle with the least weight). If the distance between the cities is not symmetric (i.e d (city 1, city 2)d (city 2, city 1), the problem is referred to as the Asymmetric Traveling Sales Man Problem (ATSP). Another approach (Bellmore and Malone) extant the solution of TSP is Branch and Bound in which first find the Hamiltonian cycle of the given network, many variations has been made in branch and bound method to solve TSP. This method is also used for optimal salesman problem with some difference we use shortest distance matrix (SD) instead of weighted matrix. In our algorithm we are also using this SD matrix. Another method (Little et al 1963) called weight matrix reduction is also could be used to solve TSP and OSP. An algorithm is also used to solve TSP for the curvature constrained. The remainder of the paper is organized as follows. Section 2 formally define the OPT problem. In section 3, we proposed the algorithm. Section 4 includes the experimental problem and section 5 discusses the conclusion.

N. Khan, et al.,

86

Definitions The simplest model used to analyze facility location problem is a graph. A graph G consists of a finite nonempty set V = V (G) of vertices together with the set E = E (G) of line segments (called edges) joining parts of distinct vertices of V. If eij E, then vi and vj are adjacent vertices, otherwise, they are non-adjacent. The degree of a vertex is its number of adjacent vertices. A path in G is an alternating sequence v0e1v1e2....... vn-1 envn of distinct vertices and edges such that eij = vi vj is an edge of G. Since each edge is uniquely determined by its end vertices, it is customary to write v0v1....... vn-1 vn v0 for the path, just described. A path v0v1....... vn-1 vn v0, where vn and v0 are joined by an edge, is called a cycle. A graph G is connected if there exists a path joining each pair of vertices in G. Thus, if a graph modeling a telecommunication network is connected, we know that it is possible for any two devices in the network to communicate. We shall restrict our attention to connected graph. The matrix representation of a graph, in which element at ith row and jth column represents length of eij, is called the Weighted matrix of that graph. The length of a path is the sum of length of edges it contains. The distance d(i,j) is the length of the shortest path joining vi and vj. The distance between vi to itself is to be considered as i.e. d(i,i) = . A matrix obtained after row and column reduction of a matrix of a graph is called reduced weight matrix, where row reduction is the subtraction of least element from each element of the row and column reduction is the subtraction of the least element from each element of the column. 3. i. Algorithm Input : weighted matrix : D, D': Reduced weighted matrix, Ti: Sets of all possible tours. ii. iii. iv. For D' : For {Ti} : Union S = Row/column penalties. Starting from a ij Selecting eij=0 if eij already visited then select next minimum eij.

2.

S = Ti

v. Redundant path removal for each k by removing all paths vi vk vj except that of min [(eik+ ekj )- eij ]. vi. Insertion of path vi vk vj If [(eik+ ekj ) < eij ]. Return vector for shortest tour.

4.

Application of the constructed model

For the given graph find the weighted matrix of the network with diagonal elements . Find the reduced weighted matrix by applying row and column reduction operation. Starting from the first vertex find all possible tour by selecting zero elements in the reduced weighted matrix, if there is no edge of zero value of available to complete such tour the edge with next most minimum value will be selected instead. Combined all the possible tours. After the combination of all the tour, start with a tour consisting of an arbitrary city and then choose in each step a city k. This city is deleted in to the existing tour between city i and j such that the deletion cost (i.e the decrease in the tour's length) (eik+ ekj )- eij is maximized. The algorithm stops when all the cities on the tour (just once), after that we insert the city between the two consecutive cities i and j such that if (eik+ ekj )< eij by using SD matrix. In this way the OSP tour of the given network is 123241 with the total cost 30, in contrast with the TSP result 13241

Searching of optimal vertices... 87

with the total cost 40. 5. Conclusion In this paper, we studied the optimal salesman problem and proposed a new algorithm in OSP, by considering the weighted matrix of the graph. First we have calculated all the possible tours, then we have combined all these tours, in the end we have applied the removal & insertion to get the optimal vertices in haphazard routes. Our example showed the superiority of OSP as compare to TSP. References Biggs, N., E.K. Lloyd, and R.J. Wilson. (1986) Graph theory, Clarendon press 1736-1936. Cirasella, J., D.S. Johnson, L.A. Mc Geoch, and W. Zhang. (2001) The asymmetric traveling salesman problem: Algorithms, instance generators, and tests. Lecture Notes in computer Science, 2153: 32-59. Diestel. R., (2000) Graph Theory. Springer-Verlag. Opns. Res. (35): 266-273. Garey M.R., and D.S. Johnson. (1990) Computers and Intractability; A Guide to the theory of Completeness. W.H. Freeman and Co., New York, USA. Hahslar M., and kurt Hernik. 25th April (2007) Infrastructure of traveling salesman problem. Jerome Le Ny, Eric Feron. (2002) An Approximation Algorithm for the curvature constrained (TSP) Traveling sales man problem. Leyla kazemi, Cyrus Shahabi, Mehdi Sharifzadeh. (2000) Dept. of Computer Science. Optimal traversal planning in Road network. University of California. Thimbleby. H.W. (2003) The directed Chinese postman problem. Softw., Pract. Exper., 33 (11): 10811096. NP-

Zhang, W. (1993) Truncated branch-and-bound: A case study on the asymmetric tsp. In Working Note of AAAI (1993) Sprig Symposium: AI and NP-Hard problems, 15 (6):160-166.

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Sindh Univ. Res. Jour. COMMON FOLK MEDICINAL AND ETHNOMEDICINAL USES OF THIRTY MEDICINAL PLANTS OF DISTRICTS DADU AND JAMSHORO, SINDH, PAKISTAN. *A.H. Memon, F.M.A. Rind, M.G.H. Laghari, U.R. Mughal, N.Memon, R.A. Gilal, M.Y. Khuhawar** and F. Almani. Faculty of Pharmacy, University of Sindh, Jamshoro, Pakistan

(Received 22nd August 2008 and Revised 19th November 2008)

Abstract

An ethnobotanical survey was carried out in Districts Dadu and Jamshoro, Sindh Province of Pakistan. In this region local people since their forefathers are using medicinal plants and their different parts for the treatment of many diseases. Although modern methods to treat disease by homeopathic and allopathic drugs reduced the use of local remedies but still countable people are using folk remedies. The knowledge about the use of medicinal plants and their remedies of these two districts are still remained untouched by ethnobotanist, ethnopharmacologist and pharmacist. Thirty (30) medicinal plants were documented which are mostly used in the treatment of many diseases by the local people of district Dadu and Jamshoro. Keywords: Folk uses, Ethnobotanical uses, Dadu, Jamshoro.

Rivers and Streams with the eastern boundary of the district daudu the Indus River flows. There is no other river in the district except numerous hill torrents known as Nais. The Gaji Nai is one of the most important Nai. Baran, Sann and Sol Nais are also present in this district. Mancher is the biggest The Kohistan part consists of rocky area natural lake in Asia. The maximum full covering which is called as Kirthar Range. It embraces the area of lake is 259 Sq. Km. and when it is empty whole western boundary of the district, having or, it occupies 52 Sq. Km. it is one of the huge breadth of 19-24 Km. The most prominent peak storage of fish, it is also a source of irrigation of named Kutay-Ji-Qabar (dog's grave) is 2096 m about twenty to twenty five thousand acres of above sea level and 244 m. A plateau known as land. However the Mancher Lake is added Dharyaro is present towards the north is drainage water through Main Nara Velly Drain immediately below it. Bleak, uneven and infertile (MNVD), the water quality, fish and funa of the *Corresponding author. [email protected] look of these hills, they afford pasturage for large **Dr. M.A. Kazi Institute of Chemistry, University of Sindh, Manchar Lake are being deteriorated due to the Jamshoro,Pakistan flocks of goats and sheep. After the rain the drainage water added into it through Main Nara Physical Feature and Topography The land of Dadu district has three characteristics areas namely, a). Kohistan or Hilly area. b). Barrage zone. c). Low lands riverian area.

Introduction District Dadu of Sindh Province of Pakistan is situated at 27-27 and 24-57 north latitudes and 67 - 09 to 68-25 east longitudes. It is situated in the south of district Larkana, in the west of districts Hyderabad, Naushero Feroze and Nawabshah, in the north of district Thatta, in north-east of district Malir and districts Lasbela and Khuzdar of Balochistan Province. The total area of the district is 19070 sq. Km. Recently this district has been devided into two districts namely Jamshoro and Dadu. The Jamshoro district is comprised of Sewan and Kotri Talkas. (http://www.dadu.sdnpk.org/index.html)

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valleys are sheltered with green grass and capable to cultivate up to a height of about 1000 m. The twisted rocky channels which drain the hills there in the form of pools are sufficiently permanent source for the survival of several species of fish. Ever shady trees, wild flowers and ferns are seen at these spots. Some natural water courses (Dhoras) and depression (Dhandhs) are found in district daudu few of them retain water throughout the year and cause a large growth of Acacia nilotica trees at their boundaries. (http://www.dadu.sdnpk.org/index.html)

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Velly Drain (MNVD). Other important lake of this area is Ganero which is situated at the distance of about 16 Km. away from Talka Mehar. (http://www.dadu.sdnpk.org/index.html) Weather The weather of the district is cold enough in winter and so hot and dry in summer. There is profound difference of climate conditions between southern and northern areas. Talka Kotri, Thano Bula Khan and most part of Sehwan which belong the southern part of districts have noderate climate while Dadu, Mehar, Johi and Khairpur Nathan Shah Talkas which belong to northern part of district have intense weather conditions. The hill range known as Gorakh Hill Range posseses the special climate in which the temperature falls to sub-zero degree during winter while it remains between 20 0C even in summer. The district receives 120 mm average rainfall annually (http://www.dadu.sdnpk.org/index.html) Flora Various types of flora are present in district different plants of medicinal values are found in Manchar Lake. The common trees available in this district are Acacia nilotica (babul), Ficus religiosa (pipal), Prosopis specigera (kandi), Aegle marmelos (belgeri) and Populus euphratica (balm). (http://www.dadu.sdnpk.org/index.html) Horticulture To fulfill the needs of local people the ample horticulture is carried out to grow different flowers, fruits and vegetables. Forestry The total covered area by forestry in district Dadu is 217,000 hectares. Amri, Abad, Bhan, Gidarji, Budhapur, Budho Dero, Dero, Gagh, Kundah Khairo Kaloo Bhori, Kandi Baghban, Kacho Magsi, Manjhand, Kamal Dero, Keti Lalya, Keti Jatoi, Kacho, Nari, Rajri, Sita, Shah Grah Dhandan, Sona Bindi, Qasim Shah, Unerpur and Soi are the important forests of district Dadu. About 239,000 Cft of timber and 78,000 Cft of firewood are yielded by the forests of this district (Bai et al., 2003). Aims of present study Many indigenous plants are used in herbal medicine to cure diseases and heal injuries. Such medicinal plants include Acacia nilotica

Lam.Willd. (Fam: Leguminosae), Alhaji maurorum Medic. (Fam: Papilionaceae) and Eclipta alba L. Hassk. (Fam: Compositae). E. alba is used in ethnomedicine as alexipharmic, anti-inflammatorry, and to treat hernia, bronchitis, asthma, leucoderma, anemia, heart and skin diseases, night blindness, syphilis, and also used for the treatment of complexion (Cheryl et al., 2001). Local people are using commonly available plants for the treatment of many diseases and maintenance of their health. However introduction of allopathic and homeopathic drugs decreased human dependency on medicinal plants for their folk uses (Bhanu et al., 1998). The folk or traditional knowledge about the uses of medicinal plants and their remedies are at the risk of everlastingly loss, if these are not documented by pharmacist, especially pharmacognosist. Therefore it is needed to manuscript the folk remedies about the consumption of medicinal plants for the cure, treatment of many diseases and maintenance of health. Dadu and Jamshoro are main districts of Sindh Province. Allopathic, homeopathic practitioners as well their pharmaceutical and homeopathical drug stores are limited in this district as compare to the population of this area. The qualities and natural origins of the medicinal plants and their dried parts are well known to the villagers and local people through individual practices and inherited remedies. Special advantage is that the herbal therapy mostly has less side effects and charge if they are used appropriately for the particular disease or disorder. Majority of medicinal plants are available in these districts where they grown naturally but due to lack of knowledge to every common person there is damage of these valuable medicinal plants by a common illiterate people of these districts. So this study is performed to document folk remedies and uses of few important and commonly available plants to save their damage and to invite pharmaceutical chemists and pharmacognosists to evaluate the ethnomedicinal and ethno pharmacological aspects of these medicinal plants scientifically.

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This documentation can be translated in local language and to make it available for common people after comparing the ethnomedicinal uses of these medicinal plants and folk remedies with scientific literature if it is available. On the other hand the most important aspect of this study is to inform about the nature of the phytochemical constituents and most potent toxic parts of plants. Because mostly local users of medicinal plants are utilizing them without any fear of dangerous side effects of these plants.This study consists information of each medicinal plant obtained on scientific and traditional bases. 2. Materials and Methods

Districts Dadu and Jamshoro were visited many times with a qualified team consisted of pharmacognosists, pharmacists, botanist, chemists and agriculture expert for the identification and collection of medicinal plants during the year of 2004. The required plants according to data were collected, preserved as herbarium sheets and submitted in the Department of Pharmacognosy, Faculty of Pharmacy, University of Sindh, Jamshoro, Pakistan. Local people above 40 years old age were interviewed for folk recepies and traditional uses of medicinal plants of this area. Two most senior, expert Hakeems (local folk practitioners) were also interviewed as they are utilizing these medicinal plants for the treatment of local people. The following questionnaire was adopted for this study.

