Bacterial Diversity

INTRODUCTION

Bacterial Diversity

The biological contamination in water is major problem of public health in developing world. WHO estimate that about 1.1 billion people globally drink unsafe water and vast majority of diarrhoeal disease in the world (88%) is attributable to unsafe water ,sanitation and hygiene (WHO, 2003). The most common and wide spread health risk associated in drinking water in developing countriesare of biological origin. Looking at 20 leading risk factors for health burden in developing region unsafe water,sanitation and poor hygenes are 3rd, behind underweight or practicing unsafe sex (WHO) . Ten major water born diseases are responsible for over 28 billions disease episodes annually in developing countries (WALSH 1990). Of these diarrhoeal diseases are big killers specially in infants . According to the WHO estimation more than 3 billion children below age 5 die annually form diarrhoeal disease contracted through drinking water in developing world. None the less ,the inadequate availability of water , poor quality at source , illmaintained water pipe lines and sewer lines , unsafe disposing of human ,animals and household waste ,unawareness about good sanitation and personal hygienic practices etc are some key factors are responsible for poor drinking water quality in rural areas of india.Also infectious diseases caused by pathogenic bacteria , viruses ,and parasites (eg:- Protozoa and Helminthes) are the most common and wide spread helth risks associated with drinking water in rural habitations . The quality of drinking water may be ascertained by its microbiological examination. The greatest risks from microbes in water is associated with consumption of drinking water that is contaminated with human and animal excreta ,all though other sources and routs of exposer may also be significant .The Colliforms bacterial group may occur in water due to fecal contamination, that is discharge of fecal by human and other animal in water. Colliforms
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includes the member of the family enterobacteriaceae , eg:Escherichia colli(E.colli). Enterobacter aerogenes ,Salmonela and klebsiella .These enteropathogenic bacteria in water are responsible for variety of diseases like Collera ,Typhoid,Dysenteries,Bacillary dysentery,etc in human and live stock(Ashbolt,2004) .The fecal indicator bacterium (E.colli) has been considered as a bioindicator of fecal contamination of drinking water. It is excreted in the feaces of all warm-blooded animal and some reptiles (Enriquez et al. 2001).The major pathogenic bacteria responsible for water-borne diseases are spread by the fecal-oral rout in which water may play an intermediate role. The public health burden is determined by the severity of the illnesses associated with pathogen , their infectivity and population exposed. There has ,therefore been an increasing interest in the application of quantitative risk assessment for microbial load in drinking water source. Water is more suitable habitat for microbial growth . Water is necessary for microbial metabolism. These microorganisms are able to grow atlow nutrient concentration. Most aquatic microbes are motile by means of either flagella or other mechanism. Water quality is the physical, biological and chemical characteristics of water. It is measure of condition of water relative to the requirements of one or more biotic species and or to any human need or purpose . It is most frequently used by reference to a set of standards against which complains can be assessed. The most common standard used to assesses water quality relate to health of ecosystem and safety of human contact and drinking water.

Physical properties of water:Each fresh water body has an individual pattern of physical and chemical characterstics which are determined largely by the climatic
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,geomorphological and geochemical condition prevailing in the drainage basin and the underlying aquifer . Summary characterstics, such as total dissolve solids ,conductivity ,and redox potential ,provide a general classification of water bodies of a similar nature .Mineral content determined by the total dissolve solids present ,is an essential feature of the quality of any water body resulting from the balance between dissolution and precipitation oxygen content is another vital feature of any water body because it greating influences the solubility of metals and its essential for all forms of biological life.

Chemical properties of water:The chemical quality of the aquatic environment where is according to local geology , the climate ,the distance from the ocean and the amount of soil cover ,etc.If surface water were totally unaffected by human activities up to 90 to 99% of global fresh water ,depending on the variable of interest would have natural chemical concentration suitable for aquatic life and human uses .Rare(between 1and 10%and between 90 and 99% of the global distribution)and very rare (<1%and >99%of the glbal distribution) chemicall conditions in fresh water , such as in occur in salt lakes, hydrothermal water ,acid ,volcanic lakes ,peat bogs ,etc., usually make the water unsafe for human use. None the less, a range of aquatic organism have adapted to these extereme environment. In many region ground water concentration of total dissolve salts , fluoride , arsenic , etc., may also naturally exceed maximum allowable concentration(MAC) . Particulate matter (PM) is key factor in water quality , regulating adsorption desorption processes . These processes depends on : The amount of PM in contact with a unit water volume .

