Bacterial Flora of Manasbal , a Freshwater Ecosystem of Valley

Dissertation Submitted in Partial Fulfillment of the Requirements for the Award of Degree of

MASTER OF PHILOSOPHY (M.PHIL.)

IN ENVIRONMENTAL SCIENCE

By Sana Shafi M.Sc. B.Ed.

Under the Joint Supervision of

Dr. Manzoor A. Shah Prof. Azra N. Kamili Sr. Assistant Professor Head/ Director Department of Botany Environmental Science/CORD (Co-supervisor) (Supervisor)

POST GRADUATE DEPARTMENT OF ENVIRONMENTAL SCIENCE Faculty of Physical and Material Sciences UNIVERSITY OF KASHMIR -190006, KASHMIR August, 2012

Phone: Office: 0194-2420078, 2420405, Ext. 2155 email: [email protected]; [email protected]

Department of Environmental Science

University of Kashmir, Srinagar -190006

No. Dated:

Certificate

This is to certify that the M. Phil. Thesis entitled “Bacterial flora of , a fresh water ecosystem of ” is the original research work carried out by Ms Sana Shafi, as whole time M. Phil. research scholar in Department of Environmental Science, University of Kashmir, Srinagar. This work has been carried out under our joint supervision and has not been submitted to this University or to any other University so far and is submitted for the first time to the University of Kashmir. It is further certified that this thesis is fit for submission for the degree of Master of Philosophy in Environmental Science and the candidate has fulfilled all the statutory requirements for the completion of the M. Phil. Programme.

Dr. Manzoor A. Shah Prof. Azra N. Kamili Sr. Assistant Professor Head/Director Department of Botany Department of Environmental University of Kashmir Science/ CORD Co-Supervisor University of Kashmir Supervisor

Head/Director Environmental Science/CORD, University of Kashmir Srinagar-190006.

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Dedicated to

My Parents

CONTENTS

Chapter No DESCRIPTION PAGE NO.

Certificate i

Acknowledgement ii-iii

ABSTRACT iv-vi 1. INTRODUCTION 1-9 1.1. Objectives 7 1.2. Study Area 8-9 2. REVIEW OF LITERATURE 10-39 2.1. Bacterial diversity 11-17 2.2. Ecological factors influencing bacterial 18-25 diversity 2.3. Bacteria as indicator of 26-32 2.4. Role of bacteria in bioremediation of aquatic systems 32-39

3. MATERIAL AND METHODS 40-47 3.1. Cleaning of glassware 40 3.2. Sterilization 41 3.3. Preparation of Media and Diluent 42-43 3.4. Dispensing Of Media 43 3.5. Culture Techniques 43 3.6. Serial Dilution Technique 43 3.7. Inoculation 44 3.8. Incubation 44 3.9. Enumeration of Colonies 44

3.10. Pure Culture Techniques 45 3.11. Identification 45-47 3.12. Motility of bacteria 47 4. RESULTS 48-82 4.1. Distribution of colonies 48-51 4.2. Total coliform 51-52 4.3. Biochemical tests 52-55 4.4. Temperature and pH 55 5. DISCUSSION 83-93 5.1. Distribution of colonies 83-84 5.2. Total coliform 85-86 5.3. Biochemical tests 86-93 5.4. Temperature and pH 93 6. CONCLUSIONS 94-96

7. BIBLIOGRAPHY 97-128

LIST OF TABLES

Table Page Description No. No. Table 1. Colony Morphology of Different Bacterial Isolates 56-57 Percentage of Gram Positive and Gram Negative Table 2. 58-59 Bacterial Strains Table 3. Shape Wise Percentage of Different Bacterial Strains 60-61 Percentage Occurrence of Gram Positive and Gram Table 4. 62 Negative Bacteria Table 5. Monthly Distribution of Bacterial Colonies (cfu/ml) 63 Table 6. Total Viable Count of Bacteria at Site I 64

Table 7. Total Viable Count of Bacteria at Site II 64

Table 8. Total Viable Count of Bacteria at Site III 64 Table 9. Total Viable Count of Bacteria at Site IV. 65 Total Coliform Count of Different Water Samples Table 10. 65 (MPN/100ml) Category Wise Distribution of Coliform Count Table 11. 66 (MPN/100ml) Biochemical Results of Bacterial Species for Table 12. 67 Enterobacteriaceae Family Biochemical Results of Bacterial Species for Gram Table 13. 68 Negative Rods Table 14. Total Viable Count of the Identified Species at Site I 69-70 Table 15. Total Viable Count of the Identified Species at Site II 71-72 Table 16. Total Viable Count of the Identified Species at Site III 73-74 Table 17. Total Viable Count of the Identified Species at Site IV 75-76 Table 18. Comparative Bacterial Analysis at Different Sites 77 Total Colony Count and Percentage Occurrence of Table 19. 78 Bacterial Species Monthly Colony Count of Bacterial Species at Different Table 20. 79 Sites Table 21. Monthly Shannon Index (H) at Different Sites 79

Table 22. Water Temperature (°C) Recorded at Four Sites 81

Table 23. pH of Water Recorded at Four Sites 82

LIST OF FIGURES

Figure No. Description Page No.

Percentage Occurrence of Gram Positive and Fig. 1 62 Gram Negative Bacteria

Monthly Distribution of Bacterial Colonies Fig. 2 63 (cfu/ml)

Total Coliform Count of Different Water Fig. 3 66 Samples (MPN/100ml)

Shannon Index (H) of Different sites in Different Fig. 4 80 Months

Site Wise Shannon index (H) of Bacterial Fig. 5 80 Communities

Fig. 6 Water Temperature Recorded at Four Sites 81

Fig. 7 pH of Water Recorded at Four Sites 82

LIST OF PHOTOGRAPHS

Photograph Description No.

1. Map showing different study sites of Manasbal Lake

Photographs showing different study sites of Manasbal 2. lake

Photographs showing different isolation techniques 3.

Photographs showing different Grams nature and shape 4. of bacteria

Photographs showing Biochemical test kit for 5. Enterobacteriaceae

Photographs showing Biochemical test kit for Gram 6. Negative Rods

List of Abbreviations

APHA : American Public Health Association BCC : Bacterial Community Composition CFU : Colony Forming Unit DOC : Dissolved Organic Carbon EMB Agar : Eosine Methylene Blue Agar Fig : Figure m.a.s.l : Meters above sea level lb/inch2 : Pound per inch square g : Gram Hrs : Hours mg : Milligrams °C : Degree Centigrade ml : Millilitre MPN : Most Probable Number NaCl : Sodium Chloride PCBs : Poly-chloro-biphenyls PCR : Polymerase Chain Reaction spp. : Species UNESCO : United Nations Educational Scientific and Cultural organisation UNICEF : United Nations International Children’s Emergency Fund USEPA : United State Environmental Protection Agency -ve : Negative +ve : Positive WHO : World Health Organisation

ACKNOWLEDGEMENTS All praises to Almighty Allah (most merciful and beneficent) who by His grace helped me to successfully complete M.Phil programme with great zeal and zest. I take this opportunity to express my deep sense of gratitude and indebtedness towards my teacher and guide Prof. Azra N. Kamili, Head, Department of Environmental Science / Director CORD, University of Kashmir, for suggesting me the work of vital interest. This study was conducted at her initiative and under her supervision. I am thankful to her for her guidance, invaluable suggestions, enthusiastic encouragement and necessary research facilities provided during the course of my studies. I must confess that without her constant endeavour in guiding me in completion of this work, it could not have been completed successfully.

I wish to express my deep sense of gratitude and indebtedness to Dr. Manzoor A. Shah, Sr. Assistant Professor Department of Botany, University of Kashmir, for his enthusiastic guidance, kind supervision, continued encouragement, generous assistance, timely cooperation, valuable suggestions and constructive criticism during the course of present study.

I also express my profound gratitude to Prof. A. R. Yousuf (former Dean Academic Affairs), Prof. A. K. Pandit (CORD/Environmental Science) and Prof. G. A. Bhat (CORD/Environmental Science) for their moral support, useful suggestions and continuous encouragement all through the course of study.

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My sincere and deep regards are due to Dr. Ruqaya Nazir, Assistant Professor (CORD) Microbiology Laboratory Incharge for her constant encouragement and kind behaviour.

Thanks are also due to other technical and non technical staff members of CORD, especially Mrs Bilquis Qadri (Scientific Officer), Ms Rubiya Dar (Sr. Technical Assistant), Ms Sumaira Tyub (Sr. Technical Assistant) and Mr Javaid A. Javaid (Jr. Technical Assistant) for their cooperation, timely help and whole hearted support.

The successful completion of the research programme was possible only because of the active cooperation of my friends and colleagues. I would like to put on record my thanks to Scholars of CORD, in general, and Microbiology Laboratory, in particular, namely Mr Suhaib A. Bandh, Mrs Samira Amjid, Ms Humera Nissa, Ms Nowsheen Shameem Mr Javaid A. Parray, Mr Bashir A. Lone and Mr Gowhar H. Dar who rendered full cooperation and support during the research programme without which conduct of field study and data analysis would have been rather difficult. I am also thankful to my friends Ms Rahila Bashir, Ms Tabasum Yaseen, Ms Aalia Ismat, Ms Sana Mehraj and Mrs Sakeena Waseem for their help and fruitful guidance.

I fail in my duty if I don’t make a special mention here of my parents and my sister, Ms Shabnum and other family members who encouraged me all throughout the research work.

Sana Shafi

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Abstract Abstract

icro-organisms have been used for a long time as indicators of and total coliform bacteria have been commonly M used to assess potential contamination of drinking and swimming water with pathogenic bacteria of intestinal origin coming from point source discharges, such as raw , storm water, combined sewer overflows, effluents from wastewater treatment plants, industrial sources and non-point source discharges, such as agriculture, forestry, wildlife, and urban run-off. The obtained data in this study reflect the importance of microbiological monitoring and reinforce the need to implement environment protection programs, especially related to pathogenic species. The majority of bacteria isolated were recognized as human pathogens or potential human pathogens. The data was obtained by the bacteriological analysis of water sample taken from Manasbal Lake on monthly basis from four different microhabitats by plating the different dilutions on a solidified culture medium in petri dishes. After incubation the bacterial colonies were divided into different types

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Abstract according to some macromorphological features like appearance, shape, size, elevation, margin, colour and some micromorphological features with the isolated strains showing marked differences in these features. On the basis of these differences they were coded with numbers ranging from MBS01 to MBS52. The different recognizable colonies were streaked and restreaked on fresh media to obtain pure cultures. The selected purified colonies of various types were identified to genus or species level using biochemical tests. Total coliforms were enumerated using multiple tube fermentation technique with lactose broth as the presumptive medium and Eosine-Methylene-Blue agar medium as the confirmatory medium and Brilliant Green Bile broth for completed test. The developed colonies on plates were enumerated by digital Qubek colony counter and the bacterial load was assessed in terms of colony forming units (cfu/ml) revealing that the total monthly bacterial population increased from March to August and then decreased from September to December with peak bacterial population in the month of August at all sites. Moreover, the density of total culturable bacteria at site II (residential hamlets around) was significantly higher in all the months compared to other sites. The overall bacterial density was maximum at site II with a cfu/ml of 203x102 in the month of August and minimum at site III (central site) with a cfu/ml of 12x102 in the month of April. The total bacterial population was higher during warm temperature months than cold temperature months for all the four sites. As far as coliform count is concerned, all the water samples collected from the Lake were positive with respect to the coliform occurrence, with their proportion ranging between a minimum value of 4 MPN/100ml and a maximum value of 460 MPN/100ml. The highest proportion of these indicator organisms was observed at site II. The category wise distribution of coliform count shows that about 95% samples lie in category II and III deeming the water unfit for drinking purposes, however, fit for bathing and swimming purposes. Moreover, the quality of water in some patches of the lake was very

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Abstract poor and unfit for any use. The water of the lake was characterised by a medium to high alkaline pH (ranging between 7.7 to 9.6) and temperature ranging between 9°C to 33.5°C. The overall Shannon diversity index (H) was highest at site I (Laar Kul) followed by site II, site III and site IV (outlet).

The bacterial isolates were then tested for Gram’s reaction and subsequently examined under microscope for their cell shape revealing that 88.5% of the bacterial strains were Gram negative and 11.5% were Gram positive, out of which 34 strains (59.6%) were Gram negative bacillus, 12 strains (28.8%) were Gram negative cocci, 4 strains (7.6%) were Gram positive bacillus and 2 strains (3.8%) were Gram positive cocci. Among Gram-negative bacteria, bacillus was the most dominant genus isolated from all sites during all months. A total of 19 bacterial strains, chosen arbitrarily were subjected to biochemical tests like Citrate utilization, Glucose, Adonitol, Arabinose, Lactose, Sorbitol, Mannitol, Rhamnose, Sucrose, Urease, Lysine utilization,

Ornithine utilization, H2S production, Phenylalanine Deamination, Nitrate utilization, Indole, Voges Proskauer’s and Methyl red revealed that 9 species viz Proteus spp. I, Proteus spp. II, Proteus spp. III, Escherichia coli, Klebsiella spp. II, Cedecea spp., Escherichia spp., Shigella spp. and Salmonella spp. belonged to Enterobacteriaceae family and 10 species viz Shigella spp. I, Shigella spp. II, Shigella spp. III, Enterobacter spp., Hafinia spp., Salmonella chloraesuis subspecies choleraesuis, Salmonella choleraesuis subspecies diarizonae, Vibrio spp., Proteus spp. IV, and Klebsiella spp. I. to Gram negative rods. During the study Proteus spp. II occurred with a highest percentage occurrence of 14.67% and Shigella spp. I with a lowest percentage occurrence of 0.21%. Overall, the study allows us to conclude that the quality of lake water has deteriorated to the extent of being unfit for drinking purposes, though it is still fit for recreational and other uses. Hence, the lake calls for urgent restoration and effective management for its sustained existence and continued provisioning of various economic goods and ecosystem services.

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Chapter: 1 Introduction Introduction

arth, the only planet in our solar system characterized and shaped by abundant water- a necessity for life that makes up 60 % of the human E body. Human beings rely on it for drinking, cooking, bathing, washing clothes, growing food, recreation, industry, and mining, as well as generation of electric power. However, it is not always accessible where it is needed, and it may not be of appropriate quality for anticipated uses. Although we commonly take for granted that clean and abundant water is as close as the nearest faucet, water resources can be contaminated with pollutants. Thus, the conservation and sustainable use of freshwater resources is of global importance. Water is the most vital resource for all kinds of life on this planet and essential for ensuring the integrity and sustainability of the earth’s ecosystems (UNESCO, 2003). The aquatic environments represent the habitat of diverse microorganisms, both beneficial and antagonistic. Microorganisms are not only the most abundant organisms in natural freshwater systems, but are also key players in ecological processes controlling water quality. Detailed

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Introduction knowledge of the diversity and functioning of microorganisms dwelling in freshwater habitats is an essential pre-requisite for the sustainable management of freshwater resources. Freshwater systems are inhabited by microbial communities that are native to this habitat type and usually do not occur in marine systems, saline inland waters and terrestrial habitats. Despite recent advances in the characterization of diversity of freshwater microorganisms, knowledge essential for a holistic understanding of their ecological roles is still somewhat lacking. Microbial ecology is the study of microorganisms in relation to their biotic and abiotic environment. Recently, microbial ecology has also been indicated to be the link between all branches of microbiology (Zinder and Salyers, 2001). It deals with the study of individual organisms, populations (of individuals), communities (of populations), and ecosystems, with a variety of approaches and tools, including microscopy, culturing, molecular biology, and biochemistry. Micro-organisms are essential to our very existence. However, microbes may also act as pathogens or consumers of plants and thereby drive negative feedbacks that maintain diversity among producers via keystone predation (Bever, 1994). Pathogenic bacteria were considered as etiological agents of infectious diseases to human and marine mammals (Baker-Austin et al., 2006 and Pereira et al., 2007). They are ubiquitous, found in common environments such as water, soil and air as well as exotic locales as diverse as deep-sea hydrothermal vents and soda lime . In these natural environments, micro-organisms have very specific jobs. They are responsible for recycling of nutrients in our soil and purifying our water bodies. In all ecosystems, bacteria are the most numerous organisms and through them flows a large fraction of annual primary production. Microbes are the food source of microbivores, such as protozoa and nematodes, and play a key role in food webs and nutrient cycling (Bouwman et al., 1994; Bloem et al., 1997; Muylaert et al., 2002). A diverse decomposer community can increase the rate of nutrient

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Introduction cycling via the variable ability of different microbes to mineralize different organic compounds; therefore high producer diversity may depend on high decomposer diversity (Naeem et al., 2000). Aquatic microbiology encompasses studies on all micro-organisms together with microscopic plants and animals, though in common practices it refers to the study of bacteria, fungi and viruses and their relation to other organisms in the aquatic environment. It is concerned with the structure, function and classification of these organisms and with ways of controlling and using their activities. The number and kind of bacteria that are found in different types of ecosystems vary and are greatly influenced by the ecosystem processes maintaining plant primary productivity (Griffiths et al., 2003). Bacteria are a diverse group of small, single-celled organisms in the kingdoms Eubacteria and Archaebacteria. Found in virtually all extreme habitats, they have existed on earth longer and are more widely distributed than any other group of organisms. Bacteria control major ecological processes, both in terrestrial and aquatic ecosystems, and directly influence plant, animal and human health. The extent to which the distribution and diversity of bacteria found in aquatic ecosystems is controlled by micro-environmental factors is poorly understood and widely debated. Understanding the factors shaping bacterial communities in aquatic systems can potentially help in assessment of the physical condition of these systems. In spite of many recent strides in microbiology, the extent to which variation in microbial communities is structured by stochastic or deterministic processes, and how these link to variation in local environmental parameters (physico-chemical environment, climate, overlying plant community and disturbance regime) or evolutionary events, is still poorly understood. Phototrophic anoxic bacteria may contribute substantially to total primary production in stratified lakes with anaerobic hypolimnion (Bieble and Pfennig, 1979). The secondary production of planktonic bacteria can be equal to or even larger than that of primary