Questionnaire Personal Bio-data Name:................................................ Father's Name:...................................... Surname:.............................................CNIC No:........................................... Age:.............................Sex:............... Occupation:.......................................... Adress:...................................................................................................... Used by, a). Himself b). Some one else c). Suugested by Hakeem d). Fore fathers e). Local person. If b,c,e then, Name of person:......................................Fathers Name:...................................... Surname:............................................. CNIC No:........................................... Age:.............................Sex:............... Occupation:.......................................... Adress:........................................................................................................ Name of disease: ........................................................................................... Condition of disease: Acute Chronic. Therapeutic effects: ........................................................................................ Side effects:.................................................................................................. Time period of therapy:.................................................................................... Dosage form:................................................................................................ Name of Plant (In local Language)....................................................................... Palce:.......................................... Month of collection:....................................... Parts used: Fresh Dried. Nature of plant Nature of plant: cultivated Wild Purchased from shop Gifted by some one Used as,

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House hold remedy Remedy prepared by expert. Botanical name:.......................................... Family:.......................................... Name of taxonomist:..................................... Designation:................................... Address:....................................................................................................... 4. Results and discussion

During the present study ethnomedicinal uses with phytochemical constituents, folk uses and recepies for thirty medicinal plants of this area were documented which are discussed below. Plant No: 01. Acacia nilotica Lam. Willd. Botanical Name: Acacia nilotica Lam. Willd. (Fam: Mimosaceae). English Name: Arabic Gum. Local Name: Babul. Parts Used: Leaves, seeds, pods, barks and gum. Chemical constituents: It contains tannins, quercetin, gallic acid, epigallocatechin, catechol, epicatechol, dicatechol, (Abdulkarim et al., 2005), (+) catechin, (-) epicatechin, (-) dicatechin and (+) leucocyanidin gallate (Bhanu et al. 1998). The presence of (+) dicatechin and (+) leucocyanidin gallate is noted for the first time in nature (Bimla et al., 2005). Calcium, magnesium, salts of ecidu acid, malic acid, polysaccharides, Larabinose, L-rhamnose, D-galactose and oxidative enzymes are present in the gum. Uses: Ethnomedicinal Uses: Astringent, anti-protozoal, anti-diarrhoeal, anti-inflammatory, anti-dysenteric agent, stomatitis, CNS depressant, demulcent, as a lotion in conjunctivitis, used in leucorrhoea, used as a gargle and mouthwash and to clear the ulcers and wounds (Eldeen et al., 2007; Anderson et al., 1967; Ghazi et al., 1999). Folk Uses and Recipe: Infusion of pods is used as anti-diarrheal for animals and flowers are used for infertility. Plant No: 02. Abutilon indicum L. Sweet. Botanical Name: Abutilon indicum L. Sweet. (Fam: Malvaceae). Local Name: Pat-tir. Parts used: Leaves. Chemical constituents: The main active constituents of Abutilon indicum leaves are flavonoids, sapogenins, steroids and carbohydrates. (Jain, 1991; Evans, 2002). Uses: Ethnomedicinal Uses: Analgesic, diuretic, aphrodisiac (Syed, 1989), hypoglycemic and laxative (Evans, 2002). Folk Uses and Recipe: Infusion of leaves traditionally is used to treat asthma, gonorrhoea and in toothache as mouthwash. But the scientific

study failed to attribute these actions to any chemical constituent present in the leaves of Abutilon indicum. Plant No: 03. Aegle marmelos L. Correa. Botanical Name: Aegle marmelos L. Correa. (Fam: Rutaceae). English Name: Bael, or Bitter Orange. Local Name: Bel geeri. Parts used: Fruit and leaves. Chemical constituents: The flavonoids, coumarins, monoterpenes and alkaloids (Subrata and Playford, 2003) are present in the fruit. Freshly picked fruits yield little vitamin C (Jagetia, 2003). Uses: Ethnomedicinal Uses: Anti-diarrhoeal, antidysentery, hypochondriasis, antifungal, antibacterial, anticancer, hypoglycemic, refreshing drink, anti-emetic, antispasmodic, carminative, laxative, deobstruent, stomachic, diuretic, expectorant, odontalgic, stimulant, vasoconstrictor. It is also used to treat toothache, intermittent fever, colds, abdominal distension, stuffy sensation in the chest, dyspepsia, constipation, prolepses of the rectum, stomach and uterus. (Subrata and Playford, 2003; Jagetia, 2003; Elizabeth et al., 2005). Folk Uses and Recipe: Fruit (ripe) is used in dysentery and eciduas. Plant No: 04. Aerva javanica Burm. F. Schult. Botanical Name: Aerva javanica Burm. F. Schult. (Fam: Amaranthaceae). English Name: Snow Bush. Local Name: Booh. Parts used: Leaves. Chemical constituents: Hentriacontane, nonacosane, nonacosanol, tritriacontane, tetratriacontane, sitosterol and oleanolic acids are isolated from the fresh leaves of the plant (Usman et al., 1982). Uses: Ethnomedicinal Uses: Anti-inflammatory and sedative. It is also used as household remedy for treatment of toothache and kidney stone (Muhammad et al., 2006). Folk Uses and Recipe: Plant fumigation and plant paste with water is used to treat sexual

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incapacity, sexual asthenia and frigidity and aphrodisiac activity.

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Plant No: 05. Alhagi maurorum Medik. Botanical Name: Alhagi maurorum Medik. (Fam: Fabaceae). English Name: Camel thorn bush. Local Name: Kandeero. Parts used: Manna. Chemical constituents: A sweet-tasting manna is exuded from the twigs at flowering time (Atta et al., 2004). It contains about 47% melizitose, 26% sucrose and 12% invert sugars (Attia et al., 2004). Uses: Ethnomedicinal Uses: Diaphoretic, diuretic, expectorant, laxative, antidiarrhoeal (Attia et al. 2004) and antinociceptive (Atta et al., 2004). Folk Uses and Recipe: Juice of leaves and flowers is used as anti-rheumatic and anti-piles. Plant No: 06. Bauhinia veriegata L. Botanical Name: Bauhinia veriegata L. (Fam: Caesalpinaceae). English Name: Mountain Ebony. Local Name: Kachnar. Parts used: Leaves. Chemical constituents: Tree contains glycosides (Rajkapoor et al., 2006), bark yields gum and tannins, seeds contain non drying oil 15% (Ciero et al., 1998). Uses: Ethnomedicinal Uses: Astringent, antibacterial (Grubben and Denton, 2004), tonic, alterative, anthelmintic, stomachic, alexipharmic, anticancer (Rajkapoor et al., 2006) and anti-worms (Ciero et al., 1998). Folk Uses and Recipe: The young leaves, flowers and fruits are boiled and eaten as a vegetable and useful in skin diseases. Plant No: 07. Brassica juncea Botanical Name: Brassica juncea L. Czern. (Fam: Cruciferae). English Name: Brown Mustard. Local Name: Saranh. Parts used: All parts. Chemical constituents: Plant contains Vitamin A and K (Grubben and Denton, 2004), essential oils (Syed et al., 1989). Seeds contain sinigrin and myrosinallylisothiosynate, fixed oils, proteins, mucilages and traces of sinapine hydrogen sulphate (Evans, 2002).

Uses: Ethnomedicinal Uses: Antimicrobial, aperient, diuretic, emetic, anodyne, lactogogue, rubefacient, stimulant and anti-inflammatory (Grubben and Denton, 2004; Srivastava et al., 1982). Folk Uses and Recipe: Oil is used in foods acts as diuretic and condiment. Plant No: 08. Cassia holosericea Fresen. Botanical Name: Cassia holosericea Fresen. (Fam: Caesalpinaceae/Leguminosae). English Name: Indian senna. Local Name: Jhangli senna. Parts used: Pods. Chemical constituents: It contains bergenin (Srinivasan et al., 2007). It yields a novel spermidine alkaloid, caesalpinine A {C25H31O3N3} resins, glucosides, anthraquinone glycosides and mucilage. The amino acids and ethylideneglutamic acid are present in the seeds of C. holosericea. The composition of fatty acid of the seed-oil is a-, b-,y-,d-,e-and j-caesalpins, caesalpin-F and 7-hydroxy-4-methoxyhomo isoflavone {bounducelline} (Khalid et al., 1989; Rai 1989). Uses: Ethnomedicinal Uses: Astringent, antipyretic, anti-periodic, diuretic, cicatrizant (Marta et al., 2006), adaptogenic, antimicrobial, anti-fatigue, antioxidant, tanning material (Howes-Kew, 2005), antidiabetic and also used for curing senile pruritus and muscle contraction. Folk Uses and Recipe: Pods in powdered form are used as cathartic and anthelmintic. Plant No: 09. Calotropis procera Aiton. Botanical Name: Calotropis procera Aiton. (Fam: Asclepiadaceae). English Name: Milkweed, Sodom Apple. Local Name: Akk. Parts used: Leaves, bark and latex. Chemical constituents: The root bark contains - and -amyrin, taraxasterol its isomer taraxasteryl isovalerate, taraxasteryl acetate. Leaves, stem, latex and roots contain cyanidin-3 rhamnoglucoside, cycloart-23-en, 25-diol, cyclosadol, multiflorenol, procesterol, quercetin3-rutinoside, -sitosterol (also present in latex of

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C. procera), -sitost-4-en-3-one, stigmasterol calactinic acid, choline and O-pyrocatechuic acid benzolisolineolone, nemzoyllineolone, long-chain fatty acids, C18 isoursane and vitamin C (Malik and Chaugtai, 1979). The flowers contain calactin, calotoxin, calotropagenin, calotropin, polysaccharides with D-arabinose, glucose, glucosamine and L-rhamnose. Flowers also contain enzymes 3-proteinase and calotropain (protease). Other chemical constituents of C. procera flowers are lupeol, uscharin, proceroside, proceragenin (cardenolide), syriogenin, taraxast-20(30)-en-3-(4-methyl-3pentenoate), 3-thiazoline cardenolide, gigantin, giganteol, isogiganteol, uscharidin, uzarigenin voruscharin a-calotropeol, 3-epimoretenol, lactuceryl acetate and -lactuceryl isovalerate (Atef et al., 1999). Uses: Ethnomedicinal Uses: Diaphoretic, expectorant, antimicrobial (Atef et al., 1999), anti-ulcer, antifertility, anti-cancer, antifungal (Larhsini et al., 1997), antipyretic, anti-inflammatory, analgesic (Ameeta et al., 2006), anthelmintic, purgative mild stimulant. It is also used in leprosy, boils, to remove the prickle of body, cures earache and toothache (Aminuddin and Girach, 1993), abdominal pains. Folk Uses and Recipe: Leaves and latex are rubbed and messaged on muscles and joints as antibacterial and anti-rheumatic. Plant No: 10. Caesalpinia bonduc L. Roxb. Botanical Name: Caesalpinia bonduc L. Roxb. (Fam: Caesalpinaceae). English Name: Fever Nut. Local Name: Kharpat. Parts used: Leaves and seeds. Chemical constituents: Seeds contain diterpenoid bondenolide (Ahmad et al., 1997), several bonducellpins, caesalpins (Jadhav et al., 2002), cassane furanoditerpene, 17-methylvouacapane-8(14),-9(11)-diene (Atul et al., 2003). Uses: Ethnomedicinal Uses: Antiviral, antifungal, anthelmintic, anti-diabetic (Roy et al., 2005), antispasmodic, febrifuge, anthelmintic, antitumor, anti-oxidant (Malaya et al., 2004), rubefacient, also used in liver and spleen disorders.

Folk Uses and Recipe: Paste of leaves and seeds is used as anti-asthmatic and in leucorrhoea. Plant No: 11. Cardiospermum grandiflorum Sw. Botanical Name: Cardiospermum grandiflorum Sw. (Fam: Sapindaceae). English Name: Baloon vine, Blister crepper. Local Name: Baihee amla. Parts used: All parts. Chemical constituents: It contains cardiospermin sulfate, saponin and sulfur containing cyanogenic glycosides (Hübel et al., 1979). Uses: Ethnomedicinal Uses: Antimicrobial, antimalarial (Kubmarawa et al., 2007; Waako et al., 2005), anti-blennorrhagic, emmenagogue, demulcent, anthelmintic, stimulant, diuretic, diaphoretic, emetic, laxative, aperient, stomachic, rubifacient, tonic in fever. Externally used on rheumatic joints, swelling, tumor. It is curative in earache and gastric complaints (Ayyanar et al., 2005) Folk Uses and Recipe: Juice of leaves is used as anthelmintic. Plant No: 12. Capparis eciduas Forssk. Edgew. Botanical Name: Capparis eciduas Forssk. Edgew. (Fam: Capparidaceae). English Name: Capper. Local Name: Kirrir. Parts used: All parts. Chemical constituents: It contains alkaloids spermidine, isocodonocarpine, anti-oxidants bioflavinoid and rutin (Viqar et al., 1985; Viqar et al., 1989). Uses: Ethnomedicinal Uses: Anti-rheumatic, diuretic, kidney disinfectant, vermifuge, tonics, reduce flatulence, improving liver functions. It is used to cure arteriosclerosis, dropsy, anemia, arthritis, gout and migraine (Jagtap et al., 2006). Folk Uses and Recipe: Decoction of young branches is used as alexipharmic and also used to cure toothache. Plant No: 13. Capparis spinosa Forssk. Botanical Name: Capparis spinosa Forssk. (Fam: Capparidaceae). English Name: Caper. Local Name: Gollaro. Parts used: Leaves.

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Chemical constituents: The root bark contains stachydrine, rutic acid. Flower buds contain glucoside, rutin, rutic acid and pectic acid (Sethi et al., 1978). Uses: Ethnomedicinal Uses: Emmenagogue, expectorant, vasoconstrictor, tonic (Ahmed et al., 1972), analgesic, anthelmintic, antihaemorrhoidal, ani-rheumatic (Khakberdyev et al., 1968), aperient, deobstruent, depurative and diuretic (Chopra et al., 1986). Folk Uses and Recipe: Infusion of leaves is used as analgesic and to cure gonorrhoea. Plant No: 14. Chenopodium album L. Botanical Name: Chenopodium album L. (Fam: Chenopodiaceae). English Name: Fat hen. Local Name: Jhill. Parts used: Flowers, Leaves and Seeds. Chemical constituents: The leaves contain Ncaffeoyldopamine, proteins 4%, fat1% and carbohydrates 9%. The seed contains about carbohydrates 50% and proteins 16% (Clarisa et al., 2000). Per 100g of leaves (Dry weight) contain minerals: Calcium: 2.3 g, Phosphorus 0.5g, Iron 0.025g, Vitamins: vitamin A (31.6 g), Thiamine (0.0007g), Riboflavin (0.0016g), Niacin (0.0025g). The major constituent is (17S, 20R, 22R)-4, 7, 20-trihydroxy-1oxowitha-2, 5, 24trienolide (Kumar et al., 2004). Uses: Ethnomedicinal Uses: Antiphlogistic, antirheumatic (Kumar et al., 2004), contraceptive, anthelmintic, laxative and odontalgic (Muhammad et al., 2002). Folk Uses and Recipe: Juice of leaves is used in hepatic and spleen disorders. Plant No: 15. Chorchorus depressus L. Botanical Name: Chorchorus depressus L. (Fam: Tiliaceae). English Name: Gunny. Local Name: Mundairie. Parts used: leaves and seeds. Chemical constituents: It contains two monodesmosidic cycloartane triterpene glycosides, i.e. depressosides E and F and two flavonols glycosides, depressonol A and B (Muhammad et al., 2002; Ahmad et al., 1998).