Bacterial Diversity

The type and character of the PM ( eg:-whether organic or inorganic) The contact time between the water and PM. The time variability of dissolved and particulate matter content in water bodies results mainly from the interaction between hydrodynamic variability, mineral solubility ,PM characterstics and the nature and intensity of biological activity.

Biological characterstics:The development of biota (flora and fauna ) in surface waters is governed by a variety of environmental conditions which determined the selection of species as well as the physiological performance of individual organisms. The primary production of organic matter ,in the form of phytoplankton and macrophytes,is most intensive in lakes and reservoirs and usually more limited in rivers.The degradation of organic substances and the associated bacterial production can be a long term process which can be important in ground water and deep lake water which are not directly exposed to sunlight. In contrast to the chemical quality of water bodies which can be measured by suitable analytical methods , the description of the biological quality of a water body is a combination of qualitative and quantitative characterization. Biological monitoring can generally be carried out 2 different levels: The response of individual species to changes in their environment .

Bacterial Diversity

The response of biological community to change in their environment Water quality classification system based upon biological characterstics have been developed for various water bodies .Where the chemical analysis of selected of selected species (eg:-mussels and aquatic mosses) and /or selected body tissues (eg :- muscle or liver) for contaminants can be considered as a combination of chemical and biological monitory. Biological quality , including the chemical analysis of biota ,has a much longer time dimention then the chemical quality of water since biota can be effected by chemaical and /or hydrological events that may have lasted only a fue days , some months or even years before the monitoring was carried out.

Water microorganism:Water microbiology is concerned with the microorganisms that livein water, or can be transported from one habitat to another by water. Water can support the growth of many types of microorganisms. This can be advantageous. For example, the chemical activities of certain strains of yeasts provide us with beer and bread. As well, the growth of some bacteria in contaminated water can help digest the poisons from the water. However, the presence of other disease causing microbes in water is unhealthy and even life threatening. For example, bacteria that live in the intestinal tracts of humans and other warm blooded animals, such as Escherichia coli, Salmonella, Shigella, and Vibrio, can contaminate water if feces enters the water. Contamination of drinking water with a type of Escherichia coli known as O157:H7 can be fatal. The contamination of the municipal water supply of
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Walkerton, Ontario, Canada in the summer of 2000 by strain O157:H7 sickened 2,000 people and killed seven people.The intestinal tract of warm-blooded animals also contains viruses that can contaminate water and cause disease. Examples include rotavirus, enteroviruses, and coxsackievirus.Another group of microbes of concern in water microbiology are protozoa. The two protozoa of the most concern are Giardia and Cryptosporidium. They live normally in the intestinal tract of animals such as beaver and deer. Giardia and Cryptosporidium form dormant and hardy forms called cysts during their life cycles. The cyst forms are resistant to chlorine, which is the most popular form of inking water disinfection, and can pass through the filters used in many water treatment plants. If ingested in drinking water they can cause debilitating and prolonged diarrhea in humans, and can be life threatening to those people with impaired immune systems. Cryptosporidium contamination of the Drinking water of Milwaukee, Wisconsin within 1993 sickened more than 400,000 people and killed 47 people. Many microorganisms are found naturally in fresh and saltwater. These include bacteria, cyanobacteria, protozoa, algae, and tiny animals such as rotifers. These can be important in the food chain that forms the basis of life in the water. For example, the microbes called cyanobacteria can convert the energy of the sun into the energy it needs to live. The plentiful numbers of these organisms in turn are used as food for other life. The algae that thrive in water is also an important food source for other forms of life. A variety of microorganisms live in fresh water. The region of a water body near the shoreline (the littoral zone) is well lighted,