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Introduction production of phyto- in the aquatic ecosystems that receive allochthonous organic input (Findlay et al., 1991). These bacteria use sulphide and other reduced compounds of sulphur as electron-donors for anoxygenic photosynthesis. Dense accumulations of these bacteria can develop, if light reaches the deep sulphide-containing layers. Although blooms of anoxic photosynthetic bacteria are observed in the pelagial of many lakes and reservoirs, their ecological significance in these systems remains unclear (Overmann, 1997). The bacterial secondary production is on an average about 20% of the primary production (Cole et al., 1988; Panzebock et al., 2000). Any chemical, biological or physical change in water quality that has a harmful effect on living organisms or makes water unsuitable for desired use is water pollution (Miller, 2002). It is one of the most important problems being faced by both developed and developing countries as a major cause of human disease, misery and death. The source of water pollution is often inadequately treated human sewage or agricultural runoff finding its way into streams or lakes; thus increasing microbial populations in drinking water distribution systems. Other factors may also influence microbial levels, in different ways. For instance, (1) wild animals are a reservoir for bacteria or protozoa that can infect humans (2) variations in turbidity or water chemistry can affect bacterial densities and (3) algal blooms may increase bacterial abundance (Geldreich, 1991). Micro-organisms have been used for a long time as indicators of water quality. Total and faecal coliform bacteria are commonly used to assess potential contamination of drinking and swimming water with pathogenic bacteria of intestinal origin. The degree of and ecological water quality have been assessed for decades by counting numbers and relative abundance of different species of algae and cyanobacteria and measurement of their activities in oxygen production- photosynthesis and biological oxygen demand- respiration (Dokulil, 2002). The water quality of natural resources has

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Introduction been deteriorating day by day due to discharge of sewage (household, industrial), laboratory chemicals, and wastes from recreational and constructional activities in the catchments areas (Kulbe et al., 1988). These changes result in nutrient character of water leading to alteration in biological characteristics such as plants, animals, and micro- organisms. The regional difference among lakes alters the community composition of the bacteria (Lindstrom and Leskinen, 2002). Recreational waters are susceptible to a variety of sources of microbial pollution, which can contain these pathogenic microorganisms that cause gastrointestinal, respiratory tract, nasal cavity and skin infections (WHO, 2003). These pathogenic microorganisms may come from point source discharges, such as raw sewage, storm water, combined sewer overflows, effluents from wastewater treatment plants and industrial sources or from non-point source discharges, such as agriculture, forestry, wildlife and urban run-off, hence can impair water quality (Griffin et al., 2001). Discriminating between two different sources of fecal pollution in order to develop remediation strategies has been a persistent challenge for conservation of water. Human health depends on safe water, more than any other thing. Basically human life is related to safe water. Most of the problems in developing countries are mainly due to the lack of safe drinking water (Parson Jefferson, 2006). Safe water supply is one of the main purposes in community development and improvement. Thus, it is evident that health of individuals depends on safe drinking water (Mahvi, 1996). is blessed with number of beautiful Lakes, which are formed in rock basins of various shapes and sizes. These play an important role as natural water reservoirs and storage of large quantity of fresh water that can be used for drinking, industrial, irrigation, aesthetic and other purposes including generation of hydro-electricity. In Kashmir, there are numerous springs, lakes, channels and rivers, whose origin lies somewhere in the heart of mountains. There are several high altitude fresh water lakes which are mostly

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Introduction oligotrophic. Kishensar and Vishensar, Tarsar and Marsar, Ganagabal and Tuliyan are the prominent ones which have undergone a lot of investigative studies. Some other Lakes, with different hydrological settings include Manasbal lake (fed mainly by groundwater), (fed mainly by fresh water streams) and (fed mainly by the river Jhelum) (Raza et al., 1978; Keller, 1985; Sharma and Bakshi, 1996; Kapoor and Das, 1997; Hussain, 1998; Khan et al., 2006). The origin of the lakes in Kashmir Valley is either tectonic (as the area is tectonically active) or fluviatile, as all the lakes lie on the flood plain of river Jhelum. The studies conducted on Kashmir lakes mostly pertain to physico- chemical parameters and amongst these Zutshi and Vass (1978), Yousuf et al. (1988) and Pandit et al. (2007) are relatively well known. But at the same time very little attention has been paid to study microbial flora of valley lakes including Manasbal Lake. Hence it was felt important to study the water quality of the Manasbal Lake viz-a-viz its use for various purposes. While some preliminary investigation of fungal flora of Manasbal Lake in relation to its water quality has been carried out (Parray et al., 2008), the present study on bacterial flora of Manasbal Lake is an attempt to relate the microbial composition with the water quality of the Lake.

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Introduction

1.1. Objectives

The objectives of the present study were to:

1. Enumerate the bacterial flora of Manasbal Lake. 2. Assess the quality of lake waters using bacterial flora as an indicator. 3. Suggest some possible remedial measures.

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Introduction

1.2. Study Area

Manasbal Lake is located about 30km north of Srinagar, Kashmir 34°14´40ʺ - 34º15ʹ20ʺ N and 74°39´00ʺ - 74º41ʹ20ʺ E at an altitude of 1,584m.a.s.l (meters above sea level) with springs as main source of water to the lake. It has predominantly rural surroundings with three villages, Kondabal, Jarokbal and Gratbal. It is the deepest (12m deep) of all the freshwater lakes fed by groundwater in Kashmir Valley (Lawrence, 1895; Raina, 1971). The lake is surrounded by moderately high mountains on its eastern and southern sides. A few limestone quarries exist towards the eastern part. The northern bank of the lake comprises a raised land or Karewas. The oblong outline of Manasbal Lake extends in a Northeast-Southwest direction with a maximum length and breadth of 3.5 and 1.5 km, respectively. The volume of water has been estimated as 12.8×106 m3 (Yousuf, 1992). The lake is also fed seasonally by an irrigational stream; Larkul, on the eastern side, which is operational only during summer season. It drains into the river Jhelum through a 1.6 km Nunnyar Nalla near Sumbal village. The lake serves as an important natural water reservoir for the local population and its water is used for drinking and agricultural purposes. It is considered as the deepest as well as 'supreme gem of all Kashmir valley lakes' with lotus (Nelumbo nucifera) no where more abundant or beautiful than on the margins of this lake during July and August (Dewan, 2004). The lake harbours the surviving population of the medicinal plant Euryale ferox and not only provides source of water but also offers facilities for navigation and transportation, fisheries, harvesting of economically useful plants, sightseeing, tourism and recreation. The lake is a good place for bird watching as it is one of the largest natural stamping grounds of aquatic birds in Kashmir (Zutshi and Vass, 1976).

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Introduction

1.3 Site Description

Site I: It is near Laar Kul- a small irrigational stream which takes off from Sindh Nallah and irrigates the agricultural fields throughout its course, drains into the lake on its eastern side. It lies between geographical coordinates 34°15´09.2˝ N and 74°41´00.2˝ E with an elevation of 1586 m.a.s.l. Although the lake has no major inflows and its water supply is chiefly derived from internal springs and precipitation.

Site II: A residential site lying between the geographical coordinates of 34°14´58.2 ˝ N and 74°40´59.5 ˝ E having an elevation of 1580 m.a.s.l is bordered with residential hamlets around. A lot of human interference like emission of domestic sewage, washing of clothes and other activities usually takes place at this particular site.

Site III: The central site of the lake lies in the open water zone between the geographical coordinates of 34°14´51.9˝ N and 74°39´41.9˝ E having an elevation of 1589 m.a.s.l. The site was deep enough with a maximum depth of 12.5 m, harbouring a lesser density of macrophytic vegetation and very less influence of anthropogenic activities. The water at this site was clear with a greater degree of visibility.

Site IV: The outlet of the lake lying between the geographical coordinates 34°14´49.4˝N and 74°39´21.3˝E with an elevation of 1599 m.a.s.l. The water at this site was turbid enough with a lesser degree of visibility due to the sediment loaded water coming out of the lake through this outlet and entering river Jhelum through Nunyar Nalla near Sumbal village.

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Chapter: 2 Review of Literature Review of Literature

icro-organisms are present in high amounts in all kinds of environments and play key roles in important ecosystem functions M such as decomposition and nutrient cycling. Assessment of water quality is necessary for understanding and documenting the occurrence and distribution of pollution indicator and human pathogenic bacteria. Microbiological indicators can therefore serve as early warnings. Bacterial community composition varies as a function of water quality. It mainly depends on temperature, pH, organic concentration and contamination sources. Dissolved organic wastes are expected to decrease bacterial diversity and simultaneously may allow the specific microbial population to flourish in the system. Natural fresh water resources are highly diverse in microorganisms.

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Review of Literature

2.1. Bacterial diversity

Microorganisms are widely distributed in nature, and their abundance and diversity may be used as an indicator for the potability of water (Okpokwasili and Akujobi, 1996). There is no official definition of a bacterial species (Colwell et al., 1995). Though Hey (2001) listed over 24 definitions of bacterial species. The traditional definition of bacterial species was based on higher plants and animals and does not readily apply to prokaryotes (Godfray and Lawton, 2001) or asexual organisms. The genetic flexibility of bacteria, allowing plasmids, bacteriophages and transposons to transfer DNA, complicates the concept of bacterial species. The widespread use of molecular biological methods has revealed an astounding diversity of microorganisms in a range of ecosystems (Curtis et al., 2002; Torsvik et al., 2002; Steele and Streit, 2005). This in turn has sparked renewed interest in understanding the distributional patterns of microorganisms and the mechanisms underlying them (Buchan et al., 2003; Lozupone and Knight, 2007; Ramette and Tiedje, 2007). In addition, microbial communities are being increasingly used to address essential ecological issues (Prosser et al., 2007), such as the determinants of community assembly (Lindstrom et al., 2005; Allgaier and Grossart, 2006) or the importance of biodiversity for ecosystem functioning (Bell et al., 2005; Dang et al., 2005) the relationship between area and species richness (Reche et al., 2005; Green and Bohannan, 2006). All of these studies were based on an understanding of microbial community structure in natural environments. Sewage discharge into the Antarctic marine environment was expected to contain enteric bacteria harboring antibiotic-resistance, as well as virulence- associated plasmids (Smith et al., 1993). Franzmann and Rohde (1991) isolated a “C-shaped”, gram-negative, non-motile bacterium from anaerobic waters of Ace Lake, Antarctica. Bell and Laybourn-Parry (1999) found that Ace Lake supports 62 distinct plankton communities; an aerobic microbial community in

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Review of Literature the upper oxygenated mixolimnion and an anaerobic microbial community in the lower anoxic monimolimnion. Loka Bharathi et al. (1999) suggested that the presence of anaerobic bacteria expresses the increased viability under reducing conditions was a strategy to counteract stress due to super-saturation of oxygen in the cold lacustrine environment. Diversity in metazoan communities often follow regular patterns across environmental gradients in nature (Rosenzweig, 1995), and there seems to be little reason to expect that the same might not be true in microbial communities. Horner- Devine et al. (2004) provided an evidence to support this contention. They show that molecular variation in bacterial communities often changes in regular ways across different kinds of habitats, within habitats that differ in their structural diversity, and across gradients of primary productivity and disturbance. Large amounts of organic wastes are expected to decrease bacterial diversity (Van Es et al., 1980, Fahy et al., 2005). The numbers and types of bacterial populations in an aquatic community are determined in part by the physical and in part by chemical properties of the water body, with temperature playing a key role (Lokoska et al., 2004). The application of molecular methods for identification and classification of microorganisms in different aquatic systems including many lakes (Glockner et al., 2000; Hiorns et al., 1997; Konopka et al., 1999; Yannarell and Triplett, 2004; Zwart et al., 2002) has significantly increased our knowledge of the taxonomic diversity of Bacteria and Archaea (Pace, 1997).

Most natural communities are highly diverse, and microbial communities appear to be no exception (Woese, 1987; Pace, 1997; Dykhuizen, 1998; Curtis et al., 2002; Kent and Triplett, 2002; Torsvik et al., 2002; Rappe and Giovannoni, 2003). Application of molecular techniques to the study of microbial diversity (Olsen et al., 1986; Pace et al., 1986; Steele and Streit, 2005) has revealed the existence of an incredible variety of genotypes and species in all known habitats-many of these types appear to be ecologically

12

Review of Literature

equivalent in terms of resource requirements (Rappe and Giovannoni, 2003; Rozen and Lenski, 2000). It was suggested by Taylor et al. (1990) that the greatest microbial densities occur in waters close to the water sediment interface. While analysing water samples from fifteen springs of Kashmir valley for coliform count Latief et al. (2003) recorded very high values of coliform from these grossly contaminated springs. Cultivation-independent molecular techniques have enabled recent investigation of freshwater bacterial species distribution across space and time. Such studies have been conducted at several different spatial distributions, horizontal (Yannarell and Triplett, 2004) and vertical (Urbach et al., 2001) within a lake, among lakes within a region (Yannarell and Triplett, 2005), among lakes of the same trophic status (Lindstrom, 2001) and of different trophic status (Lindstrom, 2000; Yannarell et al., 2003), and across continents (Lindstrom et al., 2005). Quantitative and qualitative bacteriological studies carried out by Zmyslowska et al. (2001) on vendace larvae and fry (Coregonus Albula), in tank water in the course of rearing, and in fish feeds reported the highest number of bacteria was observed for the groups of psychrophilic (TVC 20°C - total viable count of psychrophilic bacteria on broth-agar after 72 h incubation in 20°C), mesophilic (TVC 37°C - total viable count of mesophilic bacteria on broth-agar after 24h incubation in 37°C), proteolytic and ammonifying microorganisms. Furthermore, bacteria from the genera Aeromonas and Flavobacterium, and from the family Enterobacteriaceae were present in fish, while tank water contained bacteria belonging to the genera Pseudomonas, Bacillus and Flavobacterium, and fish feed from the genera Aeromonas, Pseudomonas and Bacillus. While analysing the variation with depth in the composition of bacteria in samples from alkaline, hypersaline, and currently meromictic Mono Lake in California Shaheen et al. (2003) obtained DNA samples from the mixolimnion (2 m), the base of the oxycline (17.5 m), the upper chemocline (23 m), and the

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Review of Literature monimolimnion (35 m), to assess them by sequencing randomly using selected cloned fragments of 16S rRNA genes and observed that most of the 212 sequences fell into five major lineages of the domain Bacteria and Proteobacteria (6 and 10%, respectively), Cytophaga-Flexibacter- Bacteroides (19%), high-GC-content gram-positive organisms (Actinobacteria; 25%), and low-GC-content gram-positive organisms (Bacillus and Clostridium; 19%). Twelve percent were identified as chloroplasts. The remaining 9% represented Proteobacteria, Verrucomicrobiales, and candidate divisions. Mixolimnion and oxycline samples had low microbial diversity, with only 9 and 12 distinct phylotypes, respectively, whereas chemocline and monimolimnion samples were more diverse, containing 27 and 25 phylotypes, respectively. The compositions of microbial assemblages from the mixolimnion and oxycline were not significantly different from each other, but they were significantly different from those of chemocline and monimolimnion assemblages and the compositions of chemocline and monimolimnion assemblages were not significantly different from each other. Elevated diversity in anoxic bottom water samples relative to oxic surface water samples suggests a greater opportunity for niche differentiation in bottom versus surface waters of this lake. Vertical and latitudinal differences in bacterial community composition (BCC) in Lake Tanganyika, a permanently stratified lake during the dry season of 2002 was studied by Wever et al. (2005) by means of denaturing gradient gel electrophoresis (DGGE) analysis of PCR-amplified 16S RNA fragments and represented that vertical gradients in the BCC were probably permanently present. Dominant organisms identified were the members of Cyanobacteria, Actinobacteria, Nitrospirae, green non-sulphur bacteria, and Firmicutes divisions and the Gamma and Deltaproteobacteria subdivisions. Vertical changes in BCC have been related to the thermal water column stratification, which influences oxygen and nutrient concentrations. Latitudinal variation has

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Review of Literature been related to upwelling of deep water and increased primary production in south of the lake. The increased number of bands per sample with bacterial production in the epilimnion of the lake depicted a positive diversity- productivity relationship. The artificial resource enrichment often used to find out the relative importance of carbon (C), nitrogen (N) and phosphorus (P) in stimulating bacterial production in oligotrophic (Coveney and Wetzel, 1992; Graneli et al., 2004) mesotrophic and eutrophic lakes (Schweitzer and Simon, 1995; Toolan et al., 1991). Phosphorus, rather than organic carbon, can limit bacterioplankton growth in P-deficient systems (Coveney and Wetzel, 1992). Yet, there is evidence that both quantity (Eiler et al., 2003) and quality (Van Hannen et al., 1999) of available organic substrates can promote significant change in bacterial community composition within a time span of a week to a month. Even in short-term experiments (up to 72 hours) the response of bacterioplankton to growth enhancing conditions seems to be driven by the growth capabilities of different bacterial phylogenetic lineages (Simek et al., 2006). Microbial diversity of culturable heterotrophic bacteria in tropical saline lake was assessed by Trivedi (2007). During the study population were characterised from alkaline lake of north with pH range of 7.8-8.4. The different density trend in bacterial community was justified by hydrodynamic circulation, tropism and position of sites. Total of 123 strains were isolated at different pH values. The majority of identified population were belonging to Gram positive genera Bacillus, Staphylococcus, Micrococcus, Actinomycetes and Yeast. The enzymatic versality of the observed genera suggested their importance in organic matter turnover of the oligotrophic system. Likewise, microbial analysis of agricultural soil was carried out by Bahig et al. (2008) on forty soil samples that were collected from two different sites in Sohag province, Egypt, during hot and cold seasons. Twenty samples were from soil

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Review of Literature irrigated with canal water (site A) and twenty samples were from soil irrigated with wastewater (site B). The study was aimed to compare the incidence of plasmids in bacteria isolated from soil and to investigate the occurrence of metal and antibiotic resistance bacteria, and consequently to select the potential application of these bacteria in bioremediation. The total bacterial count (CFU/gm) in site (B) was higher than that in site (A). Moreover, the CFU values in summer were higher than those values in winter at both sites. A total of 771 bacterial isolates were characterized as Bacillus, Micrococcus, Staphylococcus, Pseudomonas, Escherchia, Shigella, Xanthomonas, Acetobacter, Citrobacter, Enterobacter, Moraxella and Methylococcus. Bacterial isolates from both sites showed multiple heavy metal resistance. A total of 337 bacterial isolates contained plasmids and the incidence of plasmids was approximately 25-50% higher in bacteria isolated from site (B) than that from site (A). These isolates were resistance to different antibiotics. Approximately, 61% of the bacterial isolates were able to assimilate insecticide, carbaryl, as a sole source of carbon and energy. However, the Citrobacter AA101 showed the best growth on carbaryl. Microbiological analysis of water samples originating from a small municipal lake in Szczecin, called Syrenie Stawy carried out by Dominiak and Deptula (2009) demonstrated high content of sanitary bacteria, and hence pointed to a significant contamination of the lake. High content of psychrophilic and mesophilic bacteria were pointed to high amounts of organic substances in water. On the other hand, the high content of NPL, of coli group bacteria titres and of faecal type coli group bacteria provided evidence for drainage of communal sewage to waters of Syrenie Stawy. Effect of pollution on diversity of attached and free-living bacteria in two contrasting stations, namely, Suez Canal and outlet of West Lagoon to Lake Timsah was investigated by Bahgat (2011). Bacillus was the most abundant genus especially in West Lagoon station where higher organic agricultural and

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Review of Literature municipal loads was discharged. Bacterial species richness differed among water depths and was higher in subsurface samples. Gram negative Bacillus was more abundant in Suez Canal. The bacterial diversity of Lonar Soda Lake of was assessed by Deshmukh et al. (2011) and they isolated seventy four bacterial strains, out of which they identified 11 strains using morphological, biochemical and molecular analysis. The bacteria identified were belonging to phylum Firmicutes (8) and Proteobacteria (3). They identified Alcaniorax spp. which is well known for its oil degradation capacity. In addition they found that all the eleven strains are potential producers of industrially important enzymes, pigments and antibiotics. The microbial diversity in sediments of the mesotrophic drinking water reservoir Saidenbach, Germany, featuring a pronounced longitudinal gradient in sediment composition in the reservoir system was carried out by Roske et al. (2012). The microbial communities in two sediment depths were characterized using catalysed reporter deposition fluorescence in situ hybridization (CARD- FISH) and a bar-coded pyro-sequencing approach. Multivariate statistic was used to reveal relationships between sequence diversity and the environmental conditions. The microbial community was tremendously diverse with a Shannon index of diversity (H) ranging from 6.7 to 7.1. 18, 986 sequences that were classified into 37 phyla including candidate divisions. They found that Bacteria were more abundant than Archaea. The dominating phylum in all samples was Proteobacteria, especially Betaproteobacteria and Deltaproteobacteria. Furthermore, sequences of Bacteroidetes, Verrucomicrobia, Acidobacteria, Chlorobi, Nitrospira, Spirochaetes, Gammaproteobacteria, Alphaproteobacteria, Chloroflexi, and Gemmatimonadetes were found. The site ammonium concentration, water content and organic matter content revealed to be strongest environmental predictors explaining the observed significant differences in the community composition between sampling sites.