Uses: Ethnomedicinal Uses: Emollient, tonic, cooling agent and also used for the treatment of gonorrhoea and wounds (Kebu et al., 2006). Folk Uses and Recipe: Decoction of leaves is used in gonorrhoea. Plant No: 16. Datura alba Nees. Botanical Name: Datura alba Nees. (Fam: Solanaceae). English Name: Thorn-apple. Local Name: Chario daturo / Sufed daturo. Parts used: Seeds. Chemical constituents: The seeds contain the alkaloid daturine, atropine, hyoscine and hyoscyamine. It also contains fatty oil (25 %), which contains a new fatty acid, daturic acid (C17H34O2). It stands intermediate between palmitic and stearic acids (Michael et al., 2006). Uses: Ethnomedicinal Uses: Narcotic, mydiatric, diuretic, anesthetic, aphrodisaic, treating bruises and wounds, skin ulcers, hemorrhoids, asthma, rheumatism, whooping cough, muscle spasm, sciatica and painful menstruation (Priya et al., 2002). Folk Uses and Recipe: Powdered seeds are used as antiseptic and analgesic. Plant No: 17. Eclipta alba L. Hassk. Botanical Name: Eclipta alba. L. Hassk. (Fam: Compositae). Local Name: Bhangro. Parts used: Leaves. Chemical constituents: It contains Ecliptasaponin C, a new triterpenoid glucoside with daucosterol and stigmasterol-3-O-glucoside (Zhang and Chen, 1996), other constituents include as wedelolactone, demethylwedelolactone, isodemethylwedelolactone, -formylterthienyl, strychnolactone, -sitosterol, nonacosanol, stearic acid, lacceroic acid and 3,4-dihydoxy benzoic acid (Zhang and Guo, 2001). Uses: Ethnomedicinal Uses: Alterative, alexipharmic, anti-inflammatory (Cheryl et al., 2001), antiasthmatic, bronchodilator, used to treat hernia, night blindness, leucoderma, anemia, skin diseases, syphilis and used for complexion.

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Folk Uses and Recipe: Juice of leaves externally is used as hair restorer. Paste of leaves is used in enlargement of spleen, jaundice and asthma. Plant No: 18. Fagonia cretica L. Botanical Name: Fagonia cretica L. (Fam: Zygophyllaceae). English Name: Cretan prickly clover. Local Name: Damaho. Parts used: Leaves. Chemical constituents: It contains Sitosterol, campestral, oleonolic acid, betulic acid, docosyl docusanoate (Hamid et al., 1989), sapogenin ­ fagoenin (15, 16-dihydroxy-7-oxo-cis-enterythrox-3-ene and 16-O-acetylfagonone (AbdelKader et al., 1993), saponin-I and saponin-II (Saeed et al., 2003), betulin (Ali and Hameed, 1967; Shaker et al., 1999), nahagenin, hederagenin, ursolic acid and pinitol (Atta-urRahman et al., 1982; 1984) and 27hydroxyoleanolic acid and 27-ursolic acid (Miyase et al., 1996). It also contains glucose, rhamnose, xylose, arabinose, fatty acids, ecidu (C10H12N2) amino acids alanine, arganine, glycine, isoleucine, leucine, lysine, phenyalanine, tyrosine, and valine. Uses: Ethnomedicinal Uses: Febrifuge, antioxidant, anticancer, astringent and also used in the treatment of various types of haematological, hepatic, neurological skin lesion and inflammatory conditions (Kasture et al., 2001; Chopra et al., 1956). Folk Uses and Recipe: Aqueous infusion of leaves is used as febrifuge and prophylactic against small pox. Twigs of leaves are used in snake bite. Plant No: 19. Ficus bengalensis L. Botanical Name: Ficus bengalensis L. (Fam: Moraceae). English Name: Banyan tree. Local Name: Bharh. Parts used: Leaves, roots, seeds and bark. Chemical constituents: Its stem and bark contain three ketones, 20-tetratriacontene-2-one, 6heptatriacontene-10-one, pentatriacontan-5-one and other compounds like -sitosterol--Dglucose, meso-inositol, 6-heptatriacontene, pentatriacontan (Subramanian and Misra, 1978; Daniel et al., 1998), It also contains flavonoids

(Regi et al., 2003). The petunidin di-glycoside and quercentin 3-galactoside are present in the leaves, cyanidin rhamnoglycoside and polysaccharides in the fruit are of F. bengalensis. Uses: Ethnomedicinal Uses: Antiatherogenic, hypocholesterolaemic, antioxidant (Rimi et al., 2004), analgesic, anodyne, anti-rheumatism, tonic, anti-dandruff (Ravichandran et al., 2004), astringent and cooling. It is also used to cure toothache and abscesses. Folk Uses and Recipe: Juice of leaves and seeds is used in diabetes, leucorrhoea and lumbago. Plant No: 20. Hipophae salicifolia D. Don. Botanical Name: Hipophae salicifolia D. Don. (Fam: Eleagnaceae).English Name: Willowleaved seabuckthorn. Local Name: Beehni. Parts used: Leaves and fruit. Chemical constituents: Its fresh berries pulp oil contains total carotenoids ranged from 692 to 3420 mg/kg and total tocols from 666 to 1788 mg/kg, palmitoleic acid (32­53%) as the most abundant fatty acids followed by palmitic (25­ 35%), oleic (8­26%), linoleic (5­16%), and linolenic (0.6­2.6%) acids. The berries also contain higher amounts of lipophobic constituents such as vitamin C and flavonols (Ranjith, et al., 2006). The leaves are rich in vitamin A (25 mg/100 g), vitamin C (454 mg/100 g), flavonoids (984 mg/100 g) (Li and Schroeder, 1996) and 16 amino acids. Seabuckthorn is primarily valued for its golden-orange fruits, which are very rich in vitamins A, B1, B12, C, E, K and P (Bounous et al., 1988). Uses: Ethnomedicinal Uses: Anticancer, alexipharmic (Deepak et al., 2007), nutritional (Alam, 2004), tonic for lungs, stomach, heart, spleen, removing blood stasis and promotes blood circulation, treating burns, eczema, and radiation injury (Shandesh et al., 2006). Folk Uses and Recipe: Ripe fruit and its juice are used in jaundice, respiratory diseases. Plant No: 21. Mangifera indica L. Botanical Name: Mangifera indica L. (Fam: Anacardiaceae). English Name: Mango tree. Local Name: Anmb / Amm. Parts used: Fruit.

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Chemical constituents: Plant contains amino acids, gallotannin, gallic acid m-digallic acid, ethylgallate, quercetin, isoquercetin, -sitosterol, (±)-epicatechin, -carotene, -xanthophyll, ellagic, citric, m-trigallic and malic acids, glucogallin, meso-inositol, polysaccharides with highly branched arabinan, (1-4) linked galactouronan or glucan, mangiferin (1,3,6,7tetrahydroxyxanthone-2-glucopyranoside), riboflavin, vitamin C. The fruit of M. indica contains citronellal, diterpene, geraniol, limonene, mangiferol, mangiferone, nerol, nerylacetate, phellandrene, tannin and -,-pinenes. Its panicles, leaves and seed oil contain amyl, butyl, ethyl, methyl, propyl, isobutyl, alcohols (Ansari et al., 2004). The aromatic and aliphatic esters of gallic acid, galactose, D-arabinose and glucose are present in the leaf oil. The myrcene, limonene, isoamyl alcohol and -, -pinenes are present in the flowers (Ansari et al., 2004). Uses: Ethnomedicinal Uses: Anti-inflammatory (Ojewole, 2005). Aquoues infusion of stem-bark has analgesic, hypoglycemic (Ojewole, 2005), antidiabetic (Aderibigbe et al., 2001; Aderibigbe et al., 1999) also used in asthma, bronchitis, cough and throat troubles (Kharkhongor et al.,1981). Folk Uses and Recipe: Fruit is used as antiseptic and anti scorbutic. Plant No: 22. Malva sylvestris L. Botanical Name: Malva sylvestris L. (Fam: Malvaceae). English Name: Mallow. Local Name: Khubazzi. Parts used: Leaves, flowers and seeds. Chemical constituents: Leaves contain tannins, mucilages (Syed, 1989; Evans, 2002). Polysaccharide which is composed of L-rhamnose, D,-galactose, galacturonic acid, glucuronic acid in the molar ratio of 22:6:22:11, 8 % peptide (Gonda et al., 1990). Flowers contain anthocyanins glycosides (Farina et al., 1995). Uses: Ethnomedicinal Uses: Antiphlogistic, astringent (ükran, 2007), demulcent, diuretic, emollient, expectorant, laxative and antimicrobial (Coelho et al., 2004).

Folk Uses and Recipe: Paste of leaves is used as emollient and febrifuge. Plant No: 23. Malva parviflora L. Botanical Name: Malva parviflora L. (Fam: Malvaceae). English Name: Cheese weed. Local Name: Gogi sagh. Parts used: Leaves and seeds. Chemical constituents: It contains glycerides of cyclopropene (malvalic and sterculic), cyclopropane (dihydromalvalic and dihydrosterculic), epoxy, and conjugated dienol acids. The epoxy and conjugated dienol acids are isolated as their methyl esters. These are characterized as cis-12,13-epoxy-cis-9octadecenoic (vernolic) acid, 13-hydroxy-cis-9, trans-11-octadecadienoic (coriolic) acid (Farina et al.,1995) and contains thiamine (Mayer, 1989). Seeds contain fatty oils (Moghis et al., 2006). Uses: Ethnomedicinal Uses: Antidandruff, demulcent, emollient, pectoral, antimicrobial (Hailu et al., 2005), anti-inflammatory and analgesic(Shale et al., 2005). Folk Uses and Recipe: Aqueous infusion of leaves is used as demulcent and antiinflammatory. Plant No: 24. Melilotus indicus L. Botanical Name: Melilotus indicus L. (Fam: Papilionaceae). English Name: Yellow sweet clover. Local Name: Sinjhi. Parts used: Leaves and flowers. Chemical constituents: It contains coumarins, melilotic (hydrocoumaric), orthocoumaric acids, and melilotic anhydride, or lactone. It also contains oleanane triterpenes (soyasapogenol B, soyasapogenol G, and messagenolide), a gammacerane triterpene (gammacer-16-en-3-one), five new ecidu triterpenes (messagenic acids), (27-cis-4-hydroxycinnamoyloxy)betulinic acid, 27-(trans-4-hydroxycinnamoyloxy) betulinic acid, 20(S)-3-hydroxy-29-oxolupan-28-oic acid; 3,30dihydroxylup-20(29)-en-28-oic acid; and 3,20dihydroxylup-18(19)-en-28-oic acid, respectively) and sterols -sitosterol, ergosterol peroxide, 7hydroxysitosterol, 7-hydroxysitosterol and 7oxositosterol (Francisco et al., 1997; Nigel, 2007).

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Uses: Ethnomedicinal Uses: Antibacterial, anticoagulant, astringent, emollient, laxative, narcotic (Francisco et al., 1997). Folk Uses and Recipe: Juice of leaves is used as emollient and in infantile eciduas. Plant No: 25. Portulaca oleracea L. Botanical Name: Portulaca oleracea L. (Fam: Potulacaceae). English Name: Green Purslane. Local Name: Lunak. Parts used: Leaves and seeds. Chemical constituents: The leaves contain about carbohydrates 7%, proteins 2% and fat 0.5%, or per 100g of dried leaves, proteins 18-35g, fat 25g, carbohydrate36-63 g, fibre 9-15g, calcium0.82g, phosphorus 0.3-0.8g, iron 12-47g, sodium 0.05g, potassium 0.5-3 g (Habtemariam et al., 1993), -carotene10560-20000ug, thiamine 0.20.5mg, riboflavin 1.1- 1.6 mg, niacin 5-7mg and vitamin C 0.17-0.33 mg. -sitosterol, -sitosterolglucoside, N,N-dicyclohexylurea and allantoin (Asia et al., 2004). It contains alkaloids (oleraceins A, B, C, D and E) structurally these are known as 5-hydroxy-1-p-coumaric acyl-2,3dihydro-1H-indole-2-carboxylic acid-6-O--Dglucopyranoside, 5-hydroxy-1-ferulic acyl-2,3dihydro-1H-indole-2-carboxylic acid-6-O--Dglucopyranoside, 5-hydroxy-1-(p-coumaric acyl7'-O--D-glucopyranose)-2,3-dihydro-1H-indole2-carboxylic acid-6-O--D-glucopyranoside, 5hydroxy-1-(ferulicacyl-7'-O--D-glucopyranose)2,3-dihydro-1H-indole-2-carboxylic acid-6-O-D-glucopyranoside and 8,9-dihydroxy-1,5,6,10tetrahydro-2H-pyrrolo[2,1-]isoquinolin-3-one. It contains new compound which is identified as 1(2-hydroxy-4-methoxyphenylamino)-1-deoxy-glucoside 1,2-carbamate (1) {methoxy glucoside carbamate}) (Diana et al., 2006). Uses: Ethnomedicinal Uses: Muscle relaxant (Habtemariam et al., 1993), antibacterial, antiscorbutic (Lan et al., 2005), depurative, diuretic (Rocha et al., 2002), febrifuge, tonic, vermifuge, anti-inflammatory and analgesics (Chan et al., 1994). Folk Uses and Recipe: Leaves are cocked and taken as food to act as vermifuge and externally used in skin disease.