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2.2. Ecological factors influencing bacterial diversity

Several ecological factors shaping BCC (Bacterial Community Composition) identified so far show that the important factors influencing aquatic microbial community structure include water chemistry (Methe et al.,1999; Zwart et al., 2003; Lindstrom et al., 2005), water temperature (Pearce, 2008), meta- predation (Berdjeb et al., 2011), protistan predation (Simek et al., 2001; Simek et al., 2005; Jezbera et al., 2005), phytoplankton composition (Hofle et al., 1999), organic matter supply (Crump et al., 2003), intensity of ultraviolet radiation (Warnecke et al., 2005), habitat size (Reche et al., 2005) and water retention time (Lindstrom et al., 2005; Lindstrom et al., 2006). Although it is well known that these factors shape the overall BCC, little is known about those factors specifically shaping the dynamics of particular populations of freshwater bacteria. Agarwal et al. (1976) recorded the physico-chemical indicators of faecal pollution of River at bathing Ghats and sewage outfalls. The presence of Salmonella in fresh water indicates the pollution of surface waters being a great risk to public health. Klebsiella pneumonia was isolated from surface waters of China Lake, Cape Lake (Knittel, 1975) and some other water sources which were found to be of faecal origin. Bell and Laybourn-Parry (1999) provided some valuable evidence of the impact of natural eutrophication on Rookery Lake, as well as typical microbial community evolved which dominated the older lakes of the Vestfold Hills. A number of physical factors affect survival, persistence, and re-growth of indicator bacteria in ambient waters. Different studies have confirmed that out of various factors temperature (Howell et al., 1996; Esham and Sizemore, 1998; Noble et al., 2004) has a decisive influence on bacterial community. However, results have shown variable impact of temperature that depends on the type of experiment conducted and the nature of water analyzed.

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Furthermore, much of the research has been conducted concerning the survival of indicator bacteria focused on indicators such as faecal coliform that are not frequently used by environmental agencies to determine the sanitary quality of bathing beaches. Based upon the fact that the Enterococci group is a group of 20 different species (Torrell, 2003) the species present are likely to differ depending upon the source of faecal contamination. Therefore, there is a need to establish how particular environmental factors specifically affect survival, persistence and re-growth of Enterococcus spp. as this is the indicator used for sanitary quality in (marine) bathing waters as mandated by the United States Environmental Protection Agency (USEPA, 1998). Investigation of some selected recreational waters in North Florida USA for microbiological quality in relation to varying pH and temperature by Alvarez (1981). A correlation between various physico-chemical parameters especially temperature, pH and bacterial population was drawn by Bagde et al. (1982). Temperature or organic substrate concentrations have been found responsible for regulating bacterial growth (Pomeroy et al., 2001). Exudates produced by phytoplankton are an important organic substrate for bacteria in many aquatic ecosystems (Baines et al., 1991) although in some lakes allochthonous humic substances may be equally important (Bano, 1997). Under oligotrophic conditions, inorganic nutrients may limit bacterial growth (Chrzanowski et al., 1995). Heterotrophic nanoflagellates are often the dominant grazers on bacteria in aquatic ecosystems (Sanders et al., 1989) however in eutrophic environments; ciliates can be important grazers (Kisand and Zingel, 2000). In lakes, the metazoan Daphnia is an important grazer on bacteria (Jurgens, 1994). Temperature and water salinity are also the factors that influence the variation in number of density of the coliform and heterotrophic bacteria within the marine ecosystem (Rheinheimer, 1984). In eutrophic environments bacterial growth appears to be temperature limited during winter and resource limited during summer (Felip et al., 1996).

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Dissolved oxygen is a very important factor to the aquatic organisms, because it affects their biological processes, respiration of animal and oxidation of the organic matter in water and sediments. In latter process, complex organic substances are converted to simple dissolved inorganic salts which could be utilized by the micro and macrophyte (Okbah and Tayel, 1999). Several studies have demonstrated a relationship between BCC (Bacterial Community Composition) and oxygen concentration (Konopka et al., 1999) or temperature

(Sievert et al., 1999). The differences in thermocline depth affect nutrient concentrations in the epilimnion and result in differences in phytoplankton between the north and south of the lake (Coulter, 1991). Takacs (1999) investigated the factors affecting summer bacterial biomass and production with objectives to characterize spatial and seasonal bacterial biomass losses, and establish the potential role of inorganic nutrients, DOC (Dissolved Organic Carbon) supply, salinity, and temperature in the regulation of summer bacterial production in Fryxell, Hoare and Bonney Lakes. Butler et al. (2000) marked seasonal and inter-annual variations in the population density and productivity of the microbial plankton in an oligotrophic maritime Antarctic lake, studied for a 15-month period. Laybourn-Parry et al. (2001) recommended that primary production and bacterial production in Beaver Lake were not limited by nutrient availability, but by other factors, e.g. in the case of bacterial production by organic carbon concentrations and primary production by low temperature. Furthermore various studies (Atherholt et al., 1998; Curriero et al., 2001; Morrison et al., 2003) showed that rainfall events are the major cause of poor water quality, since raw sewage is discharged by overflows, and the rainfall can cause a resuspension of sediment that can act as a sink for indicator organisms (Crabill et al., 1999). Water with temperatures below 1080C was shown to be significantly associated with the detection of the human-specific Bacteroides marker. Light intensity, oxygen and salinity influence the survival of

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Review of Literature microorganisms in aquatic environments (Mezrioui et al., 1995; Sinton et al., 1999; Muela et al., 2000; Wait and Sobsey, 2001). Hydrogen ion concentration (pH) is the most significant factor that controls all aquatic, chemical and biological processes. Changes in pH values beyond the optimum range may affect microbial physiology (Hassanin, 2006). pH of natural waters also controls the solubility of metal ions, and affects natural aquatic life with desirable pH for fresh water in the range of 6.5 - 9.0 (Chin, 2000). The environmental factors, wind direction and wind speed are also influencing the transport of the bacteria (Canale et al., 1993). A number of studies have sought associations between molecular community fingerprints, such as those obtained by denaturing gradient gel electrophoresis, and environmental factors. In lakes, multivariate analysis showed that several factors such as the biomass of other plankton groups (Jardillier et al., 2004; Lindstrom, 2000; Lindstrom, 2001; Van der Gucht et al., 2001; Muylaert et al., 2002), pH (Stepanauskas et al., 2003; Yannarell and Triplett, 2005), nutrient concentrations (Stepanauskas et al., 2003; Van der Gucht et al., 2001; Yannarell and Triplett, 2004), temperature (Jardillier et al., 2004; Muylaert et al., 2002; Stepanauskas et al., 2003; Van der Gucht et al., 2001; Yannarell and Triplett, 2005; Yannarell and Triplett, 2004), and water flow (Jardillier et al., 2004, Stepanauskas et al., 2003) vary with bacterioplankton fingerprints. Such associations suggested that these environmental factors are important in determining the distribution of taxa. The lake bacterial community composition (BCC) varies temporarily and spatially within habitats (Lindstrom, 1998; Hofle et al., 1999; Dominik et al., 2002; Yannarell and Triplett, 2004), as well as between habitats (Yannarell and Triplett, 2004; Lindstrom, 2000; Lindstrom,

2001; Wu QL et al., 2006). Gray et al. (2004) demonstrated that redox conditions controlled the distribution of Achromatium spp. in lake sediments. Schauer et al. (2005) showed that water chemistry determined the presence of any vicarious groups of soil bacteria appeared in particular lakes same year

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Simek et al., demonstrated that populations of highly competitive R-BT065 bacteria (Beta-proteobacteria) were strongly controlled by protistan predation. As per the Lake Web model used by Hakanson et al. (2003) to quantify changes for key functional organisms and food web structures, there are close relationships between catchments area characteristics and lake characteristics. The other important factors influencing lake characteristics as per their observations are climate change, changes in epilimnetic temperatures and increased variations in lake temperature. Lake Web model was used by them. There is very little understanding and knowledge on the relative role of the different process in aquatic systems. Many short-term temporal studies have documented seasonal BCC variation within a lake or between lakes (Lindstrom, 2001; Crump et al., 2003; Kent et al., 2004). According to Ojha and Mandloi (2004) pH increases in water bodies from morning onwards and decline during evening as temperature decreases. They furthermore noticed that turbidity; suspended matters, clay, silt, colloidal organic particles, plankton and other microbes are an expression of light scattering and absorbing properties of water. However in the same year Radhika et al., reported that various abiotic as well as biotic activities of an aquatic system are regulated by water temperature. Additionally examination of literature on ecological investigations of water bodies showed that long-term monitoring and comprehensive analysis of the 23 physico-chemical parameters is crucial to a holistic approach in solving environmental problems of such systems. In year 2005 Yannarell and Triplett, found the bacterial richness associated with pH and Secchi depth in Wisconsin, USA, lakes. Statistical evaluation of the data in relation to the characteristics of the lakes showed that pH, temperature, and the theoretical hydrological retention time of the lakes were most strongly related to variations in the distribution of bacterial community composition, suggesting that pH and temperature are steering factors in the selection of particular taxa and supporting the notion that

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Review of Literature communities in lakes with short water turnover times are influenced by the input of bacterial cells from the drainage areas (Lindstorm et al., 2005). Bacterial communities in 30 lakes in Wisconsin were investigated by a fingerprinting method, automated ribosomal intergenic spacer analysis; pH was one of the factors that were most strongly related (Yannarell and Triplett, 2005). In experimental studies in which lake bacteria were inoculated into media having a neutral or alkaline pH, it was shown that growth of bacteria originating from acidic lakes was inhibited (Langenheder et al., 2005), suggesting that different taxa could be selected in acidic and alkaline environments. Lake trophic status (Lindstrom, 2000; Yannarell et al., 2003; Yannarell and Triplett, 2004), pH (Lindstrom et al., 2005), and hydrology (Yannarell and Triplett, 2005) are three important determinants of freshwater pelagic Bacterial Community Composition. Lake mixing regime is an additional important control on BCC, both over time and across lakes, as originally suggested by Yannarell et al. (2003). Multiple studies have documented the correlation between phytoplankton and bacterial community dynamics, particularly at weekly or more frequent timescales (Worm et al., 2001; Kent et al., 2004; Newton et al., 2006). Interannual seasonal patterns in benthic BCC studied by Hullar et al.

(2006) over four years in a stream related these patterns to temperature and dissolved organic carbon. In the same year Wu and Hahn, revealed, the recurrent seasonal population dynamics of planktonic freshwater bacteria was studied by and demonstrated that much of the dynamics of the investigated Polynucleobacter population could be predicted from the water temperature. Biotic factors may include the effect of virioplankton (Goddard et al., 2005) or competition and trophic interactions (Kent et al., 2004; Newton et al., 2006). These forces vary in relative importance at different spatial and temporal scales. Some are secondary to others, but all are important in shaping the dynamics of the lake bacterial community. Christner et al. (2006) stated

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Review of Literature that a sustained microbial ecosystem is present in the environment of Lake Vostok, despite high pressure, constant cold, low nutrient input, potentially high oxygen concentration and an absence of sunlight. Rogozin et al. (2009) worked on year-to-year variations of vertical distribution and biomass of anoxic phototrophic bacteria during ice periods 2003–2005 and 2007–2008 in meromictic lakes Shira and Shunet (Southern Siberia, Russian Federation) by studying the bacterial layers in chemocline of both lakes were sampled with a thin-layer hydraulic multi-syringe sampler and reported that in winter, biomass of purple sulphur bacteria varied considerably depending on the amount of light penetrating into the chemocline through the ice and snow cover. However, in relatively weakly stratified, brackish Shira Lake, the depth of chemocline varied between winters, so that light intensity for purple sulphur bacteria inhabiting this zone differed. The increased transparency of mixolimnion in winter, high chemocline position and absence of snow resulted in light intensity and biomass of purple sulphur bacteria exceeding the summer values in the chemocline of the lake. In Shunet Lake, the light intensities in the chemocline and of purple sulphur bacteria were always lower in winter than in summer, but the biomasses of green sulphur bacteria were similar. The bacterial community structure is significantly influenced by cyclonic eddy perturbations, which introduce hydrographic and chemical changes has been reported by Zhang et al. (2011). Relationship between environmental factors and bacterial communities in 41 freshwater lakes located in mountainous regions of eastern Japan were investigated by Fujii et al. (2011) was determined by polymerase chain reaction-denaturing gradient gel electrophoresis of the 16S rRNA gene and was then evaluated on the basis of physicochemical and biological variables of the lakes. Canonical correspondence analysis revealed that BCC of oligotrophic lakes was significantly influenced by dissolved organic carbon (DOC) content, but its effect was not apparent in the analysis covering all lakes including

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Review of Literature mesotrophic and eutrophic ones. The generalized linear model showed the negative association of DOC on the taxon richness of bacterioplankton communities. DOC was positively correlated with the catchment area per lake volume, suggesting that a large fraction of DOC supplied to the lake was derived from terrestrial sources. These results suggest that allochthonous DOC has a significant effect on bacterioplankton communities especially in oligotrophic lakes. The genus Polynucleobacter was detected most frequently. The occurrence of Polynucleobacter species was positively associated with DOC and negatively associated with total phosphorus (TP) levels. In addition, TP had a stronger effect than DOC, suggesting that oligotrophy is the most important factor on the occurrence of this genus. Bacterial community structure and the effects of environmental factors on the microbial community distribution were investigated in the Changjiang Estuary area and its adjacent area in the East China Sea (ECS) by Liu et al. (2012). Profiles of bacterial communities generated by denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes followed by DNA sequence analysis revealed that the dominant groups were affiliated to Gammaproteobacteria, Cytophaga–Flavobacteria–Bacteroides (CFB), Deltaproteobacteria, Cyanobacteria and Firmicutes. Effect of environmental factors on the bacterial community distribution was analyzed by the ordination technique of canonical correspondence analysis (CCA). The environmental factors significantly influencing bacterial community structure were different in the three months; dissolved organic carbon (DOC) and temperature in June and nitrite in August. No environmental variables displayed significant influence on the bacterial community at the 5% level in October. The seasonal environmental heterogeneity in the Changjiang Estuary and the adjacent ECS, such as seasonal hydrodynamic conditions and riverine input of nutrients, might be the reason for the difference in the key environmental factors determining the bacterial community in the three months.

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2.3. Bacteria as indicators of pollution

The higher the level of indicator bacteria, the greater will be the level of faecal contamination and hence the higher will be the risk of water-borne diseases (Pipes, 1981). The most widely used indicators of water pollution are the coliform bacteria, which may be the total coliform; faecal coliforms and the faecal streptococci (Kistemann et al., 2002; Pathak and Gopal, 2001; Harwood et al., 2001). Altogether, contamination of water by enteric pathogens has increased worldwide (Islam et al., 2001; Pathak et al., 1991; Craun, 1986). In 1977 WHO (World Health Organization) established a standard parameter to measure water pollution utilizing coliform, pathogenic bacteria and heterotrophic bacteria. Coliform bacteria are found in human intestines from which they are spread out and contaminate aquatic environment through human excreta (faeces). Furthermore, pathogenic bacteria in the marine or aquatic ecosystem are sourced from the human beings or warm blooded animals. WHO (1991) has estimated that some 30,000 people die every day from water related disease and less developed countries are more vulnerable to it. Due to lack of potable water and proper sanitation, over 15 million children below five years die each year (UNICEF, 1997). The presence of microbial indicators in sewage (faecal coliforms, Clostridium perfringens, and enterococci) and aquatic microbial parameters (viral direct counts, bacterial direct counts, chlorophyll a, and marine vibriophage) in injection well effluent, monitoring wells was examined by Paul et al. (1994) and results revealed that there was a transect from onshore to offshore and surface waters above these wells in two separate locations in Key Largo in August 1993 and March 1994. Further they found that effluent and waters from onshore shallow monitoring wells (1.8- to 3.7-m depth) contained two or all three of the faecal indicators, whereas deeper wells (10.7- to 12.2-m depth) at the same sites contained few or none. The presence of faecal

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Review of Literature indicators was found in two of five near shore wells (1.8 miles from shore), whereas offshore wells (2.1 to 5.7 miles from shore) showed little sign of contamination. Indicators were also found in surface waters in a canal in Key Largo and in offshore surface waters in March but not in August. Collectively, these results suggested that faecal contamination of the shallow onshore aquifer, parts of the near shore aquifer, and certain surface waters has occurred. Micro-organisms are useful indicators for ecological risk assessment and environmental monitoring because they are present in high amounts in all kinds of environments and play key roles in food webs and element cycles, like nitrogen, phosphorus, carbon, and sulphur (Domsch, 1977; Bloem et al., 1997). Thus, micro-organisms are pre-requisite for the existence of higher organisms. The small size and high surface to volume ratio of microbes cause a high affinity for very low concentrations of substances. Microbes are in intimate contact and interaction with the environment hence, are very sensitive and respond quickly to contamination and other types of environmental stress (Brookes, 1995; Giller et al., 1998). The microbial activity reflects the sum of all physical, chemical and biological factors regulating the decomposition and transformation of nutrients (Elliot, 1997; Stenberg, 1999). Microbiological indicators can therefore serve as early warnings in monitoring programmes (Jordan et al., 1995). An enormous amount of the total biomass and a considerable amount of biodiversity is present in microbes (Torsvik et al., 1990; Bloem et al., 1994). Coliform bacteria, “faecal coliforms,” and Escherichia coli have, for almost a century, been used as indicators of the bacteriological safety of drinking water (Eijkman, 1904; Mossel et al., 1973; Cliver, 1987; WHO, 1996; Marshall, 1997; APHA, 1998). Coliforms are comprised by two groups; total coliforms and faecal coliforms. Former are commonly found in faeces and also occur naturally in unpolluted soils and waters and include the faecal coliforms and other species of the genera Citrobacter, Enterobacter, Escherichia and

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Klebsiella. However later one are exclusively of faecal origin to be composed mainly of E. coli. McLellan et al. (2001) revealed that faecal pollution indicator organisms can be used to a number of conditions related to the health of aquatic ecosystems and to the potential for health effects among individuals using aquatic environments. The presence of such indicator organisms may provide indication of waterborne problems and is a direct threat to human and animal health. Coliform bacteria should not be detected in drinking water supplies (WHO, 1991) and as per the specification of drinking water distributed through pipeline distribution system, no samples should contain more than 10 coliforms per 100ml and no samples should contain faecal coliform organisms (HMG/NBSM, 2002). Determining the source of faecal contamination in aquatic environments is essential for estimating the health risks associated with pollution, facilitating measures to remediate polluted waterways, and resolving legal responsibility for remediation. Source tracking methods should enable investigators to uncover the sources of faecal pollution in a particular water body (Malakoff, 2002). Microorganisms and chemicals have been explored and re-evaluated (Field et al., 2003; Scott et al., 2002; Simpson et al., 2002) as potential tools for the identification of human faecal sources. More recently, new approaches using eukaryotic mitochondrial DNA to differentiate faecal sources in faeces-contaminated surface waters have also been explored (Martellini et al., 2005). Escherichia coli are the most frequent nosocomial pathogen and a major contaminant in catheter associated with urinary tract infections (Platt et al., 1982). The adhesion of E. coli is considered to be the first step in the pathogenesis of UTI. It would be prudent to always perform the detection of Salmonella spp. beside the traditional indicators of faecal pollution, total coliforms, faecal coliforms and faecal streptococci reported by Gabutti et al. (2004). Salmonella spp. is a definite pathogen that is of worldwide importance and transmitted mainly through water and food. The primary habitat of the Salmonella spp. is

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Review of Literature the intestinal tract of animals and humans. Over the past three decades, practically all countries in Europe have reported a sharp rise in salmonellosis incidence (including food borne outbreaks). Similar pattern was observed in a number of countries in the Eastern Mediterranean Region and south-east Asia Region (WHO, 1997). A number of studies throughout the world have investigated the incidence and survival of Salmonella in lakes, rivers, coastal water and beach sediments (Polo et al., 1998). With some strains of Salmonella to be pathogenic. Salmonella infection can be severe with diarrhea, septicemia, and bowel bleeding as seen with S.typhi and S.paratyphi infection (Bean et al., 1997). Enteric Salmonella infection is a global problem both in human and animals, and is regarded to be the most important bacterial etiology for enteric infections worldwide (Fierer and Swancutt, 2000). Microbial communities are good indicators of regional anthropogenic and global climate change in aquatic ecosystems (Paerl et al., 2003). Surface and ground water can be infected with a diversity of pathogens, however testing and monitoring for all pathogens is unrealistic, mainly because of analytical costs and technical difficulties in detecting organisms at low concentrations in chemically complex environments such as surface waters. As an alternative, indicator organisms are typically used to detect the presence of faecal contaminants in the water resource. In particular, either total coliforms or faecal coliforms (a subset of total coliforms) are measured as indicators of pathogenic microbes. However, testing for E. coli alone is becoming more prominent as it indicates the presence of only faecal contaminants, while total or faecal coliform tests may give positive results for non-faecal, naturally occurring bacterial species (Hill, 2003). Moreover, several strains of E.coli are implicated in human illness with Entero-toxigenic E.coli (ETEC) the most frequently isolated entero-pathogen in the developing world among children of the age group of 5 years or younger is also the major cause of travellers‟ diarrhea (Qadri et al., 2005). Aeromonas spp. is natural inhabitant of aquatic

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Review of Literature environments (Jennifer et al., 2006) and is ubiquitous in surface fresh and marine waters. Aeromonas has been found to have a role in a number of human illnesses including gastroenteritis, septicemia and life threatening disease (Lehane and Rawlin, 2000). The pollution indicators as well as many groups of human pathogenic bacteria and their seasonal variations in different locations in Mandovi and Zuari Rivers in the central west coast of India was carried out by Nagvenkar and Ramaiah (2009). Both the abundance and types of autochthonous and allochthonous microbial populations in the near shore environments are affected by land drainages, domestic sewage outfalls and other discharges. The overall ranges (and their mean abundance; no.ml-1) of the monitored groups of bacteria were: total coliforms: 0- 29047 (3134ml-1); total streptococci: 3-14597 (798); total vibrios: 13-42275 (2530); Escherichia coli: 0-1333 (123); Vibrio cholerae: 0-3012 (207); Salmonella spp.: 0-1646 (90); Streptococcus faecalis: 0-613 (88) and Aeromonas spp.: 0-2760 (205). In general, abundance of sewage pollution indicator bacteria such as total coliforms and total streptococci was lower than that reported from many other locations worldwide. The water quality of seven important lakes in North India during the periods of summer, monsoon and winter seasons was studied by Sharma et al. (2010). All of these studied lakes are important recreational spots of India. Water samples was analyzed for various bacteriological parameters including total viable count (TVC), total coliform (TC), faecal coliform (FC) and faecal streptococci (FS). Also physico-chemical parameters like pH, conductivity, total dissolved solids (TDS), dissolved oxygen (DO), biological oxygen demand (BOD) and chemical oxygen demand (COD) were assessed. Total viable count exceeded the maximum permissible limits in all the lakes irrespective to different seasons. The high most probable number (MPN) values and presence of faecal coliforms and streptococci in the water samples suggests

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Review of Literature the potential presence of pathogenic microorganisms which might cause water borne diseases. A direct effect of season and human activities on the pollution status was observed in all the lakes. The bacteriological quality of water collected from tropical lake system was conducted by Sadat et al. (2011). For this purpose, they monitored nine water sampling stations from Yamoussoukro lake system between February 2007 and April 2008 for Heterotrophic Plate Count (HPC), total coliforms and faecal coliforms (Escherichia coli). Varying levels of bacteriological contamination were recorded in all the stations. Bacterial indicator numbers (log means CFU/ mL) varied from 5.96 to 6.18 for HPC, 5.25 to 5.69 for total coliforms and 3.49 to 4.07 for E. coli. Results of their study showed that the water quality has deteriorated in Yamoussoukro lake system. The relationship between the traditional indicators of faecal pollution, Total coliforms (TC), Faecal coliforms (FC) and Faecal streptococci (FS), and the presence of few potentially pathogenic enteric bacteria, Vibrio cholerae (VC), Vibrio parahaemolyticus (VP), Shigella spp. (SH) and Salmonella spp. (SL) in coastal sea water was evaluated by Sudhanandh et al. (2011). The distributional statuses of these bacteria were also studied along the Southern coast. Cluster analyses were done to identify similar groups of indicator as well as enteric pathogenic bacteria. They found Kochi as highly polluted with enteric pathogens and indicator bacteria (TC of 4700, VC of 820, FC of 920 and FS of 410 CFU ml-1). In addition percentage incidence of VC (97.42%) was comparatively higher than the traditional indicator bacteria (TC 95.04%, FC of 63.64% and FS of 47.64%). VC found to be rather stable and showed significant relationship with all the traditional indicator bacteria (R² > 0.370), suggests that both quantitatively and qualitatively the abundance of Vibrio cholerae can determine faecal pollution, could be used as a faecal pollution indicator bacterium, especially in the marine environment where traditional indicator bacteria failed to survive.

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Review of Literature

The study was conducted to evaluate the status of tap water of Kashmir valley, and to monitor coliforms through multiple tube method by Rehman et al., 2012. Most Probable Number (MPN) was used to detect faecal pollution and to monitor water quality during outbreaks of water borne hepatitis A and E infection in the valley. Forty one towns of ten districts of three subdivisions were studied. The coliform count of tap water of Kashmir valley varied greatly. District Srinagar shows lowest level of MPN 2/100ml of tap water. However bacterial count of tap water of majority of the towns exceeded the recommended permissible level of WHO. Introduction of sewage into the drinking water system was the main reason for the bacterial contamination. During the study they found that tap water of Kashmir valley except Srinagar is not fit for human consumption and other similar use.

2.4. Role of bacteria in bioremediation of aquatic systems

Bioremediation is defined as the process whereby organic wastes are biologically degraded under controlled conditions to an innocuous state, or to levels below concentration limits established by regulatory authorities (Mueller et al., 1996). In presence of oxygen, for example aerobic bacteria (recognized for their degradative abilities) Pseudomonas, Alcaligenes, Sphingomonas, Rhodococcus, and Mycobacterium have often been reported to degrade pesticides and hydrocarbons, both alkanes and polyaromatic compounds with many of these bacteria using the pollutants as the sole source of carbon and energy. Toxic chlorinated compounds used as herbicides, pesticicdes and explosives are prevalent in the biosphere due to ever-growing industrial activities (Reinke and Knackmuss, 1988.). Many of these chlorinated compounds are biodegradable by a wide range of bacterial strains, some of which have been isolated and characterized for potential bioremediation applications (Reinke, 1984; Reinke and Knackmuss, 1988). The role of

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Review of Literature microbial communities in the overall eutrophication processes in aquatic environments as investigated by various researchers (Lighart and Oglesby, 1969; Mitchell, 1971; Jones 1971; Strzelczyk and Mielczarek, 1971) these communities govern a great variety of biotransformation processes which in turn relate directly to the quality and productivity of the aquatic biotopes. The ability of various groups of micro-organisms in the removal and recovery of heavy metals by biosorption has been well documented (Gadd and Griffiths, 1978; Stratton, 1987; Gadd, 1988; Volesky, 1990; Greene and Darnall, 1990; Wase and Foster, 1997). A large number of microorganisms belonging to various groups, viz. bacteria, fungi, yeasts, cyanobacteria and algae have been found to bind a variety of heavy metals to different extents. Volesky (1990) presented an exhaustive list of microbes and their metal-binding capacities. Among bacteria, Bacillus spp. has been identified as having a high potential for metal sequestration and has been used in commercial biosorbent preparation (Brierley et al., 1986). Besides, there are reports on the biosorption of metal(s) using Pseudomonas spp, Zoogloea ramigera and Streptomyces spp. (Strandberg et al., 1981; Nakajima and Sakaguchi, 1986; Mullen et al., 1989; Norberg and Persson, 1984; Norberg and Rydin, 1984). Cell walls of bacteria and cyanobacteria are principally composed of peptidoglycans which consist of linear chains of the disaccharide N- acetylglucosamine-b 1, 4-Nacetylmuramic acid with peptide chains. Cell walls of gram negative bacteria are somewhat thinner than the gram positive ones and are also not heavily cross-linked. They have an outer membrane which is composed of an outer layer of lipo-polysaccharide (LPS), phospholipids and proteins (Remacle, 1990). Glycoprotein‟s present in gram-positive bacterial cell walls have been reported to have more potential binding sites for Cd2+ than the phospholipids and hence are responsible for the observed difference in capacity. Carboxyl group modification caused a marked reduction in metal uptake by Bacillus subtilis (Beveridge and Murray, 1980). Furthermore, the

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Review of Literature

Cd2+ biosorption capacities of gram-positive and gram-negative bacteria were demonstrated by Gourdon et al. (1990). In B. subtilis, teichoic acid (Beveridge and Murray, 1980) and in B. licheniformis, teichoic acid and teichouronic acid (Hoyle and Beveridge, 1983) were found to be the prime sites for metal binding. In E. coli, peptidoglycan was found to be a potent binder of most of the metal(s) tested and carboxylate groups were the principal components involved in metal binding (Hoyle and Beveridge, 1983). The phosphoryl groups of the LPS and phospholipids have been demonstrated to be the most probable binding sites for metal cations in the outer membrane of E. coli (Strain et al., 1983; Ferris and Beveridge, 1984), with most of the metal deposition occurring at the polar head regions of constituent‟s membranes or along the peptidoglycan (Beveridge and Fyfe, 1985). In Streptomyces longwoodensis, phosphate residues were suggested to be the primary constituents responsible for uranium binding (Crist et al., 1981). Azam et al. (1983) revealed that in aquatic ecosystems, bacteria play a key role in the breakdown of organic matter and the remineralisation of nutrients. They are grazed upon by protozoa and some metazoans and hence form the base of a heterotrophic aquatic food chain. Since the development of the microbial loop concept in the early 1980s, substantial research efforts have been invested in evaluating the factors regulating bacteria in aquatic ecosystems. Bacteria also play an important role in the biogeochemical processes within coastal environments, along with being an important chain of the microbial food web, reintegrating the dissolved organic carbon (DOC) through the microbial loop. Bottom-up (resources) as well as top- down (predation) factors have been shown to regulate bacterial populations. Throughout the biosphere, organic matter is constantly being biologically transformed (Rheinheimer, 1980) and a key component of its cycling in aquatic ecosystems is microorganisms, of which bacteria play the most significant role (Kang and Seki, 1983). Naphthalene, listed as a priority pollutant by the U.S.

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Review of Literature

Environmental Protection Agency (USEPA, 1987.) a constituent of coal tar (Finalayson et al., 1997) is easily degraded by bacteria and is thus often used as a model compound for in situ biodegradation studies of polycyclic aromatic hydrocarbons (Dables, 1986) and as a result, a number of naphthalene- degrading microorganisms have been isolated and studied for mineralization (Yen and Serdar, 1998; Samanta et al., 2002). Metal sorption agent AMT- BIOCLAIMTM (MRA) has employed Bacillus biomass to manufacture granulated material for wastewater treatment and metal recovery which can accumulate metal cations with efficient removal of more than 99% from dilute solutions (Garnham et al., 1992). The quality of drinking water has been deteriorated due to discharge of wastewater into water resources as well as due to increasing environmental pollutants. The major global health problems, cross adaptation of microbial population to structurally related chemicals, may play an important role in the practical development and application of bioremediation techniques (Liu and Jones, 1995). Life on our planet is sustained in a fragile biological balance; microorganisms play an important role on nutritional chains that are an important part of this biological balance (Madigan et al., 1998). Adapting several abilities, microorganisms have become an important influence on the ecological systems, making them necessary for superior organism‟s life in this planet. Ability of microorganisms to transform and degrade many types of pollutants in soil, water, sediments and air has been widely recognized during the last decades (Saval, 1998; Autry and Ellis, 1992). Enhanced remediation of crude oil polluted soil was achieved, in situ, by accelerating the biodegradation process through seeding with adapted Azotobacter which not only acted as supplier of fixed nitrogen to the indigenous crude oil-degrading bacteria, but also performed some co-metabolic activities. This work describes the capability of Azotobacter in providing activities that are useful in the bioremediation of crude oil-polluted soil and biological nitrogen fixation when in association with

35

Review of Literature indigenous oil-degrading bacteria (Ikechukwu and Onwurah, 1999). Contaminants are often potential energy sources for microorganisms (Madigan et al., 1998) the reason why microorganisms survive in contaminated habitat because they are metabolically capable of utilizing its resources and can occupy a suitable niche. Bioremediation achieves contaminant decomposition or immobilization by exploiting the existing metabolic potential in microorganisms with catabolic functions derived through selection, or by the introduction of genes encoding such functions. The effectiveness of bioremediation is often a function of the extent to which a microbial population can be enriched and maintained in environment. When few or no indigenous degradative microorganisms exist in a contaminated area and practically does not allow time for the natural enrichment of suitable population, inoculation may be a convenient option (Kalyuzhnyi, 2000). The increasing release of organic pollutants by industries causes many health-related problems. However, increased awareness of the harmful effects of environmental pollution has led to a dramatic increase in research on various strategies that may be employed to clean up the environment and is now realized that microbial metabolism provides a safer, more efficient, and less expensive alternative to physicochemical methods for pollution abatement (Hebes and Schwall, 1987). Through evolution, microorganisms have developed effective mechanisms that help them to regulate their cellular function in response to changes in its environment (Hoch, 2000). Microbial films are important mediators of energy flux and nutrient transformation in most terrestrial and aquatic systems. In groundwater systems, where photosynthesis is absent and chemoautotrophic production can be minimal, microbial films can rely heavily on organic matter imported from the surface as an energy source (Baker et al., 2000; Findlay et al., 2003). The productivity, metabolism, and biomass of microbial films in freshwater systems are influenced from the “bottom-up” by resources and from the “top-down” by

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Review of Literature animals that consume microbes (Pace and Cole, 1996). The availability of inorganic elements (Baker et al., 2000) and organic matter (Findlay et al., 2003) can limit the activity of microbial films from the bottom-up in a diverse set of aquatic systems.

Bacteria strengthen ecosystem function in freshwater lakes by mediating most biogeochemical cycling (Cotner and Biddanda, 2002). However, surprisingly little is known of what drives bacterial community composition (BCC) and diversity in lakes. According to the report by Rooney and Kalff (2003) phosphorus, phytoplankton and heterotrophic bacteria interact in the epilimnion of lakes to determine the biogeochemical pathways and the flow of energy at the base of pelagic food webs, and macrophytes thrive well in lakes having low phytoplankton concentrations even at high phosphorus concentrations. Aquatic organisms, often considered „engineers‟ of aquatic ecosystems, not only react to physical and chemical changes in their environment, but also they can drive such changes and have important roles in cleansing and detoxifying their environment (Ostroumov, 2005). Azotobacter and Pseudomonas the members of the Pseudomonadaceae family have a significant genomic diversity and genetic adaptability in a wide range of niches. With numerous studies showing that they share many biochemical metabolic pathways such as nitrogen fixation, alginate production, and respiratory mechanisms, and they are found in similar environments (Yan et al., 2008). It was long thought that Pseudomonas spp. (Sensu stricto) do not have nitrogen fixation abilities; however, recently, it has been demonstrated that some Pseudomonas strains can fix nitrogen and that their genes related to this machinery closely resemble that of Azotobacter vinelandii (Rediers et al., 2004; Young and Park, 2007; Yan et al., 2008).