Plant No: 26. Solanum surattense Burm. F. Botanical Name: Solanum surattense Burm. F. (Fam: Solanaceae). Local Name: Aederi. Parts used: Whole Plant. Chemical constituents: Berries contain solanocarpine, solanocarpidine, solanines and carpeatral (Syed, 1989). Uses: Ethnomedicinal Uses: Digestive, alterative, expectorant, aperient, diuretic, and anthelmintic. It is also used in cough, asthma, fever, chest pain, sore throat, locally to relieve pain. Folk Uses and Recipe: Juice of leaves is used as expectorant, Anthelmintic, and anti-asthmatic. Plant No: 27. Solanum nigrum Auctplur. Botanical Name: Solanum nigrum Auctplur. (Fam: Solanaceae). English Name: Black nightshade. Local Name: Kailperoon. Parts used: Leaves and seeds. Chemical constituents: The plant contains six new steroidal saponins, solanigrosides, degalactotigonin, the 60% ethanolic extract of the dried herb contains two new pregnane saponins, i.e. solanigroside A and solanigroside B (Syed, 1989; Xin-Lan et al., 2007). Uses: Ethnomedicinal Uses: Antioxidant, anti-periodic, anti-inflammatory, perspirative, diuretic, emollient, purgative, sedative, anti-pyretic and analgesic (Kyung-Sun, et al., 2004). Folk Uses and Recipe: Ripe fruit is taken as sedative. Berries are used in piles. Plant No: 28. Solanum melongena L. Botanical Name: Solanum melongena L. (Fam: Solanaceae). English Name: Aubergine. Local Name: Wanghan / Bhengan. Parts used: Fruit and leaves. Chemical constituents: It contains hydroxyglutamic acid, 5-hydroxytryptamine, isoleucine, leucine, aspartic acid, phenylalanine, threonine, tryptophan, methionine, pipecolic acid, valine, arginine, lysine, -aminobutyric acid,

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histidine, delphinidin-3-bioside (nasunin), choline, nicotinic acid, riboflavin, vitamins A and C, fructose, glucose, sucrose, monohydroxylycopene, lycoxanthin; b-amino-4-ethyglyoxaline; caffeic, chlorogenic and neochlorogenic acids, hydroxycinnamic acid, oxalic acid; pectins; scopoletin, trigonelline, solasodine. The fruit contains arachidic, linoleic, myristic, oleic, palmitic and stearic acids, diosgenin (also in rootbark), tigogenin, -amyrin citrostadienol, cycloartanol, 24-methylenecycloartanol, 31norcycloartanol, cycloratenol, 31-norcycloartenol, cycloeucalenol, daturaolone, daturadiol, lupeol, 4-methlcholesta-8-enol, 4,24-dimethlcholesta7,24-dienol, 4-methl-24-ethylcholesta-7,24dienol, 4-14,24-trimethylcholesta-8,24-dienol, gramisterol, lanost-8-en-3-ol, 24-methylenelanost-8-en-3--ol, 31-norlanost-9(11)-enol, 24methyl-31-norlanost-9(11)-enol, lanosterol, lophenol and its 24®-ethyl and 24-methyl derivatives, obtusifoliol (seeds); chlorogenic, hydrocaffeic, protocatechuic and trans-caffeic acids,4-ethylcatechol. The leaves contain paminobenzaldehyde, ethylcaffeate, trans-ferulic acid, N-trans-feruloyltyramine, N-trans-pcoumaroyltyramine, vanillin and isoscopoletin. The root contains n-alkanes, arginine glycoside phytoalexin (lubimin), imidazoylethylamine, neotigogenin, solanina solasonin, -sitosterol and stigmasterol (Negi et al., 1993). Uses: Ethnomedicinal Uses: Anticholesterolemic, antidote for poisons, astringent, narcotic and poultice (Parmod et al., 2004; Cheryl et al., 2007). Folk Uses and Recipe: Fruit is used as food and leaves are narcotic. Plant No: 29. Vitex negundo L. Botanical Name: Vitex negundo L. (Fam: Verbinaceae). Local Name: Sunbhaloo. Parts used: Whole plant. Chemical constituents: The leaves of Vitex negundo contains new flavone glycoside (Om, et al., 2007; Sathiamoorthy et al., 2007). The constituents of the leaf oil are -pinene (35%), limonene (11%) and bicyclogermacrene (9%),

while the fruit oil contained -pinene (32%), bicyclogermacrene (14%) and limonene (11%), and the flower oil contained -pinene (14%), bicyclogermacrene (9%) and limonene (6%). Uses: Ethnomedicinal Uses: Anti-rheumatic, antiinflammatory, antifungal, antibacterial, febrifuge, expectorant, and tonic. Used to cure stomachache, eye disease, pain in body; catarrhal fever, headache and earache (Sathiamoorthy et al., 2007). Folk Uses and Recipe: Decoction of leaves is used as febrifuge and vermicide. Plant No: 30. Withania somnifera Dunal. Botanical Name: Withania somnifera Dunal. (Fam: Solanaceae). English Name: Winter cherry. Local Name: Assgandh. Parts used: Leaves and seeds. Chemical constituents: It contains Withaferin A, chemically characterized as (4, 27-dihydroxy5-6-epoxy-1-oxowitha-2, 24-dienolide), withianolide A, D, withinone (Sangwan et al., 2004). The roots of Withania somnifera contain two new sterols, namely 3-epi--sitosterol and 20, (21)-dehydrocampesterol along with -sitosterol and campesterol (Kumar et al., 2000). Uses: Ethnomedicinal Uses: Adaptogenic, anticancer (Christina et al., 2004), anti-convulsant, immunomodulatory, antioxidative. It is used to cure arthritis, geriatric, behavioural and stressrelated problems. It is also used as nervine tonic, memory and cognition improver, natural sleep inducer, anxiolytic. Relieves anxiety neurosis, physical and mental stress, and relieves general debility and depression. It is used to treat sexual impotency, oligospermia, and general weakness, fatigue, convalescence, recuperation from general debility, neurasthenia and better disease resistance (Sangwan et al., 2004). Folk Uses and Recipe: Aquoeus infusion of leaves is used as narcotic and diuretic.

A.H. Memon, et al., Fig. 1. Collected medicinal plants of Districts Dadu and Jamshoro, Sindh, Pakistan.

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Acacia nilotica.

Abutilon indicum.

Aegle marmelos.

Aerva javanica.

Alhagi maurorum.

Bauhinia veriegata.

Brassica juncea.

Cassia holosericea.

Calotropis procera.

Caesalpinia bonduc.

Cardiospermum grandiflorum.

Capparis

eciduas.

Capparis spinosa.

Chenopodium album.

Chorchorus depressus.

Datura alba.

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Fig. 2. Collected medicinal plants of Districts Dadu and Jamshoro, Sindh, Pakistan.

Eclipta alba.

Hipophae salicifolia. Fagonia cretica. Ficus bengalensis.

Malva parviflora. Mangifera indica. Malva sylvestris.

Melilotus indicus.

Solanum surattense. Portulaca oleracea. Solanum nigrum.

Solanum melongena.

Vitex negundo.

Withania somnifera.

A.H. Memon, et al., Table 1. Collected medicinal plants from Dadu and Jamshoro Districts, Sindh, Pakistan. S.# 01 02 03 04 05 06 07 08 09 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Botanical name Acacia nilotica Lam. Willd. Abutilon indicum L. Sweet. Aegle marmelos L. Correa. Aerva javanica Burm.F. Alhaji maurorum Medic. Bauhinia veriegata L. Brassica juncea L. Czern. Cassia holosericea Fresen. Calotropis procera. Aiton. Caesalpinia bonduc L. Roxb. Cardiospermum grandiflorum. Sw. Capparis decidua Forssk. Capparis spinosa L. Chenopodium album L. Chorchorus depressus L. Datura alba Nees. Eclipta alba L. Hassk. Fagonia cretica L. Ficus bengalensis L. Hipophae salicifolia D. Don. Mangifera indica L. Malva sylvestris L. Malva parviflora L. Melilotus indicus L. Portulaca oleracea L. Solanum surattense Burm. F. Solanum nigrum Auctplur. Solanum melongena L. Vitex negundo L. Withania somnifera Dunal. Local name Babul. Pat-tir. Bel geeri. Booh. Kandeero. Kachnar Saranh. Jhangli senna. Akk. Kharpat. Baihee amla. Kirrir. Gollaro. Jhill. Mundairie. Chario daturo. Bhangro. Damaho. Bharh. Beehni. Anbh. / Amm. Khubazzi. Gogi sagh. Sinjhi. Lunak. Aederi. Kailperoon. Waghan. Sunbhaloo. Assgandh. Family Leguminosae. Malvaceae. Rutaceae. Amaranthaceae Papilionaceae. Caesalpinaceae Cruciferae. Caesalpinaceae Asclepiadaceae Caesalpinaceae Sapindaceae. Capparidaceae. Capparidaceae. Chenopodiaceae. Tiliaceae. Solanaaceae. Compositae. Zygophyllaceae. Moraceae. Eleagnaceae. Anacardiaceae. Malvaceae. Malvaceae. Papilionaceae. Potulacaceae. Solanaceae. Solanaceae. Solanaceae. Verbinaceae. Solanaceae.

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Folk uses Anti-diarrheal for animals. Laxative & Diuretic. Anti-dysentery. Anti-inflammatory. Anti-rheumatic. Anti-piles. Useful in skin diseases. Diuretic. Cathartic & Anthelmintic. Anti-rheumatic. Anti-asthmatic. Anthelmintic. Alexipharmic. Analgesic. In hepatic & spleen disorders. Used in gonorrhoea. Antiseptic & Analgesic. Anti-asthmatic. Febrifuge, used in snake bite. Used in diabetic & lumbago. Used in jaundice. Antiseptic & Antiscorbutic. Emollient & Febrifuge. Anti-inflammatory. Emollient. Vermifuge & Diuretic. Expectorant & Anthelmintic. Sedative. & anti-piles. Leaves are narcotic. Febrifuge & Vermicide. Narcotic & Diuretic.

4.

Discussion The prehistoric people of all ages had knowledge of medicinal plants which they aquire as a result of trial and error. This knowledge still exists and many plants are used in traditional

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herbal remedies as whole plant or part of plant or its aqueous extract is used. Pakistan has a diverse flora containing about 6000 species of phenerograms. It is estimated that around 700 plant species are used as medicinal or aromatic plants. In Dadu and Jamshoro Districts, it is recorded that most of species are used for multiple therapies. For example aquoeus extract of leaves of Withania somnifera is used as narcotic and diuretic and juice of Solanum surattense leaves is used as expectorant, Anthelmintic, and antiasthmatic. The medicinal plants which are collected from districts Dadu and Jamshoro Sindh Provice of Pakistan are utilized by local people to treat and cure various diseases such as infusion of pods of Acacia nilotica is used as anti-diarrheal, and flowers are used for infertility in animals. Infusion of leaves of Abutilon indicum traditionally are used to cure asthma, gonorrhoea and in toothache as mouthwash. The phytochemical study is failed to attribute these therapeutic activities to any chemical constituents of Abutilon indicum leaves. Plant fumigation and plant paste of Aerva javanica plant with water is used for treatment of sexual incapacity, sexual asthenia, frigidity and aphrodisiac. Although ethnopharmacological its sedative effect is observed so there is need to stop such use of this plant by local people by informing scientific exploration. Local people had a little knowledge about proper use and phytochemicals of medicinal plants. So various awarenees programs can be adopted to inform the people from the hazardous effects of medicinal plants by informing them verbally or document the scientific research in local language so that misuse of such medicinal plants can be minimized. Refernces Abdel-Kader, M.S., A.A. Omar, N.A. Abdel-Salam and F.R. Stermitz, (1993) Erythroxan diterpenes and flavonoids from Fagonia bruguieri. Phytochemistry. (33): 718-720. Abdulkarim, A.S. Yusuf, G.O. Andrew, U. Z. Abdulkadir and E.M. Abdurahman, (2005) Evaluation of five medicinal plants used in diarrhoea treatment in Nigeria. J. Ethnopharmacol. (101): 27-30. Aderibigbe, A.O., T.S. Emudianughe and B.A. Lawal, (1999) Antihyperglycaemic effect of Mangifera indica in rat. Phytother. Res. (13) (06): 504-507. Aderibigbe, A.O., T.S. Emudianughe and B.A. Lawal, (2001) Evaluation of the antidiabetic action of Mangifera indica in mice. Phytother. Res. (15) (05): 456-458.

Ahmad, V.U., A. Ali, Z. Ali, F.T. Baqai and F.N. Zafar, (1998) Cycloartane triterpene glucosides from Corchorus depressus. J. Phytochem. (49) (03): 829-834.

Ahmad, V.U., M.S. Ali and K. Usmanghani, (1997). A new diterpenoid Bondenolide from the seeds of Caesalpinia bonduc. J. Chem. Sci. (52) (3): 410-412. Ahmed, Z.F., A.M. Rizk, F.M. Hammouda and M.M. Seif-El-Nasr, (1972) Glucosinolates of Egyptian Capparis species. Phytochemistry. (11): 251-256. AIi, M. A. and Z. Hameed, (1967) Triterpenoids, II. Another sapogenin from Fagonia cretica. Pak. J. Sci. Ind. Reso. (10) (2): 140-141. Alam, Z., (2004) Chemical and Nutritional Constituents of Sea Buckthorn Juice. Pak. J. Nut. (3) (02): 99-106. Ameeta A. and K.P. Anupam, (2006) CNS activity of Calotropis gigantea roots. J. Ethnopharmacol. (106) (1): 142-145.

Aminuddin and R.D. Girach, (1993) Observations on ethnobotany of the Bhunjia--a tribe of Sonabera plateau, Kalahandi, Orissa. J. Ethnobot. (5): 84pp. Anderson, D.M.W., S.E. Hirst and J.F. Stoddart, (1967). Studies on uronic acid materials and some structural features of Acacia arabica gum. J. Chem. Soc. 1476-1486. Ansari, S.H., M. Ali, N.A. Velasco, A. Perez and J. Maria, (2004) Characterization of Volatile Constituents of Mango 'Qalmi' (Mangifera indica L.) Fruit. J. Essen. Oil Res. (02): 122pp.

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Asia, N.R., U.A. Fatma, S. Mayadeh and O.T. Mutasem, (2004) Investigation of the active constituents of Prtulaca oleracea L. (Portulacaceae) growing in jordan. Pak. J. Pharm. Sci. (17) (01): 37-45. Atef, G.H., M.H.A. Elgamal, N.A.M. Morsy, H. Duddeck J. Kovács and G. Tóth, (1999) Two cardenolides from Calotropis procera. J. Magn. Reson. Chem. (17): 754-757. Atta, A.H. and K.A. El-Sooud, (2004) The antinociceptive effect of some Egyptian medicinal plant extracts. J. Ethnopharmacol. (95): 235-238. Atta-ur-Rahman, A.A. Ansari and L. Kenne, (1984) Hederagenin, ursolic acid and pinitol from Fagonia indica. J. Nat. Prod. (47): 186-187. Atta-ur-Rahman, A.A. Ansari, S.A. Drexiex and J. Clardyl, (1982) The isolation and structure of nahagenin. Heterocycles. (19): 217-20. Attia A.H. and S.M. Mouneir, (2004) Antidiarrhoeal activity of some Egyptian medicinal plant extracts. J. Ethnopharmacol. (92) (2-3): 303-309. Atul, N.J., N. Kaur and K.K. Bhutani, (2003) A new furanoditerpenoid marker for the distinction between the seeds of two species of Caesalpini. J. Phytochem. Anal. (14) (5): 315-318. Ayyanar, M. and S. Ignacimuthu, (2005) Traditional knowledge of Kani tribals in Tirunelveli hills, Tamil Nadu, India. J. Ethnopharmacol. 102: 246-255. Bai, W.Q., Y.L. Zhang and W.K. Bao, (2003) Landscape upper reaches of the Dadu River. J. Nat.Reso. (18) (01): 76-80. patterns and Kouthalai of in the

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Sindh Univ. Res. Jour. (Sci. Ser.) Vol: 40 (2) 109-118

SURJ

Sindh Univ. Res.