Chlorinated herbicides (e.g. s-triazines) and polychlorobiphenyls (PCBs) are persistent organic pollutants (POPs) that are widely distributed in the

37

Review of Literature environment. s-Triazine herbicides are used in agriculture and forestry in diverse regions of the world. PCBs were produced worldwide for industrial applications, and an important amount of these compounds have been released into the environment. PCBs and s-triazines are toxic compounds that could act as endocrine disrupters and cause cancer. Therefore, environmental pollution with s-triazines and PCBs is of increasing concern. Microorganisms play a main role in the removal of POPs from the environment. Bacterial degradation of s-triazines and PCBs and novel strategies to improve bioremediation of these POPs are reported by Seeger et al. (2010) and they characterised diverse bacteria that are able to degrade s-triazines and PCBs. Bacterial degradation of s-triazine herbicides involves hydrolytic reactions catalyzed by amidohydrolases encoded by the atz genes. Anaerobic and aerobic bacteria are capable of biotransforming PCBs. Higher chlorinated PCBs are subjected to reductive dehalogenation by anaerobic microorganisms. Lower chlorinated biphenyls are oxidized by aerobic bacteria.

Bioremediation of ammonia and nitrite in polluted water was carried out by Barik et al. (2011). Since high ammonia nitrogen is a key limiting factor in polluted waters or intensive aquaculture system. Removal of unionized ammonia (NH3) and nitrite (NO2) through biological activity is an important tool for changing such ecosystem. They used nitrifying bacterial inoculants that are the biologically active materials which are used in intensive aquaculture for bioremediation. In all, 12 treatments were used by them with two replications (Completed Randomized Design) factorial design in order to assess the effects on various physicochemical conditions of water. Decrease of ammonia nitrogen concentration from 10 mg l-1 to below the minimum limit (0.3 mg l-1) was obtained within 3 days after inoculation of microbial inoculums with aeration in water. Rate of nitrification was very slow in tanks without aeration. Soil at the bottom was not found to affect the nitrification process. Aeration and

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Review of Literature microbial application played an important role in increasing the nitrification. After acclimation phase nitrification rate was found to be increased. Therefore, application of bioremediators (nitrifiers) decreased ammonia and nitrate nitrogen.

Pyrene is a tetracyclic aromatic hydrocarbon with a symmetrical structure which is one of the top 129 pollutants as ranked by the U.S Environmental Protection Agency (USEPA). Degradation of pyrene by the bacteria isolated from soil of the landfills in Shiraz and the evaluation of their growth kinetic was studied by Kafilzadeh et al. (2012).The bacterial strains with better growth on the enriched mediums were isolated and then identified by standard bacteriological tests. The isolated bacteria including Mycobacterium spp., Corynebacterium spp., Nocardia spp., Pseudomonas spp., Rhodococcus spp. and Micrococcus spp. were potentially capable of degrading pyrene hydrocarbon. They showed high growth rate during screening by increasing the optical density (OD600). The pyrene biodegradation value evaluated by high performance liquid chromatography (HPLC) was 89.1%, 79.4%, 75.3%, 68.2%, 62.3% and 56.8% for each strain respectively 10 days after incubation. The highest pyrene degradation rate was found in Mycobacterium spp. and Corynebacterium sp. with 89.1% and 79.4% values; therefore these bacteria were found to be used to clean the soils which are polluted with pyrene.

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Chapter: 3 Materials & Methods Material and Methods

ater samples from four sites under consideration were collected on monthly basis from March 2011 to December 2012 in Poly W Ethylene (PET) bottles, which were previously carefully cleaned, rinsed three to four times with distilled water (APHA, 1998). All the samples were collected just below the surface of lake water by plunging the open end of each sterile bottle before turning it upright to fill. During collection of samples, extreme care was exercised to avoid contamination of the parts of bottle and collected samples were processed for microbial analysis using the methodology of APHA (1998). The steps involved in the protocol included are:

3.1. Cleaning of glassware All the glassware used was cleaned with labolene to remove oils and organic matter from it. Glassware was then rinsed under running tap water and then with distilled water. Subsequently, it was allowed to drain and dry in an oven at 110°C and then prepared for sterilization.

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Material and Methods

3.2. Sterilization The technique was used to sterilize equipments from all the living organisms including bacteria, fungi, spores, viruses etc. This method was used for glassware, culture media, suspending fluids, reagent containers and equipments. Heat sterilization was followed because it is the most common and reliable method for sterilization where the material to be sterilized is not modified by high temperature. High temperature was achieved by using either dry or moist heat sterilization.

3.2.1. Dry Heat Sterilization Dry heat was used for the sterilization of the glassware, metal instruments and other items. A hot oven equipped with thermostat was used for dry heat sterilization. The time required for sterilization was about 12 to 16 hours at 120°C. However, even after this many bacteria in a desiccated vegetative state or as spores can survive; therefore moist heat was followed for further sterilization. The factors responsible for the death of bacteria in dry heat sterilization are desiccation and coagulation. The bacterial protoplasm contains approximately 85% moisture and when this moisture is reduced various bacteria die.

3.2.2. Moist Heat Sterilization This method was used for sterilization of glassware and culture media. Moist heat was usually provided by saturated pressure in an autoclave and was found to be the most effective and reliable method for sterilization (APHA, 1998). However, it was not used for materials damaged by moisture or culture media containing compounds hydrolyzed or reactive with other ingredients at higher temperature. The temperature and length of time for sterilization with steam are different from that of dry heat. Since the vegetative cells of most bacteria and fungi are killed at 60°C within 5 to 10 minutes. Yeasts and fungi are killed only

41

Material and Methods above 80°C, while for bacterial spores about 15 minutes at 121°C and at 151 lb/inch2 pressure are suggested. This temperature and pressure is also suitable for sterilization of media, hence this method was used. Other methods of sterilization which were followed are: 3.2.3. Red Heat The articles were held on the flame till they became red hot e.g., inoculating wires, loops, tips of forceps, spatulas etc.

3.2.4. Flaming Burning articles viz, spatulas, needles and inoculating loops were sterilized by exposing them to spirit lamp flame. Prior to flaming, it was first dipped into 70% ethyl alcohol.

3.2.5. Infra Red Radiations U.V radiations were used for killing microbes in inoculation chamber (Laminar Air flow cabinet).

3.3. Preparation of Media and Diluent

Different media were used for culturing of bacteria. Nutrient agar was used for enumeration and cultivation of bacteria and Lactose broth for coliform. Ingredients of the Nutrient Agar were first dissolved in distilled water, except agar, pH was then adjusted at 7.2. Then agar powder was added followed by boiling in order to dissolve agar powder and finally sterilized in an autoclave at 121°C, 15lb/inch2 for 15-20 minutes. Normal saline (0.85%) was used as diluent. It was prepared by dissolving 0.85mg of NaCl in 100 ml of distilled water.

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Material and Methods

Ingredients of the Nutrient agar

Ingredients Quantity

Agar: 15g

Peptone: 5g

Sodium chloride: 5g

Beef extract: 3g

Distilled water: 1000ml

3.4. Dispensing Of Media The sterilized medium for bacteria was dispensed into sterilized petri plates. About 15 to 20 ml of medium was poured in each Petri plate on a laminar flow cabinet, and allowed to solidify. The petri plates were incubated overnight to check the contamination, if any, inside the media. 3.5. Culture Techniques Plate count method, which measures the number of viable cells was used for the measurement of microbial community. 3.6. Serial Dilution Technique Before inoculation, the sample was diluted to different levels, in order to get the approximate number and density of the bacteria easily. The original sample was diluted up to four folds viz, 10-1, 10-2, 10-3 and 10-4 using normal saline solution  In the first dilution 1ml of original sample was diluted by 9ml of normal saline solution (NSS).  In the second dilution, 1ml from the first dilution was further diluted with 9ml of NSS and the same procedure was continued until it reaches fourth dilution.

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Material and Methods

3.7. Inoculation Bacterial count in different samples was estimated by inoculating nutrient agar plates with 0.1ml of suitable dilutions by plate count technique which allows the microbial colonies to grow over the surface of the medium and eventually making counting becomes easier. Coliforms were detected by inoculation of samples into tubes of Lactose broth. The three-tube procedure using lactose broth (Collins and Lyne, 1976; Bakare et al., 2003) was used to detect the coliform and determine the most probable number (MPN) of coliform bacilli (Collins and Lyne, 1976). The tubes showing gas production were grown on Endo or eosine-methylene-blue agar; and one or more typical colonies were picked off into Brilliant Green Bile broth (Coyne and Howell, 1994) and studied microscopically to see whether the organisms have the morphological and staining properties of coliform bacilli. 3.8. Incubation After inoculation, the culture plates were incubated at a temperature of 30°C for 48 hrs in incubator in an inverted position to assess the growth of colonies. While as coliform analysis was done by incubating water samples of three different dilutions (viz. 10ml, 1ml, 0.1ml) into lactose broth medium (APHA, 1998) for 48 hours at 37°C. 3.9. Enumeration of Colonies Colonies that developed on agar plates were counted with digital Qubec- counter. Only plates with colonies between 30-300 were selected for counting and the counts were expressed as cfu/ml of water sample calculated by using the following formula number of colonies × d CFU/ml = volume inoculated Where, d = dilution factor CFU= colony forming units Coliform count was done by calculating MPN index using probability table of McCardy (1915).

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Material and Methods

3.10. Pure Culture Techniques Pure cultures were obtained by platinum loop through streaking technique (APHA, 1998). Microorganisms were transferred from one medium to another by sub-culturing for maintaining stock cultures. 3.11. Identification For the identification of bacteria method followed were Gram’s staining, microscopic examination and Biochemical Kits. 3.11.1. Gram’s Staining Gram’s staining technique was used to differentiate the bacteria at group levels i.e., Gram +ve and Gram –ve. The Gram’s staining method includes the following steps:

1. Preparation of bacterial smears: o Clean grease free, microscopic slide was taken. One side of the slide was gently heated in the flame of the spirit lamp to remove any grease present on it. o The slide was kept on the table, with flamed side outwards. The wire loop was heated to destroy any organisms adhered to the surface. Loop full of distilled water, placed at the centre of the slide. The wire-loop was flamed again. o The cotton plug was removed from the culture-tubes and neck of the tube was flamed. With the help of wire-loop, a minute amount of growth from the culture tube was removed. o Emulsifying the growth on the wire-loop into the drop of water placed on the slide. The suspension, then spreaded over an area of about half an inch. Again, flaming the wire-loop before settling it down on the table. o Allowing the film on the slide to dry in air or by holding high above the flame. The slide should not be hot on the back of our hand. o The last step was to ensure fixing material to be observed i.e. to make it stick to the glass slide so that the cells are not washed off during the

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Material and Methods

staining procedure. 2. Staining of smears for one minute with crystal violet solution.

3. Washing of smears with tap water.

4. Flooding of smears with Gram’s iodine solution for about one minute.

5. Washing and blot drying of smears.

6. De-colorization of smears with 95% alcohol for 30 seconds

7. Drying and counter staining of smears with safranin for 10 to 30 seconds.

8. And finally washing of smears, drying and examining under oil emulsion of microscope.

3.11.2. Microscopic Examination Gram stained bacterial smears were then microscopically examined for cell shape and Grams nature.

3.11.3. Biochemical Test Kits Biochemical test kit is a standardized colorimetric identification system utilizing seven conventional biochemical tests and five carbohydrate utilization tests. The tests are based on the principle of pH change and substrate utilization. On incubation organisms undergo metabolic changes which are indicated by a colour change in the media that can be either interrupted visually or after addition of the reagent. Two types of kits used were:

3.11.3a. Biochemical test kit for Gram Negative Rods Kits used for screening pathogenic organisms from urine, enteric specimens and other relevant clinical samples.

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Material and Methods

3.11.3b. Biochemical test kit for Enterobacteriaceae Kits used for screening of organisms belonging to Enterobacteriaceae are gram negative, oxidase negative and nitrate positive rods. 3.11.3c. Preparation of Inoculum The kits used for the identification purpose were not be used directly. The organisms were first isolated and purified, and then only pure cultures were used. The organism identified were first cultured on Nutrient Agar and then a single well isolated colony was taken for preparing homogeneous suspension in 2-3ml of normal saline. This inoculum was incubated at 35-37°C for 4-6 hours. 3.11.3d. Inoculation of kit The kit was opened aseptically by peeling off the sealing tape. Each well of the kit was then inoculated with the 50µl of the prepared inoculum by surface inoculation method. 3.11.3e. Incubation The kit was incubated at temperature of 35-37°C for 18-24 hours. 3.12. Motility of bacteria Bacterial motility is generally ascertained by microscopic examination of the culture by the use of motility agar media consisting of Peptone 3g; Yeast extract 2g; Glucose 20g; NaCl 5g; Distilled water 1000ml; pH 7.5±0.2; Agar 3g. Using a sterile inoculation needle a small amount of 18-24 hrs old broth culture was taken and stabled the motility agar medium prepared in a test tube, in a straight line. After incubation for (24 hrs) at 30°C the tubes were examined to verify whether culture has grown only along the line of inoculation (non- motile) or spread throughout the medium (motile forms) to detect its motility.

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Chapter: 4 Results Results

he results obtained in the present study are sequentially presented starting with distribution of colonies, followed by total coliform T count and biochemical tests for identification of bacterial strains. Subsequently, data on physico-chemical characteristics, such as temperature and pH is provided.

4.1. Distribution of colonies From the study it was observed that bacterial contamination was concentrated at all sites. The bacteria were isolated by plating dilutions of water in saline solution (0.9% NaCl) on nutrient agar and incubated at 37°C for 48 h. The developed colonies were enumerated in plates and the bacterial load assessed in terms of colony forming units (cfu/ml) revealed that total monthly bacterial population increased from March to August and then decreased from

48

Results

September to December as shown in Table 5; Fig. 2. The peak bacterial population was observed in the month of August. For site I the monthly distribution of colonies was maximum with a cfu/ml of 147x102 in the month of August, followed by June with 128x102 cfu/ml, July with 120x102 cfu/ml, April and September with 41x102 cfu/ml each, March with 39x102 cfu/ml, May and October with 38x102 cfu/ml each, November with 35x102 cfu/ml and in December with 33x102 cfu/ml. Following the same trend the monthly distribution of bacterial colonies for site II was maximum in the month of August with a cfu/ml of 203x102 followed by July with 191x102 cfu/ml, June with 189x102 cfu/ml, May with 158x102 cfu/ml, April with 146x102 cfu/ml, March with 133x102 cfu/ml, September with 71x102 cfu/ml, October and November with 42x102 cfu/ml and minimum was in December with 38x102 cfu/ml. For site III the distribution of colonies was maximum with 154x102 cfu/ml in August, followed by June with 94x102 cfu/ml, July with 76x102 cfu/ml, September with 57x102 cfu/ml, October with 46x102 cfu/ml, March with 31x102 cfu/ml, May with 22x102 cfu/ml, December with 18x102 cfu/ml, November with 13x102 cfu/ml and minimum was in April with 12x102 cfu/ml. However for site IV the maximum distribution of bacterial population was observed in April and July with 43x102 cfu/ml each, followed by August with 39x102 cfu/ml, March with 32x102 cfu/ml, June with 30x102 cfu/ml, September with 25x102 cfu/ml, November with 22x102 cfu/ml, December with 20x102 cfu/ml and minimum was in October with 19x102 cfu/ml. Moreover, the density of total culturable bacteria in site II was significantly higher in all the months compared to other sites. The overall bacterial density throughout the study area was maximum at site II with a cfu/ml of 203x102 in the month of August and minimum at site III with a cfu/ml of 12x102 in the month of April. The total bacterial population was much higher during summer months than winter months for all the four sites. Based on the examination of colony morphological features of bacterial colonies on nutrient agar plates after 48hrs incubation 52 morphologically different strains were isolated. The

49

Results morphological features examined were colony appearance, colony margin, elevation, size and colour. It was found that all the isolated strains showed marked differences in these morphological features and on the basis of differences they were added within code numbers ranging from MBS01 to MBS52 (Table 1). The dominant appearances of the bacterial colonies was circular with 46 of the strains showing this appearance, 4 strains showing irregular appearance and 2 strains showing rhizoidal appearance. 44 strains showed entire margin, 4 showed filamentous margin and scalloped, serrate, undulate, lobate margins were shown by one strain each. The elevation of the colonies also varied. The strains showed convex (39), Flat (2), Umbonate (2), Raised (1) and Fluffiform (1) elevations. In addition to this, the isolated strains differ in their size and the maximum strains were moderate (25) in size, 18 were small, 4 were large and 5 were pinpoint. These isolates were then tested for Gram’s reaction and subsequently examined under microscope for their cell shape. Among Gram-negative bacteria, Bacillus was the most frequently isolated genus from all sites during all months. Out of 52 bacterial isolates, 31 were Gram negative bacillus, 15 Gram negative cocci, 4 Gram positive bacillus and 2 were Gram positive cocci as shown in Table 3. It was also observed that 88.5% of the bacterial strains were Gram negative and 11.5% were Gram positive (Table 2), out of which 34 strains (59.6%) were Gram negative bacillus,12 strains (28.8%) were Gram negative cocci, 4 strains (7.6%) were Gram positive bacillus and 2 strains (3.8%) were Gram positive cocci (Tables 2, 3, 4; Fig. 1). Total viable count of bacteria at different dilutions assessed on monthly basis (Table 6, 7, 8, and 9) showed an increase in the colony count from March to August and a decrease thereafter with the peak colony count in the month of August at all the sites and all the dilutions. Among all the dilutions the highest bacterial colony count was observed at 10-1 dilution in all the water samples collected from all the sites. With a higher number during warm temperature months (June, July, and August) and lower number during cold temperature months (September, October, November and March). From

50

Results all the dilutions 10-2 was taken for future calculations as the number of bacterial colonies in the petri-plates were lying in the recommended range of 30-300 colonies per plate.

4.2. Total coliform

As far as coliform count is concerned, all the samples were positive with respect to the coliform occurrence, though the count was variable. All the samples tested showed coliform counts above the permissible limits. The data also revealed that the highest proportion of these indicator organisms was in the samples of site II. The distribution was highly variable with significant differences existing between the sampling sites. The concentration ranged between 4 and 460MPN/100 ml (Table 10; Fig. 3). For the site I maximum coliform count 460 MPN/100 ml was observed in December, followed by 93 MPN/100 ml in March and April each, 43 MPN/100 ml in June, 21 MPN/100 ml in July and October, 9 MPN/100 ml in August and November each, 4 MPN/100 ml in each September and May. For site II maximum coliform count 240 MPN/100 ml was observed in March and September each, 75 MPN/100 ml in June, 43 MPN/100 ml in April, July and August, 28 MPN/100 ml in October, 23 MPN/100 ml in November, 20 MPN/100 ml in May and 9 MPN/100 ml in December. For site III the maximum 240 MPN/100 ml was observed in June and August followed by 93 MPN/100 ml in September and July, 9 MPN/100 ml in October and November, 4 MPN/100 ml in March, April, May and December. For site IV the maximum concentration 460 MPN/100 ml was observed in September followed by 210 MPN/100 ml in June, 150 MPN/100 ml in April, 75 MPN/100 ml in July and August, 28 MPN/100 ml in March, October and November, 15 MPN/100 ml in December and 4 MPN/100 ml in May. The category wise distribution of coliform count (Table 11) into four categories with MPN range of 0 (zero) for category I, 4-50 MPN/100ml for category II, 51-400 MPN/100ml for category III and 401-1100 MPN/100ml for category IV shows that the maximum (62.5%) water samples

51

Results

lie in category II, followed by 32.5% samples lying in category III, 5% samples in category IV and 0% samples were lying in category I. The observation of the results indicates that none of the samples was fit for drinking purpose with an excellent water quality with respect to this particular parameter. Most of the water samples (95%) obtained from the lake was fit for bathing and swimming with a good or fair quality. However, some patches of the lake were having very much poor quality, hence unfit for any use.

4.3. Biochemical tests After observing a positive growth and on conformation that each plate has only one type of bacteria, Gram’s test was performed. The Gram’s test was repeated many times, until satisfied that results were correct i.e. only one type of colony is present. A total of 19 strains, chosen arbitrarily were subjected to Biochemical tests and was identified using these Test Kits (Hi-Media) following the procedures as described in the instruction manual. It is a striking tool that does not require the development of specific reagents or special methodology and can be integrated into bacterial identification procedure. The biochemical test kits taken were for Gram Negative rods and Enterobacteriaceae. Each kit has a standardized colorimetric identification system (Appendix I & II) utilizing four conventional biochemical tests and eight carbohydrate utilization tests based on the principle of pH change and substrate utilization. After overnight incubation organisms undergo metabolic changes which were indicated as a colour change in the media that was interpreted visually in case of Citrate utilization, Glucose, Adonitol, Arabinose, Lactose, Sorbitol, Mannitol, Rhamnose, Sucrose, Urease,

Lysine utilization, Ornithine utilization and H2S production, while in others such as Phenylalanine Deamination, Nitrate utilization, Indole, Methyl red and Voges Prokauer’s test the reaction was observed after addition of the respective reagents. Results revealed that out of the 19 strains taken for biochemical identification, 9 species were belonging to Enterobacteriaceae family and 10 strains were belonging to Gram negative rods. The Enterobacteriaceae were

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Results tested for their reaction with Indole, Methyl red, Voges Proskauer’s, and Citrate utilization, Glucose, Adonitol, Arabinose, Lactose, Sorbitol, Mannitol, Rhamnose and Sucrose (Table 12). However strains belonging the Gram negative rods were assessed for their reaction with Citrate utilization, lysine utilization, Ornithine utilization, Urease, Phenylalanine deamination, Nitrate

Reduction, H2S production, Glucose, Adonitol, Lactose, Arabinose and Sorbitol (Table 13). On the basis of Biochemical tests the species identified were Proteus spp. I, Proteus spp. II, Proteus spp. III, Escherichia coli, Klebsiella spp. II, Cedecea spp., Escherichia spp., Shigella spp. and Salmonella spp. all these species belong to Enterobacteriaceae. The species belonging to gram negative rods were Shigella spp. I., Shigella spp. II, Shigella spp. III, Enterobacter gergoviae, Hafinia spp., Salmonella chloraesuis subspecies choleraesuis, Salmonella choleraesuis subspecies diarizonae, Vibrio spp., Proteus spp. IV, and Klebsiella spp. I. Total viable count of identified species at four sites assessed on monthly basis given in Tables 14, 15, 16 and 17, shows that the number of identified bacterial species was following the trend of increased number in warmer months for all sites. The comparative analysis of different bacterial isolates at study sites reveals that Proteus spp. I, Proteus spp. II, Proteus spp. III, Cedecea spp., Klebsiella spp. II and Salmonella choleraesuis subspecies choleraesuis were present at all sites except Klebsiella spp. II and Salmonella choleraesuis subspecies choleraesuis that were absent at site IV and site I respectively. Site I showed the presence of E. coli, Shigella spp. I, Salmonella choleraesuis subspecies diarizonae, Proteus spp. IV and Enterobacter spp. Also, Shigella spp. II, Escherichia spp. and Vibrio spp. were found only at site II while as Shigella spp. III was present at sites (I and II) and Hafinia spp. and Shigella spp. IV at sites (I and III). However, Salmonella spp. and Klebseilla spp. I were present only at site IV (Table 18). A total of 1391 colonies of 19 identified strains as presented in Table 19 were obtained during the study. Out of this number 204 colonies with a percentage occurrence of 14.67% of

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Results

Proteus spp. II followed by 136 (9.78%) colonies of Shigella spp. IV, 130 (9.35%) colonies of Cedecea spp., 117 (8.41%) colonies of Shigella spp. III, 100 (7.76%) colonies of Proteus spp. III, 92 (6.62%) colonies of Proteus spp. I, 90 (6.47%) colonies of Escherichia spp., 65 (4.67%) colonies of Salmonella choleraesuis subspecies choleraesuis, 60 (4.31%) colonies of Enterobacter spp., 56 (4.02%) colonies of Shigella spp. II, 55 (3.96%) colonies of Salmonella spp., 55 (3.95%) colonies of Klebseilla spp. II, 52 (3.75%) colonies of Proteus spp. IV, 47 (3.37%) colonies of Vibrio spp., 49 (3.53%) colonies of Hafinia spp., 24 (1.72%) colonies of Salmonella choleraesuis subspecies diarizonae, 22 (1.58%) colonies of E. coli, 26 (1.87%) colonies of Klebseilla spp. I and 3 (0.21%) colonies of Shigella spp. I. The total colony count of different identified species at different sites as presented in the Table 20 indicates that the colony number was highest for site II followed by site I and III and lowest for site IV. Furthermore the colony count was much higher in the month of August for all sites and lowest for the month of November.

Shannon index (H) of the different months presented in Table 21 and Fig. 4 reveals that the overall higher bacterial diversity was found at site II (H=2.316) in the month of August. For site I the diversity index was highest in the month of September (H=2.187), followed by May, April (H=2.16), August (H=2.111), July (H=2.048), June (H=2.009), December (H=1.733), March (H=1.72), November (H=1.56) and October (H=1.517). For site II the diversity index was maximum for the month of August (H=2.316) followed by July (H=2.306), June (H=2.231), September (H=2.216), May (H=2.005), April (H=1.639), December (H=1.561), November (H=1.55) March (H=1.541) and lowest in the month of October (H=1.494). For site III maximum diversity index was found in the month of July (H=1.761) followed by September (H=1.748), August (H=1.723), June (H=1.669), April (H=0.6931), May (H=0.6931), October (H=0.6931) and for the month of November, December and March the index was 0. For site IV the diversity index was highest for the

54

Results month of July (H=1.567) followed by August (H=1.39), April (H=1.085), September (H=0.9433), June (H=0.6853), March (H=0.6829), October (H=0.673), November and December (H=0.6365) and May (H=1.085). Overall Shannon index was highest for site I followed by site II, site III and Site IV respectively (Fig. 5).

4.4. Temperature and pH The temperature and pH of the water samples recorded on spot (Table 22 and 23, Fig. 6 and 7) indicates that lake water was characterized by medium to high alkalinity with pH ranging between 7.7 and 9.6 for all the sites in all the months. The water temperature ranged from 9°C to 33.5°C with a low temperature in cold temperature months and a higher temperature in warm temperature months. All sites had similar ranges of low and high temperatures which occurred during winter and summer months respectively.

55

Results

Table 1. Colony Morphology of Different Bacterial Isolates

Isolate S.No Appearance Margin Elevation Colour Size Name

1 MBS01 Circular Entire Convex Yellow Moderate

2 MBS02 Circular Entire Convex Creamish Moderate

Light yellow with 3 MBS03 Circular Entire Convex Small white outline

4 MBS04 Circular Entire Convex White Large

5 MBS05 Circular Entire Convex Light yellow Moderate

6 MBS06 Irregular Lobate Flat Creamish Moderate

7 MBS07 Circular Scalloped Flat Creamish Moderate

8 MBS08 Circular Entire Convex Light Creamish Moderate

9 MBS09 Circular Entire Convex White Pinpoint

10 MBS10 Circular Entire Convex Red Moderate

11 MBS11 Circular Entire Convex Dark yellow Moderate

12 MBS12 Circular Entire Convex Pink Moderate

13 MBS13 Irregular Filamentous Flat White Large

14 MBS14 Circular Entire Convex Creamish Moderate

15 MBS15 Circular Entire Flat Transparent Pinpoint

16 MBS16 Circular Entire Convex Yellow Pinpoint

Creamish outline 17 MBS17 Circular Entire Convex with white inner Moderate white

18 MBS18 Circular Entire Flat Creamish Small

19 MBS19 Circular Entire Convex Creamish Moderate

20 MBS20 Circular Entire Convex White Moderate

21 MBS21 Circular Entire Convex Creamish Small

22 MBS22 Circular Entire Convex Pink Moderate

23 MBS23 Circular Entire Convex Brown Small

24 MBS24 Circular Entire Convex Yellow light Small

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Results

25 MBS25 Rhizoid Entire Convex White Small

26 MBS26 Circular Entire Convex Yellowish Moderate

27 MBS27 Circular Entire Convex Peach Small

28 MBS28 Rhizoid Filamentous Flat White Large

29 MBS29 Circular Entire Umbonate White Small

30 MBS30 Circular Entire Convex Brown Pinpoint Creamish with 31 MBS31 Circular Entire Convex Moderate green dot at centre 32 MBS32 Circular Entire Convex Brown Moderate

33 MBS33 Circular Serrate Raised Greenish yellow Moderate

34 MBS34 Irregular Filamentous Flat White Moderate

35 MBS35 Circular Entire Umbonate Yellow Moderate

36 MBS36 Circular Entire Convex Grey Small

37 MBS37 Circular Entire Convex Light yellow Small

38 MBS38 Circular Entire Convex Red Small

39 MBS39 Circular Entire Fluffiform White Small

40 MBS40 Circular Entire Convex Light orange Moderate

41 MBS41 Circular Entire Convex White Small

42 MBS42 Circular Filamentous Flat White Small

43 MBS43 Circular Entire Convex Creamish Small

44 MBS44 Irregular Undulate Flat Yellow Moderate

Circular/ 45 MBS45 Entire Convex Creamish Moderate hollow

46 MBS46 Circular Entire Convex Pink Small

47 MBS47 Circular Entire Convex Light yellow Moderate

48 MBS48 Circular Entire Convex Yellow Moderate

49 MBS49 Circular Entire Convex Creamish Small

50 MBS50 Circular Entire Convex Opaque Pinpoint

51 MBS51 Circular Entire Convex White Large

52 MBS52 Circular Entire Convex White Small

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Results

Table 2. Percentage of Gram Positive and Gram Negative Bacterial Isolates Isolate Gram’s S.No Cell Shape Percentage Name Reaction 1. MBS01 -ve Bacillus 2. MBS02 -ve Cocci 3. MBS03 -ve Bacillus 4. MBS04 -ve Bacillus 5. MBS05 -ve Cocci

6. MBS06 -ve Bacillus 7. MBS07 -ve Bacillus 8. MBS08 -ve Cocci 9. MBS09 -ve Bacillus 10. MBS10 -ve Bacillus 11. MBS11 -ve Cocci 12. MBS12 -ve Bacillus 13. MBS13 -ve Bacillus 14. MBS15 -ve Bacillus 15. MBS17 -ve Bacillus 16. MBS18 -ve Bacillus 17. MBS20 -ve Bacillus 18. MBS22 -ve Bacillus 19. MBS23 -ve Bacillus 20. MBS24 -ve Bacillus 21. MBS25 -ve Bacillus 22. MBS26 -ve Cocci 23. MBS27 -ve Bacillus 46(88.5%) 24. MBS28 -ve Cocci 25. MBS29 -ve Bacillus 26. MBS30 -ve Bacillus 27. MBS31 -ve Cocci 28. MBS32 -ve Bacillus

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Results

29. MBS33 -ve Bacillus 30. MBS34 -ve Bacillus 31. MBS35 -ve Bacillus 32. MBS36 -ve Bacillus 33. MBS38 -ve Bacillus 34. MBS40 -ve Bacillus 35. MBS41 -ve Cocci 36. MBS42 -ve Bacillus 37. MBS43 -ve Bacillus 38. MBS44 -ve Bacillus 39. MBS45 -ve Cocci 40. MBS46 -ve Bacillus 41. MBS47 -ve Bacillus 42. MBS48 -ve Cocci 43. MBS49 -ve Bacillus 44. MBS50 -ve Cocci 45. MBS51 -ve Bacillus 46. MBS52 -ve Bacillus 47. MBS14 +ve Bacillus 48. MBS16 +ve Bacillus

49. MBS19 +ve Cocci

50. MBS21 +ve Bacillus 6(11.5%) 51. MBS39 +ve Bacillus 52. MBS37 +ve Cocci Total 52(100%)

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Results

Table 3. Shape Wise Percentage of Different Bacterial Strains

S. No. Isolate type Gram’s reaction Cell shape Percentage 1. MBS02 -ve Cocci 2. MBS05 -ve Cocci 3. MBS08 -ve Cocci

4. MBS11 -ve Cocci 5 MBS26 -ve Cocci 6 MBS28 -ve Cocci 7 MBS31 -ve Cocci 8 MBS40 -ve Cocci 9 MBS41 -ve Cocci 12(23.17%) 10 MBS45 -ve Cocci 11 MBS48 -ve Cocci 12 MBS50 -ve Cocci 13 MBS01 -ve Bacillus

14 MBS03 -ve Bacillus

15 MBS04 -ve Bacillus

16 MBS06 -ve Bacillus

17 MBS07 -ve Bacillus

18 MBS09 -ve Bacillus

19 MBS10 -ve Bacillus 20 MBS12 -ve Bacillus 21 MBS13 -ve Bacillus 22 MBS15 -ve Bacillus

23 MBS17 -ve Bacillus

24 MBS18 -ve Bacillus

25 MBS20 -ve Bacillus

26 MBS22 -ve Bacillus 34(65.38%) 27 MBS23 -ve Bacillus

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Results

28 MBS24 -ve Bacillus 29 MBS25 -ve Bacillus 30 MBS27 -ve Bacillus 31 MBS29 -ve Bacillus 32 MBS30 -ve Bacillus 33 MBS32 -ve Bacillus 34 MBS33 -ve Bacillus 35 MBS34 -ve Bacillus 36 MBS35 -ve Bacillus 37 MBS36 -ve Bacillus 38 MBS38 -ve Bacillus 39 MBS42 -ve Bacillus 40 MBS43 -ve Bacillus 41 MBS44 -ve Bacillus 42 MBS46 -ve Bacillus 43 MBS47 -ve Bacillus 44 MBS49 -ve Bacillus 45 MBS51 -ve Bacillus 46 MBS52 -ve Bacillus 47 MBS19 +ve Cocci 48 MBS37 +ve Cocci 2(3.83%) 49 MBS14 +ve Bacillus

50 MBS16 +ve Bacillus

51 MBS21 +ve Bacillus 4(7.62%) 52 MBS39 +ve Bacillus

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Results

Table 4. Percentage Occurrence of Gram Positive and Gram Negative Bacteria

Gram’s S.No Shape Number %age Reaction

1 -ve Bacillus 34 65.38% 46 88.5% 2 -ve Cocci 12 23.17%

3 +ve Bacillus 4 7.62% 6 11.5% 4 +ve Cocci 2 3.83%

Cocci +ve Bacillus +ve 4% 8%

Cocci -ve 23% Bacillus -ve 65%

Fig. 1. Percentage Occurrence of Gram Positive and Gram Negative Bacteria

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Results

Table 5. Monthly Distribution of Bacterial Colonies (cfu/ml)

Months Site I Site II Site III Site IV

Mar 39x102 133x102 31x102 32x102

Apr 41x102 146x102 12x102 43x102

May 38x102 158x102 22x102 41x102

Jun 128x102 189x102 94x102 30x102

July 120x102 191x102 76x102 43x102

Aug 147x102 203x102 154x102 39x102

Sept 41x102 71x102 57x102 25x102

Oct 38x102 42x102 46x102 19x102

Nov 35x102 42x102 13x102 22x102

Dec 33x102 38x102 18x102 20x102

250 Site I 200 Site II Site III 150 Site IV

cfu/ml 100

50

0 Apr May Jun Jul Aug Sep Oct Nov Dec Months

Fig. 2. Monthly Distribution of Bacterial Colonies (cfu/ml)

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Results

Table 6. Total Viable Count of Bacteria at Site I

Dilutions Mar Apr May Jun Jul Aug Sep Oct Nov Dec

10-1 169 191 175 ˃300 ˃300 ˃300 180 87 68 188

10-2 39 41 38 128 120 147 41 38 35 33

10-3 1 2 14 21 94 153 0 1 0 8

10-4 0 1 3 1 58 129 0 0 0 0

Table 7. Total Viable Count of Bacteria at Site II

Dilutions Mar Apr May Jun Jul Aug Sep Oct Nov Dec

10-1 ˃300 303 276 ˃300 >300 >300 268 89 80 71

10-2 133 146 158 189 191 203 71 42 42 38

10-3 27 115 36 21 94 70 0 0 0 5

10-4 2 37 0 2 23 38 0 1 0 0

Table 8. Total Viable Count of Bacteria at Site III

Dilutions Mar Apr May Jun Jul Aug Sep Oct Nov Dec

10-1 69 47 52 ˃300 194 ˃300 186 172 39 48

10-2 31 12 22 94 76 154 57 46 13 18

10-3 0 3 1 46 42 54 1 5 0 0

10-4 0 6 14 9 5 0 0 1 0 0

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Results

Table 9. Total Viable Count of Bacteria at Site IV

Dilutions Mar Apr May Jun Jul Aug Sep Oct Nov Dec

10-1 68 82 152 109 107 111 41 34 30 42

10-2 32 43 41 30 43 39 25 19 22 20

10-3 8 20 0 3 8 11 0 0 5 0

10-4 2 3 0 1 5 3 0 0 0 0

Table 10. Total Coliform Count of Different Water Samples (MPN/100ml)

Sites Mar Apr May Jun Jul Aug Sep Oct Nov Dec

I 93 93 4 43 21 9 4 21 9 460

II 240 43 20 75 43 43 240 28 23 9

III 4 4 4 240 93 240 93 9 9 4

IV 28 150 4 210 75 75 460 28 28 15

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Results

500 Site I 450 Site II 400 Site III 350 Site IV 300 250 200 MPN/100ml 150 100 50 0 Mar Apr May Jun Jul Aug Sep Oct Nov Dec Months

Fig. 3. Total Coliform Count of Different Water Samples (MPN/100ml)

Table 11. Category Wise Distribution of Coliform Count (MPN/100ml)

Categories MPN range % age Usage Grade

Category I 0 0 Drinking Excellent

Category II 4-50 62.5 Good

Bathing and

Category III 51-400 32.5 Swimming Fair

Category IV 401-1100 5 Unfit Poor

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Results

Table 12. Biochemical Results of Bacterial Species for Enterobacteriaceae Family

Isolate I MR VP C G Ad Ar L Sr Mn Rh S MT Species Identified

MBS40 + + _ _ + ______+ M Proteus spp. I

MBS46 V + _ _ + _ + V + + + + M Escherichia coli

MBS47 _ + + + + + + V V + + V NM Klebsiella spp.II

MBS01 _ + V + + _ _ V _ + _ + M Cedecea spp.