BPEL SERVICES COLLABORATION A. G. Memon, and M. S. Chandio Institute of Mathematics and Computer Science, University of Sindh, Jamshoro, Pakistan

[email protected], [email protected]

(Received 07th February 2009 and Revised 14th February 2009) Abstract Today collaboration services technologies for interoperability; dynamic discovery and collaboration of complex service have become popular. This may be used in different mechanism such as P2P, Grid-to-Grid, network-to-network and Internet. Complex services ensure the ability exchange of different applications running on heterogeneous platforms over the Internet. Nowadays these types of services are gaining attention in the world. Industry standards for Web Service composition, such as BPEL technology, provide the notation and additional control mechanisms for the execution of business processes in Web service collaborations. The function of BPEL technology consists of locations, containing dynamic documents and -calculus processes which communicate with each other. Here, we have used process equivalence to analyze various communication patterns used in different query systems by servers, and suggest a novel model connecting mobility. The BPEL, which is a simple and conventional addition of the -calculus, we introduce it as a theoretical and foundational model for collaborative services management. We proposed BPEL model and implemented in formalization of location and relocation with cloning framework for multiple web collaborative services. It validates a range of access types to resources with mixture of relocation, similar execution, and BPEL declaration. Our model simplifies some semantical and pragmatical aspects, in particular regarding temporization, gaining a better understanding of the basic issues. Keywords: BPEL, -calculus, Services-oriented, Collaboration Services.

1. Introduction and related work The rise of web service has spurred automation of cooperation among organizations. Organizations process interoperability is an important topic for business currently. Loosely coupled organizations services are gaining importance in many business domains. The distinct characteristics of inter-organizational process, such as heterogeneity and autonomy of the participating applications have lead to the development of several new agent-based technologies supporting inter-organizational cooperation. The organizations shall contribute with their core competence and thereby generate a "best-of-breed" construct in respect of required expertise. The propagated benefits are higher flexibility and quicker provision of customized services. Besides, bespoke customer solutions, dissolution of conventional organizational structures by concentrating on core competences, and the rapid development of technologies to collaborate and communicate between remote locations are further trends that render, this concept conceivable and promising. It also speaks of different languages for exemplifying a process that collaboration open communications and composes a new one. The aim of collaboration communication is to ease and to automate business process collaborations across organizations boundaries. In paper (Memon

2005), we presented encoding and translation for organization-to-organization communication for share services which is based upon the -calculus. Now this paper is extensive the work which based upon BPEL. It defines information of control and data flow among jobs, such as invocation to a communication mechanism. The main reason here for using the -calculus for formalization is collaboration services composition language, like XLANG, BPEL and WS-CDL claim to be based on it, and they should therefore allow rigorous mathematical treatment. However, no interesting relation with process algebras has really been proved for any of

A. G. Memon, and M. S. Chandio

interoperability of Client/Server interactions. We proposed BPEL model and implemented in formalization of migration and cloning framework for composite web collaboration services (Yin, 2000). A BPEL is a protocol that has the ability to nomadic from one position to different positions (Memon 2005, Ishikawa, 2004). It validates a range of access types to resources with combination of relocation, similar execution, and BPEL announcement. This paper aims to make process calculus that is well suited express the conventions, uniform organizational resources and other shared idiosyncrasies that hold large organizational communication and collaborations methods together.

110

neither them, nor an effective tool for analysis and reasoning, either theoretical or software based, has been released. Therefore, we see a gap that needs to be filled and we want to address the problem of composing services starting directly from the -calculus. In this paper, we mainly refer to BPEL, the most likely candidate to become a standard among workflow-based composition languages. Other languages have been introduced, among them WS-CDL (Kavantzas, 98) which claims to be in some relation with the fusion calculus (Parrow, 98). Researchers discuss formal semantics of compensable activities in this context. The calculus in (Butler, 2004) is inspired by BPBeans (Chessel, 2001) the t-calculus (Bocchi, 2003) focuses on BizTalk; (Bruni, 2003) deals with short-lived transactions in BizTalk; (Bruni, 2005) also presents the formal semantics for a hierarchy of transactional calculi with increasing expressiveness. This paper focuses on formal definition which framework based on Mobile Ambients (Bugliesi, 2004, Cardelli, 2000) which is similar to -calculus (Parrow, 2001). We studied the interoperability of Client/Server and explored the approach to supporting high

-calculus The -calculus (Yin, 2005) is built around two classes of identifiers, which are Channels and Levels. Channel names (Milner, 97) are drawn from infinite supply. A specification language must be powerful enough to express the mapping of a simulation model to any protocol on any target agent architecture. The -calculus expresses the move of data but also of code. This offers a solid base to make use of the Agent generation. The BPEL paradigm is a construct where mobility is achieved by allowing agents to be passed as values in a communication. This model of concurrent computation based upon the notion of naming. The most primitive entity in calculus is a name which refers to a link or a channel. Semantics is done in terms of a reduction system and a version of labeled transitions called commitment. In the BPEL not only names, but also agents of arbitrarily high-order, can be transmitted. The syntax of the BPEL is an extension of the syntax of the -calculus. Let be a, b, .......and x, y,....... a set of names and P, Q,...... a set of processes.

2.

· · ·

0 is inaction; it is a process that can do nothing.

| denotes parallel composition,

in the composition P | Q , the two components can proceed independently and interact via shared names or processes. is called the restriction and means that the scope of name b is restricted to P.

(v.b) P

· · · · · · · · · ·

null

( x).P

New input action

a[ P]

in M

M=

out M

def

ambient

Enter into M

Masseges

Exit of M

Loc

M .M

Loc

·

open M

M .P

open M

Capability actions

M

asynchronous output action

·

!P replicated input

on channel

The following grammars define the languages of processes in between P2P, Grid-to-Grid, network-tonetwork and Internet.

Channels: Host Net Ch ::= | ( ) @host Ch ::= | ( ) @host | @Net

host , net ,........ Level 111

Names: Meta-Variable Levels Net, places and agents

BPEL Services collaboration

def

message types and port types which represent the The Net consists of three places. Client is a operations supported by the service and the interaction place where customer for enquiring information of modalities. This information is then used by BPEL for particular goods will resides. Serveri is different specifying the flow of actions to perform. A BPEL organizations where it hold an agent that gives a document is an XML-based document that can be visiting agent an advice for shops selling a executed by an orchestration engine which is the central particular goods and Server is required shop where coordinator. The engine will read the BPEL document and product available. will invoke the necessary collaboration services in the 3. Formal Model of BPEL Process within specified order. The process itself will be offered as a BPEL collaboration service and can be invoked in the same way. 3.1 WS-BPEL BPEL is, in practice, a layer on top of WSDL. The information contained in WSDL documents defines

Net =Customer | Client | Serveri | Server(Required_server)

<process name = "The Process"> . <partnerLinks> for listing the external web service invoked from within the workflow . <variable>, <assign>, <copy> and XPath queries for data storage and manipulation . <corelarionSets> for specifying binding for a set of operations to a service instance . <invoke> and <invoke> ..... < receive> for consuming synchronous and asynchronous web services . <scope> and <faultHandlers> for execution management . <flow> and <sequence> for parallel execution paths . <switch> for conditional branching . </process>

To make more intelligible the syntax, elements will be expressed by using BNF formalisms instead of XML-Schema (in the same style of the BPEL specification). The BPEL Technology Framework This section outlines the main features of the BPEL, a distributed model of computing for large-scale open networks. We briefly introduce BPEL, discuss the objectives of the work and present the abstractions of the framework, which we exploit for reporting data travelers' attitudes. In Section 2 shows the syntax and basic communication of BPEL. Common reductions and A. G. Memon, and M. S. Chandio notions of concurrent process algebrian. Name and boundary are used earlier work (Memon, 2005). BPEL can be generally described as resource access control with hierarchical locations mobility. Our aim is not to provide distributed high level-programming model but only we explore hiding localities in distributed programming and BPEL enables representation of synchronization/asynchronization programming constructs. Another view of BPEL is as advanced distributed services, which is implementing in higher-level languages. BPEL is thus expressive enough to define the attitudes of agent as processes P,Q. It can be expressed as ambient host and ambient agent like (host[agent[P|Q| ...]...] ). In the light of BPEL task takes place for the lowest common denominator among various database organizations applications. Specially complicated services that require higher degrees of lucidity can be built-on. Moreover it is assumed that goings-on

def

3.2

internal communication remote messaging with other external communication. In other terminology may any interactions occur outside but not inside of hosts. This logically implies that observable action of each host or agent do not change even manifold hosts or agents actions are distributed. Such collaborative services are needed distributed Memory management, location independently, secure messaging and channels with guaranteed quality of service. So BPEL can provide a model of mobility, which subsumes message passing, remote evaluation as well as process migration, along with models user mobility.

112

The BPEL Interaction We first characterize interaction actions for remote that provides a service as activity process. Name-value procedure handles information the form of identifiers by BPEL. BPEL using the terminology of records [Luca Cardelli, 1998] directly in Name-values. As is received record that has rec r (l1 = v1 ,..., ln = vn ) consists of pairs of variables labels li with value vi, get r l ( x).P obtain a data of the variable l in the record and continues as P, and at last set r l x.P sets data of the variable l in the record and continues as P. We denote l1 = x, ln = vn as l = v when the label Names and data with reductions an

recr (l1 = v1 ,..., ln = vn )

3.2.1

The values of Name-values in the BPEL process are lagent and lhost which contains special entries. In order to get the Names, we communicate as bellow.

| setr li x.P recr (l1 = v1 ,..., l1 = x, ln = vn ) | P .

getloc ( agentc , hostc ).P =get Mem agent ( agentc ).getr Mem host ( hostc ).P

Asynchronous send and synchronous receive are representing remote messaging. External services activities are swap of action messages in the similar process.

Remote Input

cmp send ( M , x ) = pktinput / output [ M . x ] send ( M , x ).P =cmp send ( M , x ) | P

def def

Remote Output

cmp recv ( M , x , n ) = pktinput / output [ M .( x ).n[ M -1 . x ] recv ( M , x ).P = ( vx ) cmprecv ( M , x , n ) | !( open n ) | ( x ).P

def def

P and Q process launched I/O components,!open pktinput/output to make I/O actions embedded in pktinput/output work.

agent1[ send (out agent1 .in ch, n).P] | recv(out agent2 .in ch, x).Q] | ch [!open pktinput / output ] agent1[ P] | agent2 [Q{x n}] | ch[!open pktinput / output ]

In our work, an agent activity is contained a remote I/O action in a host. As seen in the following

Activity Remote Input

send (ch, x) =getloc(agentc , hostc ). send (out agentc .out hostc .in ch, x)

def

Activity Remote Output

recv(ch, x) =getloc( agentc , hostc ). recv(out agentc .out hostc .in ch, x)

def

Service locator for each BPEL process We reveal a large-scale locator communication for each BPEL process, that have record location (loc) which returns a path to the mediator that has the most recent address of Name. We explore that each mediator has its own service space Memory as we prove earlier that

3.2.2

represent real service swap in between clientserver. Virtually only required service should be sent to one mediator to different mediator by analyzing service dependencies in linking different activities. In the following figure mediator performs setv for locating service.

Service Access

cmpsetv ( M , l , M , n) = pktloc [ M .setr loc l M .n[ M -1 ] i.e M is roaming Loc,M is Update Loc and M -1 is reverse Loc setv( M , l , x, M ).P =(vn)(cmpsetv ( M , l , M , n) | open n.setr Mem l x.P) cmpgetv ( M , l , x, n) = pktloc [ M .getr loc l M . pktMem [ M .getr Mem l ( x).n[( M 1 ) -1 .M -1 . x ]] getv( M , l , x).P =(vn)(cmpgetv ( M , l , x, n) | open n.( x).P)

def def def def

All Agents and locator services must control !open pktinput/output and !open pktMem.

BPEL Services collaboration

In our work all above said actions are communicate by the following methods. We are to embody action in BPEL as a process interface with I/O remote processes and remote variable access.

Remote Input

cmp send ( M , x ) = pktinput / output [ M . x ] send ( M , x ).P =cmp send ( M , x ) | P

def def

113

Remote Output

cmprecv ( M , x , n ) = pktinput / output [ M .( x ).n[ M -1 . x ] recv ( M , x ).P = ( vx ) cmprecv ( M , x , n ) | !( open n ) | ( x ).P

def def

P and Q process launched I/O components,!open pktinput/output to make I/O actions embedded in pktinput/output work.

i.e., agent1[ send (out agent1 .in ch, n).P] | recv(out agent2 .in ch, x).Q] |

ch [!open pktinput / output ] agent1[ P] | agent2 [Q{x n}] | ch[!open pktinput / output ]

In our work, an agent activity is contained a remote I/O action in a host. As seen in the following Activity Remote Input Activity Remote Output

send (ch, x) =getloc(agentc , hostc ). send (out agentc .out hostc .in ch, x)

def

recv(ch, x) =getloc(agentc , hostc ). recv(out agentc .out hostc .in ch, x)

def

We are to embody an activity in BPEL as a process interaction with remote I/O processes and remote variable access.

4. BPEL Mapping Activity to Process

BPEL WSDL, UDDI

Transactions Coordination

BPEL

Cryptographic Security Service

Bellow we explore the BPEL interaction activity process and expressions consequent to those in BPEL.

BPEL Process and BPEL First we judge the expression of a BPEL process. A host is activated in BPEL with the help of BPEL expressions. We show bellow.

[< process > activities < / process >] ::= [(v agent (l1 ... ln , ch1 .... chn ).sv)

4.1

agent[[actvities] | rec Mem(l1 =,......., ln =lagent = agent lhost = host ) | !open pktMem

| ch[out agent.out host.!open pktinput / output ) | ..... | chn [out agent.out host.!open pktinput / output ] | sv[rec loc(l1 =,......., ln =)]]

The Every new agent contains own Memory as well as processes communication channel (channels ch1,...,chn ) for action. Service locator sv to be activated the host Name. With existing processes P, as (host[ P | agent[...]]) are contained in the host mutually. Activation and completion of control messages expressed as exchange control by structured activities (SA) in BPEL process. In section 4.2, shows the communicating of progressive movement from

- 0 SA.

first to last channel ci for action activityi. In BPEL structured activities (switch, while, and pick) are expressions of branch and iteration [16 Luca Cardelli, 98]. All nested flow activities are activates simultaneously. Theorem 3.1 Every BPEL events can be uttered as a process P, BPEL events are related to SA. Bellow said BPEL events processes to be satisfied.