MBS26 + + _ _ + _ _ + V + V + M Escherichia spp.

MBS50 _ + _ _ + _ V _ _ _ _ + M Proteus spp. II

MBS38 _ + _ _ + _ + V _ + V _ NM Shigella spp.I

MBS45 _ + _ _ + _ _ _ _ _ V + M Proteus spp. III

MBS51 _ + _ + + _ + _ V + + _ M Salmonella spp.

I=Indole; MR=Methyl red; VP=Voges Proskauer’s; C=Citrate utilization; G=Glucose; Ad=Adonitol ; Ar=Arabinose; L=Lactose; Sr=Sorbitol; Mn=Mannitol; Rh=Rhamnose;S=Sucrose ; MT=Motility Test; M=Motile; NM=Non-motile; V=11-89% Positve; +=Positive (more than 90%); -=Negative (more than 90%) .

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Results

Table 13. Biochemical Results of Bacterial species for Gram Negative Rods

Isolate C LY O U PD N HP G Ad L Ar Sr MT Species Identified

MBS49 _ _ _ _ _ + _ + _ _ V _ M Shigella spp.II

MBS42 _ _ _ _ _ + _ + _ _ + + M Shigella spp.III

MBS09 + + + + _ + _ + + _ + _ M Enterobacter spp

MBS52 _ + + _ _ + _ + _ _ + _ M Hafnia spp.

Salmonella choleraesuis MBS13 + + + _ _ + + + _ _ + + M subspecies choleraesuis Salmonella choleraesuis MBS43 + + + _ _ + + + _ V + + M subspecies diarizonae

MBS30 _ + _ _ _ + _ V _ _ + _ M Vibrio spp.

MBS15 + _ _ + + + _ + _ _ _ V M Proteus spp. IV

MBS18 _ _ _ _ _ + _ + V _ + V NM Klebsiella spp. I

MBS22 _ _ _ _ _ + _ + _ _ V V M Shigella spp. IV

C=Citrate utilization;LY=lysine:O=Ornithine;U=urease:PD=Phenylalanine Deamination:N=Nitrate; HP=H2S Production G=Glucose; Ad=Adonitol; L=Lactose; Ar=Arabinose; Sr=Sorbitol; MT=Motility Test; M=Motile; NM=Non-motile: V=11-89% Positive; +=Positive (more than 90%); -=Negative (more than 90%)

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Results

Table 14. Total Viable Count of the Identified Species at Site I

Species Name Mar Apr May Jun Jul Aug Sep Oct Nov Dec Total

Cedecea spp. 0 2 0 9 14 8 2 2 1 1 39

E. coli 0 0 1 8 10 3 0 0 0 0 22

Escherichia spp. 0 0 0 0 0 0 0 0 0 0 0

Enterobacter spp. 3 3 2 14 12 18 2 3 1 2 60

Hafinia spp. 2 3 0 0 0 0 4 4 2 1 16

Klebseilla spp. I 0 0 0 0 0 0 0 0 0 0 0

Klebseilla spp. II 0 1 1 0 6 1 2 0 0 0 11

Proteus spp. I 0 0 2 4 13 12 1 0 0 0 32

Proteus spp. II 2 2 2 0 0 0 1 0 2 2 11

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Results

Proteus spp. III 0 2 3 15 0 14 0 0 0 0 34

Proteus spp. IV 2 2 1 18 15 13 1 0 0 0 52

Salmonella choleraesuis 0 0 0 0 0 0 0 0 0 0 0 subspecies choleraesuis Salmonella choleraesuis 0 0 0 0 0 23 0 0 0 1 24 subspecies diarizonae

Salmonella spp. 0 0 0 0 0 0 0 0 0 0 0

Shigella spp. I 0 0 1 0 0 0 2 0 0 0 3

Shigella spp. II 0 0 0 0 0 0 0 0 0 0 0

Shigella spp. III 1 2 2 12 15 6 1 2 0 0 41

Shigella spp. IV 1 2 1 13 15 16 3 1 2 1 55

Vibrio spp. 0 0 0 0 0 0 0 0 0 0 0

Total 11 19 16 93 100 114 19 12 8 8 400

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Results

Table 15. Total Viable Count of the Identified Species at Site II

Species Name Mar Apr May Jun Jul Aug Sep Oct Nov Dec Total

Cedecea spp. 9 26 18 10 5 5 2 0 0 0 75

E. coli 0 0 0 0 0 0 0 0 0 0 0

Escherichia spp. 7 19 21 12 9 15 2 3 1 1 90

Enterobacter spp. 0 0 0 0 0 0 0 0 0 0 0

Hafinia spp. 0 0 0 0 0 0 0 0 0 0 0

Klebseilla spp. I 0 0 0 0 0 0 0 0 0 0 0

Klebseilla spp. II 0 3 3 0 7 13 3 0 0 0 29

Proteus spp. I 0 0 2 5 6 12 2 0 0 0 27

Proteus spp. II 13 34 12 15 16 24 1 2 2 2 121

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Results

Proteus spp. III 0 0 11 16 12 21 2 0 0 0 62 0 Proteus spp. IV 0 0 0 0 0 0 0 0 0 0

Salmonella choleraesuis 0 0 0 5 9 7 2 1 2 1 27 subspecies choleraesuis Salmonella choleraesuis 0 0 0 0 0 0 0 0 0 0 0 subspecies diarizonae Salmonella spp. 0 0 0 0 0 0 0 0 0 0 0

Shigella spp. I 0 0 0 0 0 0 0 0 0 0 0

Shigella spp. II 0 0 4 13 21 14 4 0 0 0 56

Shigella spp. III 4 13 15 18 12 9 2 1 1 1 76

Shigella spp. IV 7 19 12 13 8 11 8 1 1 1 81

Vibrio spp. 0 0 0 18 7 16 6 0 0 0 47

Total 40 114 98 125 112 147 34 8 7 6 691

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Results

Table 16. Total Viable Count of the Identified Species at Site III

Species Name Mar Apr May Jun Jul Aug Sep Oct Nov Dec Total

Cedecea spp. 0 0 0 0 1 0 1 0 0 0 2

E. coli 0 0 0 0 0 0 0 0 0 0 0

Escherichia spp. 0 0 0 0 0 0 0 0 0 0 0

Enterobacter spp. 0 0 0 0 0 0 0 0 0 0 0

Hafinia spp. 0 1 0 8 12 10 1 1 0 0 33

Klebseilla spp. I 0 0 0 4 12 9 1 0 0 0 26

Klebseilla spp. II 0 0 1 0 5 7 2 0 0 0 15

Proteus spp. I 0 0 0 1 4 7 1 0 0 0 13

Proteus spp. II 0 1 0 4 14 17 1 1 0 0 38

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Results

Proteus spp. III 0 0 0 4 0 0 0 0 0 0 4

Proteus spp. IV 0 0 0 0 0 0 0 0 0 0 0 Salmonella choleraesuis 0 0 1 5 8 6 0 0 0 0 20 subspecies choleraesuis Salmonella choleraesuis 0 0 0 0 0 0 0 0 0 0 0 subspecies diarizonae Salmonella spp. 0 0 0 0 0 0 0 0 0 0 0

Shigella spp. I 0 0 0 0 0 0 0 0 0 0 0

Shigella spp. II 0 0 0 0 0 0 0 0 0 0 0

Shigella spp. III 0 0 0 0 0 0 0 0 0 0 0

Shigella spp. IV 0 0 0 0 0 0 0 0 0 0 0

Vibrio spp. 0 0 0 0 0 0 0 0 0 0 0

Total 0 2 2 26 56 56 7 2 0 0 151

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Results

Table 17. Total Viable Count of the Identified Species at Site IV

Species Name Mar Apr May Jun Jul Aug Sep Oct Nov Dec Total

Cedecea spp. 0 0 0 0 6 8 0 0 0 0 14

E. coli 0 0 0 0 0 0 0 0 0 0 0

Escherichia spp. 0 0 0 0 0 0 0 0 0 0 0

Enterobacter spp. 0 0 0 0 0 0 0 0 0 0 0

Hafinia spp. 0 0 0 0 0 0 0 0 0 0 0

Klebseilla spp. I 0 0 0 0 0 0 0 0 0 0 0

Klebseilla spp. II 0 0 0 0 0 0 0 0 0 0 0

Proteus spp. I 0 0 1 0 4 10 5 0 0 0 20

Proteus spp. II 12 9 0 0 3 2 1 2 1 4 34

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Results

Proteus spp. III 0 8 0 0 0 0 0 0 0 0 8

Proteus spp. IV 0 0 0 0 0 0 0 0 0 0 0

Salmonella choleraesuis 0 0 1 9 6 2 0 0 0 0 18 subspecies choleraesuis Salmonella choleraesuis 0 0 0 0 0 0 0 0 0 0 0 subspecies diarizonae

Salmonella spp. 9 6 12 7 7 3 4 3 2 2 55

Shigella spp. I 0 0 0 0 0 0 0 0 0 0 0

Shigella spp. II 0 0 0 0 0 0 0 0 0 0 0

Shigella spp. III 0 0 0 0 0 0 0 0 0 0 0

Shigella spp. IV 0 0 0 0 0 0 0 0 0 0 0

Vibrio spp. 0 0 0 0 0 0 0 0 0 0 0

Total 21 23 14 16 26 25 10 5 3 6 149

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Results

Table 18. Comparative Bacterial Analysis of Different Sites

Species name Site I Site II Site III Site IV

Cedecea spp. + + + +

E. coli + - - -

E. spp. - + - -

Enterobacter spp. + - - -

Hafinia spp. + - + -

Klebseilla spp. I - - + -

Klebseilla spp. II + + + -

Proteus spp. I + + + +

Proteus spp. II + + + +

Proteus spp. III + + + +

Proteus spp. IV + - - -

Salmonella choleraesuis subspecies choleraesuis - + + +

Salmonella choleraesuis subspecies diarizonae + - - -

Salmonella spp. - - - +

Shigella spp. I + - - -

Shigella spp. II - + - -

Shigella spp. III + + - -

Shigella spp. IV + + - -

Vibrio spp. - + - -

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Results

Table 19. Total Colony Count and Percentage Occurrence of Bacterial Species Site I Site II Site III Site IV Grand Total Species Name Number %age Number %age Number %age Number %age Number %age Cedecea spp. 39 30 75 57.69 2 1.55 14 10.76 130 9.35 E. coli 22 100 0 0 0 0 0 0 22 1.58 Escherichia spp 0 0 90 100 0 0 0 0 90 6.47 Enterobacter spp. 60 100 0 0 0 0 0 0 60 4.31 Hafinia spp. 16 32.65 0 0 33 67.35 0 0 49 3.53 Klebseilla spp. I 0 0 0 0 26 100 0 0 26 1.87 Klebseilla spp. II 11 20 29 52.73 15 27.27 0 0 55 3.95 Proteus spp. I 32 34.8 27 29.34 13 14.13 20 21.73 92 6.62 Proteus spp. II 11 5.4 121 59.31 38 18.62 34 16.67 204 14.67 Proteus spp. III 34 31.48 62 57.41 4 3.70 8 7.41 108 7.76 Proteus spp. IV 52 100 0 0 0 0 0 0 52 3.75 Salmonella choleraesuis 0 0 27 41.54 20 30.77 18 27.69 65 4.67 subspecies choleraesuis Salmonella choleraesuis 24 100 0 0 0 0 0 0 24 1.72 subspecies diarizonae Salmonella spp. 0 0 0 0 0 0 55 100 55 3.96 Shigella spp. I 3 100 0 0 0 0 0 0 3 0.21 Shigella spp. II 0 0 56 100 0 0 0 0 56 4.02 Shigella spp. III 41 35.05 76 64.95 0 0 0 0 117 8.41 Shigella spp. IV 55 40.44 81 59.56 0 0 0 0 136 9.78 Vibrio spp. 0 0 47 100 0 0 0 0 47 3.37 Total 400 28.76 691 49.67 151 10.86 149 10.71 1391 100

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Results

Table 20. Number of Colonies of Identified Species at Different Sites

Sites Mar Apr May Jun Jul Aug Sep Oct Nov Dec Total

Site I 11 19 16 93 100 114 19 12 8 8 400

Site II 40 114 98 125 112 147 34 8 7 6 691

Site III 0 2 2 26 56 56 7 2 0 0 151

Site IV 21 23 14 16 26 25 10 5 3 6 149

Total 72 158 130 260 294 342 70 27 18 20 1391

Table 21. Shannon Index (H) of Different Sites in Different Months

Mar Apr May Jun Jul Aug Sep Oct Nov Dec Sites

1.72 2.16 2.22 2.009 2.048 2.111 2.187 1.517 1.56 1.733 Site I Site

1.541 1.639 2.005 2.231 2.306 2.316 2.216 1.494 1.55 1.561 Site IISite

0 0.6931 0.6931 1.669 1.761 1.723 1.748 0.6931 0 0 Site III Site

0.6829 1.085 0.5091 0.6853 1.567 1.39 0.9433 0.673 0.6365 0.6365 Site IVSite

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Results

2.5 Site I Site II Site III 2 Site IV

1.5

1 Shannon index H index Shannon

0.5

0 Mar Apr May Jun Jul Aug Sep Oct Nov Dec

Fig. 4. Shannon Index (H) of Different Sites in Different Months

2.5

2

1.5

annon index H annon 1 h S

0.5

0 Site I Site II Site III Site IV

Fig. 5. Site Wise Shannon Index (H) of Bacterial Community

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Results

Table 22. Water Temperature (°C) Recorded at Four Sites

Months Site I Site II Site III Site IV

Mar 15.5±1.1 16.5±1.1 16±2.0 16.4±1.1

Apr 19.1±0.65 18±1.0 19.5±1.1 19.8±1.1

May 30.8±0.15 30±0.9 31.8±0.1 27±1.0

Jun 30±0.2 26.4±0.95 25.2±1.1 26.5±1.1

Jul 27.5±1.1 27±1.0 27.2±1.1 27±1.0

Aug 29.8±0.9 33.5±0.15 29.6±0.8 29.6±0.9

Sep 24.6±1.5 25.1±1.2 24±1.0 27.1±0.76

Oct 17.8±0.9 17.3±0.9 17.2±0.9 17±1.0

Nov 15.6±0.9 15.6±0.9 15±1.0 15.7±1.1

Dec 9.5±0.7 9.4±0.6 9±1.0 9.2±0.7

40 Site I Site II 35 Site III Site IV 30

25

20 Temperature er t 15 Wa

10

5

0 Mar Apr May Jun Jul Aug Sep Oct Nov Dec Months

Fig. 6. Water Temperature Recorded at Four Sites

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Results

Table 23. pH of Water Recorded at Four Sites

Months Site I Site II Site III Site IV

Mar 8.5±0.1 8.8±0.15 8.84±0.065 8.53±0.2

Apr 8.3±0.2 8.5±0.26 8.5±0.2 8.6±0.43

May 9.4±0.2 9.4±0.3 8.57±0.07 8.66±0.14

Jun 9.3±0.15 9.4±0.1 8.9±0.26 8.9±0.2

Jul 9.26±0.32 9.3±0.2 8.83±0.25 8.7±0.1

Aug 9.65±0.07 9.53±0.47 8.7±0.2 8.8±0.2

Sep 8.49±0.35 8.7±0.112 8.7±0.26 8.76±0.11

Oct 8.63±0.15 8.7±0.2 8.2±0.1 8.16±0.37

Nov 8.4±0.2 8.43±0.25 8.1±0.1 8.26±0.2

Dec 8±0.1 8.26±0.15 7.8±0.20 7.7±0.057

Site I 12 SiteII Site III 10 Site IV

8

6 pH

4

2

0 Mar Apr May Jun Jul Aug Sep Oct Nov Dec Months

Fig. 7. pH of Water Recorded at Four Sites

82

Chapter: 5 Discussion Discussion

he analysis of bacterial diversity in aquatic environments subjected to different anthropogenic pressures has been of considerable interest in Tthe recent times, mainly in view of potential management implications of such studies (Aguilo-Ferretjans et al., 2008). Bacteria are most widely present in water and their diversity is used as an indicator for the potability of water (Okpokwasili and Akujobi, 1996). Bacterial pathogens and indicator species may be introduced into water from both point and non-point sources (Metcalf and Eddy, 1991; Moe, 1997).

5.1 Distribution of colonies The results of present study revealed that distribution of colonies (cfu/ml) increased from winter months to summer months with the bacterial

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Discussion density much higher for site II in comparison to other sites that can be attributed to anthropogenic pressure such as sewage coming from the surrounding catchment of the lake. The results of the present study draw support from the findings of Sharma et al. (2010) who have worked on the bacterial indicators of faecal pollution and physico-chemical assessment of North Indian lakes and have reported that the places with greater anthropogenic pressure experience a comparatively higher bacterial load. The dominance of gram negative bacilli bacteria with a percentage occurrence of 65.38% in the lake waters is a cause of concern, as these gram negative bacteria are mostly of pathogenic in nature, although some of them are beneficial in the aquatic environments and can be attributed to the human activities taking place in the catchment area. The abundance of the gram negative bacteria observed during the study at the different sites may be attributed to the increased addition of the excretory substances to the water. Gram negative bacteria having a reservoir in the intestines of man and other warm blooded animals are excreted in faeces and are known to survive in the environment but do not reproduce (Feachem et al., 1983). However, in tropical environments there are evidences that the enteric bacteria can survive as well as multiply (Rivera et al., 1988). These revelations are also confirmed by a study carried out by Gandotra (2009) in river Tawi in Jammu city confirming the dominance of gram negative bacilli in the river water samples. The overall increase in total viable count of bacterial colonies at all the sites in temperature months may be attributed to an increased water temperature itself and our results are in consonance with the results of Murphy (2000) who showed that bacteria grow faster at higher temperatures, while as at lower temperatures their growth rate slows down dramatically. Another study carried out by Lokoska et al. (2004) also supports our findings by showing that bacterial community composition in any aquatic system is determined by various physical and chemical characteristics of the water, with temperature playing a key role.