A. G. Memon, and M. S. Chandio - if P P and P SA then P SA.

'

114

- if P SA then ract.P SA where ract {send (ch, x), recv(ch, x), setv( sv, l , x), getv(sv, l , x)}.

- if P, Q SA' then P | Q SA' .

An BPEL events executed block is expressed as P SA. and also as comparable composition behavior belongs to SA. Mobility: Mobility allows a code to move on other execution platform in proactive mode: a copy of the code and its current state is moved to the destination platform for being started from a specific point (weak mobility). Unlike the clonation, the code that performs the move primitive comes destroyed if the execution of the

def

movement primitive succeeded. Communication directly results from the clonation primitive. After a clone operation will be created an exclusive communication channel between cloning and cloned codes. Only way to have processes migrate in the BPEL is by locality migration. Migrated channel P is executed as block associate, where P communicates to the block. The migrated action we communicate as bellow.

migrate ch.P =(vb)(recv(Ch, th).getloc(agentc , hostc ).out .hostc .in th.setr Mem lhost th.open b | b[ P])

In predefined channel, first of all arrived Name of th (objective host) then roaming from the existing host to th, action will be updated in host Memory, and lastly starts execution of behavior in the block are invoked the platform We have the following reduction.

host1[ agent[ migrate ch.P | Q | rec Mem (lhost = host1 , l = v )] | R ] | host 2 [ S ] | ch[!open pktinput / output ] | send (in ch, host 2 )

host1 [ R ] | host2 | agent[ P | Q | rec Mem (lhost = host2 , l = v )]]] | ch[!open pktinput / output ]

Clonation: A clone is a set of functions containing all projections and closed under composition. Clonation allows duplicating the code and the state of a running code. After a

def

clonation there will be two identifiable codes in execution. The smallest clone containing a set of functions P is denoted by [P]. The clonation action is formulizing as bellow.

clone.P = (v agent )( recv (ch, c).getloc ( agentc , hostc ). agent[out agentc .P | rec Mem(lagent = agent , lhost = hostc , l =| !open pkt Mem ]

Above said actions are predefined channel that is fired by the invoked platform, and makes a newfashioned agent containing P outside of the inventive agent. We have the bellow reduction.

host1 [agent1[clone.P | Q | rec Mem (lagent = agent1 , lhost = host , l = v )] | ch[!open pktinput / output ] | send (in ch,)

(v agent ) ( host[ agent1[Q | rec Mem (lagent = agent1 , lhost = host , l = v )] | agent[ P | rec Mem (lagent = agent , lhost = host , l =)

| !open pktMem ]] | ch[!open pktinput / output ]

Communication: Communication (through exchange of messages) is possible only between cloning and cloned. Messages are sent-received in asynchronous-synchronous fashion. The virtual machine relies on a calculus which describes the semantics of the minimal set of operation isolated to characterize a platform supporting mobile code [Jianwei Yin, 2005]. The calculus for modeling BPEL technology is summarized here below.

def

4.2 Barbed Equivalence In section 3.2.1 and 3.2.2, we classify barbs that hold initiate mechanism of remote I/O events. Though general barbs Theorem 3.1. catch the primitive actions which is based on observable behaviors of processes[M. Vigliotti, 2002]. As we use the barbed bisimulation notion.

P send ( ch , x )

def

= P (vm ....m

1

n

)( cmp send (in ch , x ) | P )

P setv(sv, l, x) = M , n such that P (vm1....mn )(cmpsetv (in sv, l, out sv.M , n) | P)

P recv (ch, x ) = n such that P (vm1 ....mn )(cmprecv (in ch, x, n) | P )

def

P getv(sv, l, x) = n such that P (vm1....mn )(cmpgetv (in sv, l, x, n) | P)

def

BPEL Services collaboration

115

These barbs exited the agent, host and to go through channel or sv. The Name (n) is for synchronization process for launching mechanism, and M is the return process path. In barbs n and M are ignored. So the following equivalence doesn't concern with host and agent components.

P M =P *Q and Q M

def

Conditional relation R with bisimulation

if PRQ then

if P M then P M

if P P then for some Q, Q Q and PRQ

If there exists bisimulation R is satisfying

PRQ, then P Q.

4.3 Barbs Equivalence BPEL primary lemmas

Barbed Bisimulation satisfies the following

In this section, our work is focused on internal and external actions (host and agent), which behave the

Location

location activities.

P0 host1[agent1[ P | rec Mem (l = v )]] host2 [agent2 [ P | rec Mem (l = v )]]

The bisimulation of two processes are added to inner processes. Different activities can be held to obtain similar actions recognizable from outside, conditionally the particular entries are correctly set and the mechanism take the related actions after the receiving each existing agent or host.

Composition

Same way, two inner equivalent composition processes indicates barbs equivalence of parallel composition. It is fact that without channels or locator services remote events cannot finish their events outside of hosts or directly process never accesses in its environments.

host1[agent1[ P | rec Mem (lagent = agent1, lhost = host1 , l = v )]] host2 [agent2 [ P2 | rec Mem (lagent = agent2, lhost = host2 , l = v )]] 1 host1[agent1[Q1 | rec Mem (lagent = agent1, lhost = host1 , l = v )]] host2 [agent2 [Q2 | rec Mem (lagent = agent2, lhost = host2 , l = v )]] host1[agent1[ P | Q1 | rec Mem (lagent = agent1 , lhost = host1 , l = v )]] host2 [agent2 [ P2 | Q2 | rec Mem (lagent = agent2, lhost = host2 , l = v )]] 1

where P, Q, P , Q SA and

ract {send (ch, x), recv (ch, x), setv ( sv, l , x), getv ( sv, l , x)

5. BPEL Technology Approach We now present a formal description of participants (Net, Customer, Client and Server) and how they can be composed in e-Business scenario.

Net( ) := (ac)(ap)(c1)(p1)(c2)(p2) (c3)(p3) (Customer(ac, ap) |Client(ac,ap,c1,p1,c2,p2, c3,p3) | Serveri ((ci,pi)

The process Net( ) composes the various participants to the scenario; first of all, it creates some global channels, used by the processes to interact together: the channels ac and ap are the Client interfaces exposed to the Customers. So, they are passed as arguments both to the Customer (ac,ap) and to the Client (ac,ap,c1,p1,c2,p2,c3,p3) processes. The first one is used to require a price 5.2 Customer

list, while the second one to emit a purchase orders. The other global channels are the set of pair ci and pi, which are respectively the query and the purchase interface of the ith Server. Those names are passed as arguments to the Client (ac,ap,c1,p1,c2,p2,c3,p3) and Serveri(ci, pi) processes. We also assume the Net as a closed system, in the sense that (fn( ) Net( )) = Ø.

% % % % % % %% % Customer(a c , a p ) := (q 1 )(q 2 )(q 3 )(a r )(a s )(a f )(a c áq 1 ,q 2 ,q 3 ,a r ñ | ar (l%%,l%).a p áq1 ,q2 ,q3 ,as ,a f | as ().S() | a f ().F()) 1 ,l2 3

The Customer process first browses a price list. When it receives an answer, it emits a purchase request, and waits for the outcome. To do %% % this it creates these names: q1 ,q2 and q3 which contain the two item preferences, the channel ar, which is the restricted reply channel used by the Client to inform the Customer about the price list consultation, and the two channels as (success) and

A. G. Memon, and M. S. Chandio

consultation interface. This message carries the items description and the reply channel. This first phase ends with the receipt of the reply message a (l%%, l%), , which carries three names, ,l %% and l% encoding the features of the requested , l ,l items. When the Customer process receives one of this message, the purchase transaction has completed and it goes on with the appropriate task

r 1 2 3

1 2 3

116

af (failure), which signal respectively the outcome of the purchase transaction. Then the Customer process sends the message ac q1 ,q2 ,q3 ,ar to the Client %% % 5.3 Client

identified by S( ) or F( ). Moreover, it is guaranteed that either all the items have been bought, or the appropriate compensations have been emitted.

%% % %% % Client (ac , a p , c1 , p1 , c2 , p2 , c3 , p3 ) := ac (q1 , q2 , q3, ar ). ( BPEL(a p ,c1 , p1 ,c2 , p2 ,c3 , p3 ,q1 ,q2 ,q3 ,ar )

| Client (ac ,a p ,c1 , p1 ,c2 , p2 ,c3 , p3 ))

% % % % % % BPEL (a p ,c1 , p1 ,c2 , p2 ,c3 , p3 ,q1 ,q2 ,q3 ,ar ) := Query ( c1 ,c2 ,c3 ,q1 ,q2 ,q3 ,ar )

| PURCHASE (a p , p1 , p2 , p3 )

%%% % % % Query (c1 ,c2 ,c3 ,q1 ,q2 ,q3 ,ar ) := (r1 )(r2 )(c1 q1 ,r1 | c2 q2 ,r2 | c3 q3 ,r3 | (r1 (q1 ,l% r2 (q2 ,l% r3 (q3 ,l% ar l%% l% % 1 ). % 2 ). % 3 ). 1 , l2 , 3 1 1

% 1 1 % 2 2 % 3 3 %% % Purchase( a p , p1 , p2 , p3 ) := a p ( q1 , q2 , q3 , as , a f ).( rs1 )( rf1 )( rs2 )( rf2 )( rs3 )( rf3 ) | p1 q1 , rs , rf | p2 q2 , rs , rf | p3 q3 , rs , rf

| WAIT ( rs1 , rf1 , rs2 , rf2 , rs3 , rf3 , as , a f ))

The Client process exposes a service that can be used by a Customer to query some distributed price lists, and subsequently to purchase the items. When it receives a request ac ( q1 , q2 , q3, ar ), it executes a managing process -- ( BPEL %% %

%% % %% % ( a p , c1 , p1 , c2 , p2 , c3 , p3 , q1 , q2 , q3 , ar )). the ( BPEL(a p , c1 , p1 , c2 , p2 , c3 , p3 , q1 , q2 , q3 , ar )) process

executes two sub-

processes Query(c1 , c2 , c3 , q1 , q2 , q3 , ar ) and Purchase ( a p , p1 , p2 , p3 ). %% %

these subtasks, % % % Q u e r y ( c 1 , c 2 , c 3 , q 1 , q 2 , q 3 , a r ), receives the consulting channels c1 and c2, the Customer preferences %% % q1 , q2 and q3 and the reply channel ar. It emits in parallel the various price list consultations with the messages c1 q1 , r1 , c2 q2 , r2 and c3 q3 , r3 that % % % contain the Customer preferences and the private channels r1 and r2 on which it will wait for a reply. Those replies contain the outcomes of the queries executed on the Server's databases -- encoded with names l%%and l% When the Client receives ,l . them, it forwards them to the Customer application with the message a l%%, l%, and it waits ,l for a purchase order on the channel a ( q , q , q , a , a ). % % % The process Purchase(ap, p1, p2) is called with the channel ap, on which it will wait for the Customer's order, and the Servers' channels p1 and p2. At this point, it creates a pair of success/failure reply channels rs and rf for each Server, and emits the purchase requests % % % p q , r , r , p q , r , r and p q , r , r . When the requests have been emitted, the process Purchase (ap, p1, p2, p3) executes the process WAIT (r , r , r , r , r , r , a , a ) , which will manage the purchase transactions' outcomes. Due to space limitation, the following specified activates process are omitted such as CPi, PPi, DBPi.

1 2 3 1

The

first

of

channels, the Server creates three sub-processes, CPi(ci, dbc), PPi(pi, dbp) and DBPi(dbc, dbp). The first two processes manage the consultation and the purchase orders emitted by the Customer, while the third one represents a database process. 6. Conclusion The BPEL is a programming language suited for modeling Internet applications. BPEL captures their inherent parallelism, the repartition of computational elements amongst multiple locations, the mobility of resources and locations. We discussed a reference implementation and using BPEL based communication innovated in XML. The pattern-based design is the aim that a similar model can be implemented in any language with any collaboration services framework. Our propose in our calculus work to intend concrete contribution from the point of view of the orchestration language specifications in terms of unambiguous semantics and of the feasible implementations of BPEL orchestration engines. We introduced BPEL, a simple extension of the -calculus with long running transactions. Some considerations about the choice of a -calculus based language are needed. One peculiarity of such language is mobility: it is possible to transmit channel names that then can be used by any process receiving them. This aspect is essential in the formalization of BPEL and in particular plays an important role in the formalization of interaction with request­response services. Indeed, the invoker must send to the service a channel name to be used to return the response, thus we have to exploit name mobility This proposed model representation in a calculus in which test and formal proofs could be performed, or into a executable representations in BPEL languages. We deem that given formalization will help us to explore the practical aspects of our framework. This work contributes a

r

1

2 1

3 1

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f

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1

1 s

1 f

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2

2 s

2 f

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3 s

3 f

1 s

1 f

2 s

2 f

3 s

3 f

s

f

5.4 Server Serveri(ci, pi) := (dbc)(dbp)(CPi(ci, dbc) | PPi(pi, dbp) |DBPi(dbc, dbp)) The generic ith Server receives two names as arguments, ci and pi. When the Server process begins its execution, it creates a pair of channels, which are used to interact with a database process. The channel dbc is used to invoke a price list consultation service exposed by the database; the channel dbp is used to emit a purchase order to the same database. After the creation of these

BPEL Services collaboration

117

simple, concise until now dominant and communicative formal language, with a concrete semantics that allows formal interpretation. The language shows a clear relation with the calculus, and the actual encoding of it with the calculus is a feasible task, while it would be quite harder to get such an encoding for BPEL and other collaboration services composition languages. References Bocchi, L., C. Laneve, and G. Zavattaro, (2003) "A Calculus for Long-running Transactions". In FMOODS'03. ©Springer-Verlag. Bruni, R., C. Laneve, and U. Montanari, (2003) "Orchestrating Transactions in Join Calculus". In CONCUR'02. © Springer-Verlag. Bugliesi, M., G. Castagna, and S. Crafa, January (2004) "Access Control for Mobile Agents: The Calculus of Boxed Ambients", ACM Transactions on Programming Languages and Systems 26(1): 57-124. © ACM Press. Butler, M., and C. Ferreira., (2004) "An Operational Semantics for StAC, a Language for Modelling Long-running Business Transactions". In COORDINATION' 04: © Springer-Verlag. Bruni, R. H. Melgratti, and U. Montanari. (2005) "Theoretical Foundations for Compensations in Flow Composition Languages". To appear in POPL-. Cardelli, L., and A.D. Gordon, (1998) "Mobile ambients", in: M. Nivat (Ed.), Lecture Notes in Computer Science, Vol. 1378, 140­155. © Springer, Berlin, Germany. Cardelli, L., G. Ghelli, and A.D. Gordon, (2000) "Secrecy and group creation". In CONCUR'2000: © Springer-Verlag. Chessel, M., D. Vines, C. Griffin, V. Green, and K.Warr, January (2001) "Business Process Beans: System Design and Architecture

Document". Technical report. IBM UK Laboratories. Ishikawa, F., Y. Tahara, N. Yoshioka, and S. Honiden, July, (2004) "Behavior Descriptions of Mobile Agents for Web Services Integration", 2004 IEEE International Conference on Web Services (ICWS 2004) 342-349. Kavantzas, N., G. Olsson, J. Mischkinsky, and M. Chapman, "Web Services Choreography Description Languages". [otn.oracle.com/tech /webservices/ htdocs/ spec/cdl v1.0.pdf. Milner, R., October (1991) "The polyadic -calculus: a tutorial. Laboratory for Foundations of Computer Science , Computer Science Department, University of Edinburgh, The King's Buildings, Edinburgh, UK. Memon A.G., J. Yin, J.D. Maree, and Mujeeb-uRehman," (2005) Deploying -calculus Technology in Inter-organizational Process ",WAIM2005© Springer-Verlag. Parrow. J., (2001) "An Introduction to the -calculus" Handbook of Process Algebra,ed. Bergstra, Ponse and Smolka, pages 479­543,. © Copyright Elsevier. Parrow, J., and B. Victor, "The Fusion Calculus: Expressiveness and Symmetry in Mobile Processes". In LICS'98:. © IEEE Computer Society Press. Sangiorgi, D., (1997) "The Name Discipline of Receptiveness". In Autornata, Languages and Programming: ICALP 97. © Springer-Verlag, Vigliotti, M., and I. Phillips, (2002) "Barbs and congruences for safe mobile ambients". In F-WAN: Foundations of Wide Area Network Computing. Yin, J., A.G. Memon, J. D. Maree, and Mujeeb-uRehman," (2005) RBT-calculus Protocol for Web service Long Running Transactions", (ISCIT05).© IEEE.

Sindh Univ. Res. Jour. (Sci. Ser.) Vol: 40 (2) 119-126 (2008)

SURJ

Sindh Univ. Res. Jour. CITRIC ACID FERMENTATION OF HYDROLYSED RAW STARCH BY ASPERGILLUS NIGER IIB-A6 IN STATIONARY CULTURE S. Anwar, S. Ali and A. A. Sardar*

Biotechnology Research Centre, Govt. College University, Lahore, Punjab, Pakistan *Corresponding author: Email: [email protected] (Received 13th January 2008 and Revised 2nd March 2008)

Abstract

In the present investigation, better citric acid production (23.87±2.75g/l) was obtained with sweet potato starch hydrolysate at an initial sugar concentration of 200 g/l by Aspergillus niger IIB-A6. The product formation kinetic parameters viz., Qp (0.1 g/l/h) and qp (0.02 g/g/h) were higher for sweet potato when compared to maize starch hydrolysate. Incubation period (264h), initial pH (3.0), volume of fermentation medium (50 ml/250 ml Erlenmeyer flask) and inoculum size (2.0 %) were also optimized. The optimal citric acid production was obtained when methanol (1.5 %, v/v) as a stimulant was added into the medium 24 h after the inoculation. Different metal complexing agents such as ethylene diamine tetra acetic acid (EDTA) and potassium ferrocyanide were used to reduce heavy metal ions during the fermentation process. The maximum amount of citric acid (45.90±4.20 g/l) was achieved when 200 ppm of K4Fe (CN)6 was added into the medium just before inoculation under hot conditions. EDTA however, showed insignificant results.

Keywords: Citric acid, Aspergillus niger, IIB-A6 stationary culture.

1.

Introduction Citric acid is used as acidulate, flavour enhancer, preservative, antioxidant and stabilizer. There are three principle methods which are available for microbial production of citric acid i.e., surface culture, submerged and solid-state fermentation (Kristiansen et al., 1999). The surface culture technique is a conventional method but it is still being extensively employed. Although it is labour intensive but the energy requirements are less compared to the submerged or solid-state fermentation. One of the prerequisites for abundant citric acid production is Aspergillus niger (Maddox and Brooks, 1998) and has dominated others both in laboratory and industrial scale. Citric acid fermentation by A. niger was successfully carried out with crude carbon sources such as starch hydrolysates or incompletely refined sucrose. It has been observed that starch hydrolysate gives better yield of citric acid (Wayman and Mattey, 2000). The initial sugar concentration has been found important to determine the amount of citric acid and other organic acids produced in the culture broth. Normally strains of A. niger need a fairly

higher initial sugar concentration in the medium (Grewal and Kalra, 1995). The optimal incubation period for the maximum citric acid production varies both with the organism and fermentation conditions. Attempts have been made to decrease fermentation time period by altering the cultural conditions (Rojas et al., 1995). An appropriate initial pH is critical for successful fermentation process and it varies from strain to strain (Hess et al., 2000). The stimulatory effect of methanol permits its application in the commercial production of citric acid. The alcohol addition reduces mycelial growth, inhibits sporulation and increases the efficiency of the fermentation. A. niger needs a variety of divalent trace elements such as Fe+2, Cu+2, Zn+2, Mn+2 and Mg+2, etc. for growth and citric acid production (Majolli and Aguirre, 1999). Potassium ferrocyanide reacts with the heavy metals causing their precipitation. It removes not only metals of vegetative influence but also some of the microelements. Therefore, the concentration of these heavy metals should be regulated for the optimal fungal growth (Walish et al., 1983). The present study deals with the citric acid

S. Anwar, et al.,

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fermentation of hydrolysed raw starch by Aspergillus niger IIB-A6 in stationary culture. 2. Materials and methods Organism and culture maintenance Aspergillus niger strain IIB-A6 was obtained from the available stock culture of Biotechnology Research Centre, GCU Lahore. The culture was maintained on potato dextrose agar (PDA) slants, pH 5.6. The slants were inoculated by transferring small amount of conidia by an inoculum needle and incubated at 30°C in an incubator (3-5 days) for maximum sporulation. The slant cultures were stored at 4°C in a cold-cabinet.

Preparation of conidial inoculum The conidial suspension was prepared by adding 10 ml of sterilized 0.005 % (w/v) sodium salt of bis-2-ethylhexyl sulfosuccinate to a 4-6 day old slant culture of A. niger having profuse conidial growth on its surface. A sterile wire-loop was gently used to break the conidial clumps and shaken vigorously to make a homogeneous suspension. Preparation of starch hydrolysate Sweet potatoes obtained from local market were cut into small pieces after washing and peeling. The pieces were blended in distilled water to form a homogenous mixture and placed at 4C. The starch settled down at the bottom was separated from liquid and oven dried at 60C, overnight. Maize starch was obtained from the local market. A starch solution of 250g/l was prepared and autoclaved. To liquefy starch, alpha amylase (2.0 U/ml) was added and heated at 95C in a water bath for 15 min. For saccharification, amyloglucosidase (2.0 U/ml) was added and heated at 55C while constant stirring for 4h. The reducing sugars were determined by dinitrosalicylic acid (DNS) method (Miller 1959). Fermentation technique

Surface culture technique was employed for the production of citric acid by A. niger. Fifty millilitre of the fermentation medium containing (g/l): starch hydrolysate 150, NH4NO3 2.5, KH2PO4 1.0, MgSO4. 7H2O 0.25 and CaCl2 0.5 was transferred to 250 ml conical flasks and cotton plugged. The flasks were autoclaved at 15 psi (121°C) for 15min. Sterilized ferrocyanide (free conc. 200 ppm) was added to each flask aseptically while the medium was hot. After cooling at room temperature, the flasks were inoculated with 1.0 ml of the conidial suspension (1.35×106 conidia). The addition of methanol (1.0 ml of 1.5 %) was made 24h after the inoculation and incubated at 30°C for 264h. All the experiments were run parallel in triplicates. Assay methods The mycelial morphology was determined on an aliquot extended on the petriplates and dry cell mass was determined according to Haq and Daud (1995). The sugar contents were estimated after Miller (1959). Citric acid was estimated following pyridineacetic anhydride method (Marrier and Boulet, 1958). The ferrocyanide concentration was estimated by colorimetric method (Marrier and Clark, 1962). The kinetic parameters for citric acid production were studied according to the procedures of Pirt (1975). Statistical analysis Treatment effects were compared by the protected least significant difference method (Spss-10, Version-4.0, USA) after Snedecor and Cochran (1980). Significant difference among the replicates has been presented as Duncan's multiple ranges in the form of probability (p) value. 3. Results

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The maximum citric acid production (15.81±2.60 g/l) was obtained with sweet potato starch hydrolysate containing initial sugar concentration of 200 g/l which is 1.5 fold higher than maize starch hydrolysate (Fig.1) in (Fig.2) is depicted the comparison of sweet potato and maize starch hydrolysates during time course study (24-360 h) on citric acid production by A. niger IIB-A6. The maximum citric acid production (23.87±2.45g/l) from sweet

potato starch hydrolysate was achieved 264h after incubation which is 1.41 fold higher than maize starch hydrolysate. The sugar consumption and dry cell mass were 121.3±3.10

and 26.02±1.63g/l, respectively. Table-1 shows the kinetic parameters during time course study by using different sugar concentrations of sweet potato and maize starch hydrolysates. The maximum growth in terms of specific growth rate (µ) was only marginally different in both hydrolystes containing different sugar concentrations. When substrate consumption parameters were monitered, the volumetric substrate uptake rate Qs (0.46g/l/h), volumetric cell formation rate Qx(0.1g cells/l/h) and specific substrate uptake rate qs(0.46g/g cells/l/h) showed enhancement in sweet potato starch hydrolysate containing 200g/l sugar concentration over to other levels.

Fig. 1. Comparison of sweet potato and maize starch hydrolysates on citric acid production containing different sugar concentrations by A. niger IIB-A6*

Sugar c ons umed, dry c ell mas s , c itric ac id (g/l) 160 140 120 100 80 60 40 20 0 100

Sugar c ons um ed, dry c ell m as s , c itric ac id (g/l)

125 150 175 200 225 250 275

160 140 120 100 80 60 40 20 0 100

125

150

175

200

225

250

275

Sugar conc. of sw eet potato starch hydrolysate (g/l)

Sugar conc. of maize starch hydrolysate (g/l) Sygar consumed (g/l) Dry cell mass (g/l) Citric acid (g/l)

Sugar consumed (g/l)

Dry cell mass (g/l)

Citric acid (g/l)

*Fermentation period 240 h, incubation temperature 30°C, initial pH 3.5. Y-error bars indicate standard deviation (±SD) of means among the three parallel replicates. The values differ significantly at p0.05.

Fig. 2: Comparison of sweet potato and maize starch hydrolysates during time course study on citric acid production by A. niger IIB-A6 *

180 160 140 120 100 80 60 40 20 0 0 24 48 72 96 120 144 168 192 216 240 264 288 312 336 360 384 Rate (h) of sw eet potato starch hydrolysate Sugar consumed (g/l) Dry cell mass (g/l) Citric acid (g/l)

180 Sugar consumed, dry cell mass, citric acid (g/l) 160 140 120 100 80 60 40 20 0 0 24 48 72 96 120 144 168 192 216 240 264 288 312 336 360 384 Rate (h) of maize starch hydrolysate Sugar consumed (g/l) Dry cell mass (g/l) Citric acid (g/l)

*Sugar concentration 200 g/l, initial pH 3.5, incubation temperature 30°C. Y-error bars indicate standard deviation (±SD) of means among the three parallel replicates. The values differ significantly at p0.05.

Sugar consumed, dry cell mass, citric acid (g/l)

S. Anwar, et al.,

Table - 1. Kinetic parameters of citric acid production by A. niger IIB-A6 at different sugar concentrations of sweet potato and maize starch hydrolysates* Kinetic parameters Specific growth rate µ (h-1) Substrate consumption parameters Qs (g/l/h) qs (g/g cells/l/h) Qx (g cells/l/h) Citric acid formation parameters Qp (g/l/h) qp (g/g cells/h)

122

Sugar concentration (g/l) Sweet potato starch Maize starch 175 200 225 175 200 225 0.08 0.10 0.11 0.06 0.08 0.1 0.44 0.41 0.08 0.045 0.008 0.46 0.46 0.1 0.1 0.02 0.54 0.48 0.11 0.046 0.008 0.35 0.32 0.06 0.032 0.006 0.42 0.42 0.08 0.064 0.012 0.45 0.50 0.1 0.026 0.005

*Qs =g substrate consumed/l/h, qs =g substrate consumed/g cells/h, Qx = g cells formed/l/h, Qp =g citric acid produced/l/h, qp =g citric acid produced/g cells/h. The value differs significantly at p 0.05.

Sugar consumed, dry cell mass, citric acid (g/l)

The maximum citric acid production (20.60±1.70 g/l) was obtained at pH 3.0. The sugar consumption and dry cell mass were 107.96±1.56 and 15.27±0.89 g/l, respectively (Fig. 3). Fig. 4 exhibits the effect of different volumes (25-100 ml/250 ml flasks) of fermentation medium on citric acid production by A. niger IIB-A6 in stationary culture. The citric acid production (13.43±2.53g/l) was lower at 25ml volume. The maximum citric acid production (28.66±1.76g/l) was obtained when 50 ml of the medium was used. The effect of different alcohols (methanol, ethanol and butanol; 1.0 %, v/v) on citric acid production by A. niger IIB-A6 was studied (Fig.5a). The maximum citric acid production (36.80±2.54 g/l) was obtained with methanol. The effect of different concentrations of methanol (0-2.0 %) and its addition at different time intervals was also studied (Fig. 5b c). The maximum amount of citric acid (40.23±2.05 g/l) was produced when 1.5% methanol was added into the medium 24h after inoculation.

Fig. 3. Effect of different initial pH on citric acid production by A. niger IIB-A6*

160 140 Sugar consum dry cell m ed, ass, citric acid (g/l)

120

100

80

60

40

20

0 1.5 2 2.5 3 pH

Sugar consumed (g/l) Dry cell mass (g/l) Citric acid (g/l)

3.5

4

4.5

5

*Sugar concentration 200 g/l, fermentation period 264 h, incubation temperature 30°C. Y-error bars indicate standard deviation (±SD) of means among the three parallel replicates. The values differ significantly at p0.05.