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Discussion

5.2 Total coliform count For further assessing the degree of potability, water samples were tested for coliform group, indicators of water pollution. An indicator of faecal pollution comprises the bacteria which are normally taken as indicators of the degree of purity of the water. Feng et al. (2007) suggested that Escherichia coli is a faecal bacterium which is found in the intestinal canal of man and warm- blooded animals and is discharged with faeces. Enterotoxigenic E. coli (ETEC), (organism is a common cause of "traveler's diarrhea" in developing countries) infects only humans, with transmission occurring through water and food contaminated with human waste. Though, faecal indicators are common in water samples from non agricultural or pristine watersheds, their long periods of survival especially in the sediments indicate the reality that if they are transported to streams, they may effectively degrade water quality of stream. All the samples were analysed by Multiple Tube Fermentation technique and the tubes with both gas and acid production were taken as positive. The MPN index observed for the water samples revealed that maximum of samples were crossing the permissible limit set by WHO (1993) indicating gross pollution of the lake and its transition towards eutrophic status. The high MPN values in most of water samples collected can be attributed to the agricultural runoff and sewage drained into the lake from the catchment area via Laar Kul. The results are in conformation with the results of Geldrich (1972) who observed an increase in the total coliform count of water bodies due to increased use of animal wastes as manure in the agricultural fields. The occurrence of coliforms in the samples is also confirmed by a local study conducted by Lateef et al. (2003) reporting high coliform count in fifteen springs of Kashmir valley. Due to change in agricultural methods, diminishing of livestock farms, intensive farming operation, increased concentration of animal wastes results in an increased pollution of rivers and streams (Gelt, 1998). Because of the animal wastes entering the water, occurrence of several new pathogens of concern in

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Discussion drinking water have known or suspected animal hosts, concern has grown over the potential for direct or indirect animal-to-human transmission through drinking water supplies contaminated with animal wastes containing these pathogens. As per the results of present study depicted in Table 11, only 5% were found to fall in a higher range which is unfit for any purpose. While considering best use of water body, Manasbal Lake can be categorised in category III and designated as satisfactory for the purpose of bathing (Pandey and Sharma, 1999). Therefore, control must be implemented to minimize bacterial transport to natural systems such as Manasbal Lake.

5.3 Biochemical tests From the results of the tests performed it can be suggested that all the strains isolated were of pathogenic nature. Since microorganisms are widely distributed in nature and can be transported to the lake system through the rains mainly with the particulate matter with attached bacteria. A wide variety of bacteria present in the soil itself can be washed as runoff to the lake, the species present are related to man’s activities on the land. Anthropogenic activities such as agricultural practices and excessive addition of domestic sewage destroy the capacity of self purification of the water body (Geldreich, 1972; Sharma et al., 2010). The access of faecal pollution to water body results in the addition of variety of intestinal pathogenic organisms discharged from warm- blooded animals. Salmonella spp., Shigella spp., enteropathogenic Escherichia coli, Vibrio spp., entero-viruses, Klebseilla spp. have been frequently detected in the polluted fresh water bodies and estuarine environment (Geldreich, 1972). These organisms might reach to an infective dosage level for a significant number of bathers and become a serious risk to public use of this recreational water.

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Discussion

5.3.1 Salmonella spp. Three different species of Salmonella which differed in one or other biochemical tests in the study were isolated. Salmonella species are Gram- negative rods, motile (with a few exceptions), facultatively anaerobic, producing acid from glucose usually with the production of gas, and are oxidase negative (Le-Minor, 1984). Most of the species produce hydrogen sulphide except S. paratyphi and S. typhi, which is a weak producer. Salmonella spp. is of concern because of its pathogencity that is of worldwide importance and transmitted mainly through water and food. The primary habitat of the Salmonella spp. is the intestinal tract of animals and humans. All countries in Europe have reported a sharp rise in salmonellosis incidence (including food borne outbreaks) over the past three decades. According to WHO (1997) the similar pattern was observed in a number of countries in the Eastern Mediterranean Region and south-east Asia Region. The occurrence of Salmonella spp. in Manasbal lake water is confirmed by number of studies revealing the incidence and survival of Salmonella in lakes, rivers, coastal water and beach sediments (Polo et al., 1998). In these environments some, but not all, strains of Salmonella are pathogenic. Carraminana et al. (1997) reported that Salmonella spp. has long been regarded as non-haemolytic microorganisms. Salmonella infection can be severe with diarrhoea, septicaemia, and bowel bleeding as seen with S.typhi and S. paratyphi infection (Bean et al., 1997). 5.3.2 Shigella spp. Species of the genus Shigella are among the bacterial pathogens most frequently isolated from patients with diarrhoea. Shigella is responsible for 5- 15% of diarrheal case including 1.1 million fatal ones (Kotloff et al., 1999). They are Gram negative rods, facultative anaerobes, non-motile, oxidase negative, urease-negative, do not decarboxylate lysine and all except S. dysenteriae are catalase-positive (Rowe and Gross, 1984). The species may be

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Discussion differentiated by biochemical tests and serology of their lipo-polysaccharides (Emmerson and Gillespie, 1997). Shigella species are highly infective and the infective dose is particularly low with S. dysenteriae, which may require as few as 10-100 organisms to cause infection (Emmerson and Gillespie, 1997). S. sonnei strains ferment lactose upon extended incubation, but other species do not utilise this substrate in conventional medium. Shigellosis remains a common gastrointestinal disease in developing and industrialized countries. It occurs mostly in children less than 5 years of age; S. sonnei is the most frequently isolated species (Mead et al., 1999). 5.3.3 Escherichia coli Escherichia coli common inhabitants of the small intestine and large intestine of mammals are often the most abundant facultative anaerobes in environment. The human colon sustains a microbial density approaching 1012 organisms per gram of faeces, representing a perfectly balanced system. The commensal microbiota consists of more than 400 species and lives in perfect harmony with the human intestine (Hooper and Gordon, 2001). E. coli can be found secondarily in water and soil as the result of faecal contamination. Typically, detection of E. coli has been used as an indicator of poor water quality. From biochemical, physiological and genetic perspectives, E. coli is one of the best understood and characterized living organisms. Several E. coli pathotypes have been implicated with diarrhoeal illness, a major public health problem worldwide, with over two million deaths occurring each year (Kosek et al., 2003). They can occasionally be isolated in association with the intestinal tract of number of mammalian animals and insects. The presence of E. coli in the environment is usually considered to reflect faecal contamination and not the ability to replicate freely outside the intestine. There is evidence however to suggest that E. coli may freely replicate in tropical fresh water (Bermudez and Hazen, 1988). E. coli are Gram-negative, non-spore forming bacilli. Biochemical tests reveal that E. coli is positive for Indole production

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Discussion and methyl red test (IM). Most strains are oxidase, citrate, urease, and hydrogen sulphide negative. The classic differential test to primarily separate E. coli from Shigella and Salmonella is the ability of E. coli to ferment lactose, which the latter two genera fail to do. Most E. coli strains are capable of growing over a wide range in temperature (approximately 15–48°C). The growth rate is maximal in the narrow range of 37–42°C. E. coli can grow within a pH range of approximately 5.5–8.0 with best growth occurring at neutrality. Some diarrheagenic E. coli strains have the ability to tolerate exposure to pH 2.0. Such an acid shock mimics transit through the stomach and induces expression of sets of genes involved in survival and pathogenesis. There are six species, of which four are known to cause human disease. The most commonly isolated is E. coli, which contains numerous serotypes, some of which are associated with specific diseases. A number of strains of E. coli may produce entero-toxins or other virulence factors, including those associated with invasiveness. E. coli can respond to environmental signals such as chemicals, pH, temperature, osmolarity, etc., in a number of very remarkable ways considering it is a unicellular organism (Cristina, 2006). 5.3.4 Klebsiella spp. The occurrence of different Klebsiella spp. in the study is confirmed by the study of Christina et al. (2011), who in his study revealed that Klebsiella spp. are environmental organisms commonly found in manure and organic bedding and are ubiquitous in nature. The genus Klebsiella contains five species and four subspecies. Four species, previously named pneumoniae, ozaenae, rhinoscleromatis and aerogenes are now classed as subspecies of K. pneumoniae. K. pneumoniae subspecies aerogenes is the most frequently isolated species. All grow readily on ordinary media, are non-motile and capsulated, Gram-negative rods, facultatively anaerobic and are non spore forming. Klebsiella spp. have been recognised as a major cause of hospital- associated infections. They generally produce lysine decarboxylase but not

89

Discussion ornithine decarboxylase and are generally positive in the Voges-Proskauer test (Edwards and Ewing, 1986). 5.3.5 Enterobacter spp. All species of bacteria belonging to family Enterobacteriaceae are oxidase-negative, Gram-negative, straight rods, with some of them being motile. Most species grow well at 37°C, although some species grow better at 25 - 30°C. They are facultatively anaerobic and catalase-positive (except Shigella dysenteriae). They are distributed worldwide and may be found in water, soil, plants and animals. There are eleven species, but only eight have been isolated from clinical material. They grow readily on ordinary agar, ferment glucose with the production of acid and gas, and are motile by peritrichous flagella. Some strains with a K antigen possess a capsule. E. gergouiae is a gram-negative, oxidase-negative, nitrate-positive, fermentative, rod-shaped organism that is peritrichous when motile. Enterobacter species are common causes of nosocomial infections in humans. Apart from E. hormaechei, E. asburiae and E. aerogenes, which represent the most frequently encountered Enterobacter species in clinical specimens, there are several further Enterobacter taxa associated with human disease (Nazarowec and Farber, 1999). 5.3.6 Proteus spp. Proteus is a genus of Gram-negative Proteobacteria, with four species of which three P. vulgaris, P. mirabilis and P. penneri are pathogenic to humans, causing chronic urinary tract infections, bacteraemia, pneumonia, focal lesions and wound infections. They exhibit characteristic swarming and are part of the normal flora of the gastrointestinal (GI) tract. These species only become pathogenic if present outside the GI tract. Proteus species can easily adhere to the kidney urothelium, which facilitates the upper urinary tract. Proteus also hydrolyzes urea, which alters the pH of urine and may lead to the formation of kidney stones. Proteus species can resemble non-motile Salmonella

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Discussion biochemically; however all are highly motile with peritrichous flagella. Some Proteus species are motile, and all are oxidase negative, urease positive, aerobic, rod shaped bacilli that do not ferment lactose. P. vulgaris is usually found to inhabit the intestinal tract of animals, however it can be found in water, soil, faecal matter, and raw meat. P. mirabilis is the most frequently isolated member of the Proteus genus and is most oftenly associated with infection (Matsen et al., 1972). 5.3.7 Hafnia spp. The genus Hafnia contains a single species, H. alvei growing readily on ordinary media and is generally motile. Motility is more pronounced at 30°C than 37°C. H. alvei can resemble non-motile salmonella biochemically, and can agglutinate in polyvalent salmonella antisera. The genus Hafnia was first recognised by Moeller (1954) while studying amino acid decarboxylase patterns in members of the family Enterobacteriaceae and named this new group the ‘Hafnia group’. Distinguishing phenotypic features of this taxon included a positive Voges-Proskauer reaction, production of lysine and ornithine decarboxylases, and a negative arginine dihydrolase test. It is suspected to cause a variety of intestinal disorders, including gastroenteritis

(Ridell et al., 1994). H. alvei were positive for the fermentation of D-glucose and L-arabinose, decarboxylation of lysine and ornithine, and are negative for fermentation of sucrose, D-sorbitol, raffinose, myoinositol, D-adonitol, and melibiose; hydrogen sulfide (H2S) production; indole production; and TDA; and are variable for fermentation of L-rhamnose, urease, arginine dihydrolase, esculin (Rodriguez et al., 1999).

5.3.8 Cedecea spp. Cedecea strains are gram-negative straight rods, motile, peritrichous when they are motile, non-capsulated, non-spore forming, aerobic and facultative anaerobs. They grow on nutrient agar at 37ºC forming convex

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Discussion colonies about 1.5mm in diameter. They grow at a pH range of 7-9 in peptone water containing 0-4% NaCl and in tryptic soy broth. Cedecea strains are catalase positive, oxidase negative, arabinose negative, arginine positive reduce nitrates to nitrites, blacken esculin iron agar, and produce acid from D-arabitol, cellobiose, maltose, D-mannitol, D-mannose salicin and trehalose. Isolates of Cedecea are often negative in the Voges –Proskauer test. Cedecea spp., which are the member of Enterobacteriaceae family, is frequently isolated from sputum, but their clinical importance is not clear. C. davisae and C. neteri (Dalamaga et al., 2008b; Aguilera et al., 1995) were reported to cause bacteremia, ulcer, abscess, wound and ophthalmic infections and C. lapagei was reported to cause pneumonia (Farmer, 1982; Dalamaga et al., 2008a; Yetkin et al., 2010). 5.3.9 Vibrio spp. Vibrio spp. are small, curved (comma-shaped), Gram-negative bacteria with a single polar flagellum. Vibrio cholerae is the only pathogenic species of significance from freshwater environments. There are a number of pathogenic species, including V. cholerae, V. parahaemolyticus and V. vulnificus. Non- toxigenic V. cholerae is widely distributed in water environments, but toxigenic strains are not distributed as widely. Humans are an established source of toxigenic V. cholerae; in the presence of disease, the organism can be detected in sewage. Toxigenic V. cholerae has also been found in association with live copepods as well as other aquatic organisms, including molluscs, crustaceans, plants, algae and cyanobacteria. Numbers associated with these aquatic organisms are often higher than in the water column. Non-toxigenic V. cholerae has been isolated from birds and herbivores in areas far away from marine and coastal waters (Ogg et al., 1989; Rhodes et al., 1985). The prevalence of V. cholerae decreases as water temperatures fall below 20 °C. Contamination of water due to poor sanitation is largely responsible for disease transmission. The presence of the pathogenic V. cholera serotypes in drinking-

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Discussion water supplies is of major public health importance and can have serious health and economic implications in the affected communities. It is highly sensitive to disinfection processes (Kaper et al., 1995; WHO, 2002).

5.4 Temperature and pH As the bacterial population are known to vary in response to the variable physical parameters such as temperature (Pearce, 2008) and pH (Hassanin, 2006), the pH of the water samples recorded was found in the range of 7.7-9.6, which is within the desirable pH range (6.5 to 9.0) for fresh water aquatic life as proposed by Chin (2000).

93

Chapter: 6 Conclusion Conclusions

he knowledge of the microbial diversity in inland waters especially in freshwater habitats has changed fundamentally. It has been T observed that fresh water systems are populated by indigenous bacterial communities, not being found in other environments such as terrestrial and marine habitats. Research studies have shown that determining water quality using microbes as indicator of pollution is an important means for ensuring the supply of safe drinking water. While waterborne diseases may be considered to be problems of underdeveloped countries with inadequate sanitary practices, there is increasing recognition that industrialized, developed countries also have significant public health problems caused by use of untreated, partially treated, or inadequately treated domestic water supplies. The detection and identification of microbial contaminants in drinking water supplies therefore need to be viewed as a high priority. Hence need arises to reduce runoff containing pathogenic microbes into water supplies, at the same time to strength water treatment programs and public health surveillance for infectious diseases. From the present study it can be concluded that:

94

Conclusions

• Bacteriological analysis of water is a powerful and foremost tool in order to foreclose the presence of microorganisms that might constitute a health hazard.

• Most of the bacterial species isolated and characterised were Gram- negative bacillus, a cause of concern due to their high pathogencity

• The MPN index revealed the high coliform count indicating that the lake water is not potable.

• Among the strains identified, Proteus spp. and Cedecea spp. were present at all the study sites with highest percentage occurrence (14.73%) of Proteus spp.

• The highest bacterial load was found at site I and site II comparable to rest two sites.

• The primary data generated on bacterial composition of the lake waters fills up the important knowledge gap regarding the trophic status of the system.

Based on these conclusions, attention has to be paid for protecting water quality of the lake including microbial flora and fauna especially those responsible for the outbreak of diseases that take us back that we cannot overlook traditional public concerns. In developing countries, inadequate sewage disposal and drinking water treatment facilities are primary causes of waterborne infectious disease. This includes inappropriate sanitation for the density of the community, no facilities to dispose grey water, poor operation and maintenance of services, sewer blockages, littering, and poor design of waste removal services. As a result of recreational activities, many individuals may contract diseases that range from self-limiting gastrointestinal disturbances to severe and life-threatening infections. The disease incidence is dependent on several factors: the extent of water pollution, time and type of exposure, the

95

Conclusions immune status of users and other factors. These issues tend to exacerbate or directly cause the physical problems. Important institutional concerns re- addressing the problems are: a lack of funds within the Local Authority; a lack of capacity to maintain the services; and a diversion of resources to other priorities.

The remedial measures suggested are:

• The quality of water in some of these areas was fair or poor on the basis of results obtained from the coliform test. So it is necessary to prevent pollution of the lake. • To prevent water pollution, there must be a suitable wastewater treatment system, considering the population density in catchment hamlets, topographical and economic condition. • Prevention of storm flooding into springs and also doing enough sanitation. • Regular monitoring of the Lake is recommended to detect any unacceptable variability of pollutants and adopt appropriate remedial measures. In order to meet the potability of water it is recommended that continuous, effective treatment combined with constant monitoring is essential to ensure that it meets the standards of drinking water.

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Chapter: 7 Bibliography Bibliography

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Photo Plates

A. Site I (Laar Kul) B. Site II (Residential hamlets around)

C. Site III (Centre) D. Site IV (Outlet)

PLATE 2. Photographs showing different study sites of Manasbal Lake

A. Spread Plate Technique

B. Streak Plate Technique

PLATE 3. Photographs showing different isolation techniques

A. Gram Negative Bacillus B. Gram Positive Bacillus

C. Gram Negative Cocci D. Gram Positive Cocci

PLATE 4. Photographs showing Grams nature and shape of bacteria

Before After

Incubation

A. Enterobacteriaceae

PLATE 5. Photographs showing Biochemical test kit for Enterobacteriaceae

Before After

Incubation

A. Gram Negative Rods

PLATE 6. Photographs showing Biochemical test kit for Gram Negative Rods

Plate 1. Map showing different study sites of Manasbal Lake

Appendix Appendix: I

Appendix: II