Fig. 4. Effect of different initial volume of fermentation medium on citric acid production by A. niger IIB-A6*

140 120

100

80

60

40

20

0 25 50 Volume (ml)

Sugar consumed (g/l) Dry cell mass (g/l) Citric acid (g/l)

75

100

*Sugar concentration 200 g/l, fermentation period 264 h, initial pH 3.0,incubation temperature 30°C. Y-error bars indicate standard deviation (±SD) of means among the three parallel replicates. The values differ significantly at p0.05.

Citric acid fermentation of hydrolysed...

Fig 5a. Effect of different alcohols on citric acid production by A. niger IIB-A6*

140 Sugar consum dry cell m ed, ass, citric acid (g/l) 120

123

100

80

60

40

20

0 Control Methanol Ethanol Butanol Alcohols (1.0 %, v/v)

Sugar consumed (g/l) Dry cell mass (g/l) Citric acid (g/l)

*Sugar concentration 200 g/l, fermentation period 264 h, initial pH 3.0, incubation temperature 30°C. Y-error bars indicate standard deviation (±SD) of means among the three parallel replicates. The values differ significantly at p 0.05.

Fig 5b. Effect of different methanol concentrations on citric acid production by A. niger IIB-A6*

140 Suga co m d cell m r nsu ed, ry ass, citric a (g cid /l) 120

The effect of different concentrations of potassium ferrocyanide (50-300 ppm) and its addition at different time intervals (0-96 h) was studied by A. niger IIB-A6. A slight difference was observed in the production of citric acid by increasing the ferrocyanide concentration (Fig. 6a). The maximum citric acid (45.90±1.272 g/l) was produced when 200 ppm K4 Fe (CN)6 was added into the medium before inoculation, under hot conditions. The sugar consumption and dry cell mass were 130.7±2.44 and 27.22±2.17g/l, respectively (Fig. 6b). The effect of addition of different concentrations of EDTA (0-200 ppm) and its addition at different time intervals (0-96 h) was also examined (Fig. 7). The maximum citric acid (41.50±1.10 g/l) was produced when 50 ppm of EDTA was added into the medium prior to the time of inoculation, under hot conditions.

Fig 6a. Effect of different concentrations of ferrocyanide on citric acid production by A. niger IIB-A6*

160 140 Sugar consumed, dry cell mass, citric acid (g/l)

100

80

60

40

120

20

100

0 0 0.5 1 1.5 2 2.5 Methanol concentration (%)

Sugar consumed (g/l) Dry cell mass (g/l) Citric acid (g/l)

80

60

*Sugar concentration 200 g/l, fermentation period 264 h, initial pH 3.0, incubation temperature 30°C. Y-error bars indicate standard deviation (±SD) of means among the three parallel replicates. The values differ significantly at p0.05.

40

20

0 0 50 100 150 200 250 300 350 Ferrocyanide conc. (ppm)

Sugar consumed (g/l) Dry cell mass (g/l) Citric acid (g/l)

Fig 5c. Effect of varying the time of methanol addition on citric acid production by A. nige IIB-A6*

140 Sugar consum dry cell m ed, ass, citric acid (g/l) 120

*Sugar concentration 200 g/l, fermentation period 264 h, initial pH 3.0, incubation temperature 30°C, methanol concentration 1.5 %. Y-error bars indicate standard deviation (±SD) of means among the three parallel replicates. The values differ significantly at p0.05.

100

80

Fig 6b. Effect of varying the time of ferrocyanide Addition on citric acid production by A. niger IIB-A6*

140

60

120

40

Sugar consum dry cell m ed, ass, citric acid (g/l)

100

20

80

0 0 12 24 36 48 60 Time of addition (h)

Sugar consumed (g/l) Dry cell mass (g/l) Citric acid (g/l)

60

40

*Sugar concentration 200 g/l, fermentation period 264 h, initial pH 3.0, incubation temperature 30°C, methanol concentration 1.5 %. Y-error bars indicate standard deviation (±SD) of means among the three parallel replicates. The values differ significantly at p 0.05.

20

0 0 24 48 72 96 120 Time of addition (h)

Sugar consumed (g/l) Dry cell mass (g/l) Citric acid (g/l)

S. Anwar, et al.,

*Sugar concentration 200 g/l, fermentation period 264 h, initial pH 3.0, incubation temperature 30°C, methanol concentration 1.5 %, ferrocyanide conc. 200 ppm. Y-error bars indicate standard deviation (±SD) of means among the three parallel replicates. The values differ significantly at p 0.05.

124

Fig 7. Effect of different concentrations and arying the time of addition of EDTA on the production of citric acid by A. niger IIB

140 Sugar consumed, dry cell mass, citric acid (g/l) 120 100 80 60 40 20 0 0 50 100 150 200 250 EDTA concentration (ppm) Sugar consumed (g/l) Dry cell mass (g/l) Citric acid (g/l)

140 Sugar consumed, dry cell mass, citric acid (g/l) 120 100 80 60 40 20 0 0 24 48 72 96 120 Time of EDTA addition (h) Sugar consumed (g/l) Dry cell mass (g/l) Citric acid (g/l)

effects of different cultural conditions and nutritional requirements such as sugar concentration, rate synthesis, initial pH, volume of fermentation medium, type and size of alcohols and ferrocyanide concentration on citric acid production were studied. Sweet potato and maize starch containing different sugar concentrations (100-250 g/l) were examined for citric acid production. A lower concentration of sugar leads to lower yield of citric acid as well as accumulation of oxalic acid (Kovats, 1960). In the present study, the maximum amount of citric acid (15.81±1.60 g/l) was produced with sweet potato starch hydrolysate at a concentration of 200g/l which was 1.52 fold higher than maize starch hydrolysate. Gradual reduction in citric acid formation was observed when the sugar concentration of medium was further increased. It might be due to the over growth of mycelial cells which resulted in the increased viscosity of medium and hence, mass transfer limitations (Benuzzi and Segovia, 1996). An increase in dry weight of mycelia with the increase in the sugar concentration is in agreement with the work reported earlier by (Haq et al. 2003). The rate of citric acid fermentation (24-360 h) was carried out using sweet potato and maize starch hydrolysates containing 200g/l sugar concentration. Increase in incubation period resulted in the increased citric acid production. The maximum amount of citric acid (23.87±2.45g/l) was obtained 264h after inoculation by using sweet potato starch hydrolysate. In stationary culture, the production starts after a lag phase of approximately 2-3 days and reaches maximum at the onset of stationery phase (Vergano et al., 1996). The incubation period beyond 264 h did not show any enhancement in citric acid production. It might be due to the decreased available nitrogen in fermentation medium, age of fungi; inhibitors produced by fungi itself and the depletion of sugar contents. This finding is in agreement with the observations of Rajoka et al., (1998). On the basis of a comparison of kinetic parameters namely the volumetric product formation rate (Qp) and specific product

*Sugar concentration 200 g/l, fermentation period 264 h, initial pH 3.0, incubation temperature 30°C, methanol concentration 1.5 %. Y-error bars indicate standard deviation (±SD) of means among the three parallel replicates. The values differ significantly at p 0.05.

4.

Discussion Citric acid is an important metabolite produced by fermentation with specific moulds, especially with Aspergillus niger (Hang and Woodams, 1998). Various carbohydrate materials may be used in citric acid production but only a few workers have used starch hydrolysate (Mourya and Jauhri, 2000) and got comparatively better results. The

Citric acid fermentation of hydrolysed...

125

formation rate (qp), sweet potato starch hydrolyste was found to be the best substrate for citric acid production. The findings suggested that sweet potato starch hydrolysate possesses enhanced ability for citric acid production compared to maize starch hydrolysate. The pH of the basal medium has a direct influence on mould metabolism (Roskosu and Anenil, 1980). The maximum citric acid production (20.60±1.70g/l) was obtained in the fermentation medium when pH was adjusted at 3.0. It might be due to proper cell division, appropriate nutrients supply and adjustment of fungus according to physiological conditions. Similar kind of work has also been reported by Pirt (1975). The study is directly substantiated by the findings of Rajoka et al., (1998). Maximum values for Yp/s, were several folds improved over those of the previous workers (Pirt 1975; Benuzzi and Segovia, 1996; Kristiansen et al., 1999). The effect of different volumes (25-250 ml/250 ml Erlenmeyer flasks) of fermentation medium on citric acid production was also studied. The maximum citric acid production (28.66±2.20 g/l) was obtained when 50 ml of the medium was used (depth 1.5cm). At this volume of the medium, growth of cell mass was optimal due to a better oxygen supply, resulting in the increased citric acid production. In the present study, the maximum amount of citric acid (40.23±2.05g/l) was produced when methanol at 1.5 % (v/v) level was added into the medium 24 h after inoculation. It might be due to that methanol increased the permeability of cell membrane which resulted in a better citric acid excretion from mycelial cells (Ali et al., 2002). In addition, methanol markedly depressed cell proteins in the early stages of cultivation and also increased the enzyme metabolic activity (Pazouki et al., 2000). Addition of ethanol or butanol, however, did not enhance citric acid production. A little enhancement in citric acid production over control was observed with EDTA. The maximum citric acid production (45.920±1.27 g/l) was achieved when 200 ppm (w/v) potassium ferrocyanide was added before inoculation, under hot conditions. It might be due to that the insoluble complexes of ferrocyanide with heavy metals acted as metal buffers in the medium, which made metal ions available at a concentration suitable for the optimal citric acid yield. In addition, the ferrocyanide ions check mycelial growth and inhibit aconitase activity (Shankaranand and Lonsane, 1993). Conclusion In a comparison between sweet potato and maize starch hydrolysates by A. niger IIB-A6 using stationary culture, the maximum amount of citric acid (23.87±2.27g/l) was obtained with sweet potato starch hydrolysate 264 after inoculation. The initial sugar concentration (200 g/l), pH (3.0), effect of alcohols (1.5 % methanol) and the effect of ferrocyanide (200 ppm K4Fe(CN)6) were also optimized. After the optimization of cultural conditions and nutritional requirements, an overall improvement of 3.45 fold in citric acid production was achieved, which is highly significant (p0.05). However, further studies on the effect of various levels of bis-2-ethylhexyl sulfosuccinate on citric acid production is prerequisite before scale up in a lab scale stationary shelf-bioreactor. 6. References Benuzzi, D.A., and R.F. Segovia, (1996) Effect of the copper concentration on citric acid productivity by an Aspergillus niger strain. Appl. Biochem. and Biotechnology., 61 (3): 393-397. Grewal, H.S., and K.L. Kalra, (1995) Fungal production of citric acid. Biotechnol. Adv., 13(2): 209234. Hang, Y.D. and E.E. Woodams, (1998) Production of citric acid from corncobs by Aspergillus niger. Biores. Technol., 65 (3): 251-253. Haq, I., S. Ali and J. Iqbal, (2003) Direct production of citric acid from corn starch niger. Process Biochem., (38): 921-924. by Aspergillus J. 5.

Haq, P.B., and D.A. Daud, (1995) Process of mycelial dry weight calculation for citric acid. Biotechnol., (9): 31-35.

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Hess, S.J., G.J. Ruijter, C. Dijkema and Jour. Visser, (2000) Measurement of intracellular pH by 31P NMR in Aspergillus niger. Jour. Biotechnol., (77): 5-15. Kovats, J. (1960) Studies on submerged citric acid fermentation. Acta. Microbiol., 9: 275-285. Kristiansen, B., M. Mattey and J. Linden, (1999) Citric Acid Biotechnology. Taylor and Francis Inc. Philadelphia, 55-62. Maddox, I.S., and J.D. Brooks, (1998) Application of a multiplayer packed bed reactor to citric acid production in solid state fermentation using Aspergillus niger. Process Biochem., 33 (2): 117-123. Majolli, M.V.I., and S.N. Aguirre, (1999) Effect of trace metals on the cell morphology, enzymic activity and citric acid production in a strain of Aspergillus wentii. Rev. Argent. Microbiol., 31(2): 65-71. Marrier, J.R., and D.S. Clark, (1962) An improved colorimetric method for determining ferrocyanide concentration and its application to molasses. Analyst, (85): 574pp. Marrier, J.R., and M. Boulet, (1958) Direct determination of citric acid in milk with an improved pyridine, acetic anhydride method. Jour. Dairy Sci. (41): 1683pp. Miller, G.L. (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem., 31(3): 426-428. Mourya, S., and K.S. Jauhri, (2000) Production of citric acid from starch-hydrolysate by Aspergillus niger. Microbiol. Res., 155 (1): 37-44. Pazouki, M., P.A. Felse., J. Sinha and T. Panda, (2000) Comparative studies on citric acid production by Aspergillus niger and Candida lipolytica using molasses and glucose. Bioprocess Engin., 22 (4): 353-361. Rajoka, M.I., M.N. Ahmad, R. Shahid, F. Latif and S. Parvez, (1998) Citric acid production from sugar-cane molasses by cultures of Aspergillus niger. Biologia, 44 (1): 241-253. Rojas, G.M., J. Cordova, R. Auria, S. Revah and T.E. Favela, (1995) Citric acid and polyols production by Aspergillus niger at high glucose concentration on inert support. Biotechnol. Lett., 17(2): 291-292. Roskosu, A.A., and C.A. Anenil, (1980) Effect of various conditions on the production of citric acid during fermentation of molasses by Aspergillus niger. J. Microbiol. Technol., 2 (1): 61-62. Shankaranand, V.S., and B.K. Lonsane, (1993) The use of sugarcane as substrate for the production of citric acid by solid-state fermentation. W. J. Microbiol. and Biotechnol., (9): 377-380. Snedecor, G.W. and W.J. Cochran, (1980) Statistical methods. 7thed., Lowa State University, USA, 32-43. Vergano, M.G., N. Femandez, M.A. Soria and M.S. Kerber, (1996) Influence of inoculum preparation on citric acid production by Aspergillus niger. Jour.. Biotechnol., 12 (6): 655-656. Walish, S., K. Kediziora and G. Prusk, (1983) Effect of trace metals (Nickel, Cobalt, Molybdenum) on process of citric acid biosynthesis using Aspergillus niger on a molasses medium. Przem. Spozyw., 31 (1): 31-33. Wayman, F.M., and M. Mattey, (2000) Simple diffusion is the primary mechanism for glucose uptake during the production phase of the Aspergillus niger in citric acid process. Biotechnol. and Bioengin., 67(4): 451-456.

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