Bacterial Diversity

shallow, and warmer than other regions of the water. Photosynthetic algae and bacteria that use light as energy thrive in this zone. Further away from the shore is the limnitic zone. Photosynthetic microbes also live here. As the water deepens, temperatures become colder and the oxygen concentration and light in the water decrease. Now, microbes that require oxygen do not thrive. Instead, purple and green sulfur bacteria, which can grow without oxygen, dominate. Finally, at the bottom of fresh waters (the benthic zone), few microbes survive. Bacteria that can survive in the absence of oxygen and sunlight, such as methane producing bacteria, thrive. Saltwater presents a different environment to microorganisms. The higher salt concentration, higher pH, and lower nutrients, relative to freshwater, are lethal to many microorganisms. But, salt loving (halophilic) bacteria abound near the surface, and some bacteria that also live in freshwater are plentiful (i.e., Pseudomonas andVibrio). Also, in 2001, researchers demonstrated that the ancient form of microbial life known as archaebacteria is one of the dominant forms of life in the ocean. The role of archaebacteria in the ocean food chain is not yet known, but must be of vital importance. Another microorganism found in saltwater are a type of algae known as dinoflagellelates. The rapid growth and multiplication of dinoflagellates can turn the water red. This "red tide" depletes the water of nutrients and oxygen, which can cause many fish to die. As well, humans can become ill by eating contaminated fish. Water can also be an ideal means of transporting microorganisms from one place to another. For example, the water that is carried in the hulls of ships to stabilize the vessels during their ocean voyages
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is now known to be a means of transporting microorganisms around the globe. One of these organisms, a bacterium called Vibrio

cholerae, causes life threatening diarrhea in humans.

Drinking water is usually treated to minimize the risk of microbial contamination. The importance of drinking water treatment has been known for centuries. For example, in preChristian times the storage of drinking water in jugs made of metal was practiced. Now, the anti-batreatment cterial effect of some metals is known. Similarly, the boiling of drinking water, as a means of protection of water has long been known. Chemicals such as chlorine or chlorine derivatives has been a popular means of killing bacteria such as Escherichia coli in water since the early decades of the twentieth century. Other bacteria-killing treatments that are increasingly becoming popular include the use of a gas called ozone and the disabling of the microbe's genetic material by the use of ultraviolet light. Microbes can also be physically excluded form the water by passing the water through a filter. Modern filters have holes in them that are so tiny that even particles as miniscule as viruses can be trapped. An important aspect of water microbiology, particularly for drinking water, is the testing of the water to ensure that it is safe to drink. Water quality testing can de done in several ways. One popular test measures the turbidity of the water. Turbidity gives an indication of the amount of suspended material in the water. Typically, if material such as soil is present in the water then microorganisms will also be present. The presence of particles even as small as bacteria and viruses can decrease the clarity of the water. Turbidity is a quick way of indicating if water quality is

Bacterial Diversity

deteriorating, and so if action should be taken to correct the water problem. In many countries, water microbiology is also the subject of legislation. Regulations specify how often water sources are sampled, how the sampling is done, how the analysis will be performed, what microbes are detected, and the acceptable limits for the target microorganisms in the water sample. Testing for microbes that cause disease (i.e., Salmonella typhymurium and Vibrio cholerae) can be expensive and, if the bacteria are present in low numbers, they may escape detection. Instead, other more numerous bacteria provide an indication of fecal pollution of the water. Escherichia coli has been used as an indicator of fecal pollution for decades. The bacterium is present in the intestinal tract in huge numbers, and is more numerous than the disease-causing bacteria and viruses. The chances of detecting Escherichia coli is better than detecting the actual disease causing microorganisms. Escherichia coli also had the advantage of not being capable of growing and reproducing in the water (except in the warm and food-laden waters of tropical countries). Thus, the presence of the bacterium in water is indicative of recent fecal pollution. Finally, Escherichia coli can be detected easily and inexpensively.

Resources Books Chapelle, F.H. Ground Water Microbiology and Geochemistry. New York: John Wiley & Sons, 2000.

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Madigan, M.M., J. Martinko, and J. Parker. Brock Biology of Microorganisms. 8th ed Upper Saddle River, NJ: Prentice-Hall, 2000. Periodicals Karner, M.B., E.F. DeLong, and D.M. Karl. "Archae Dominance in the Mesopelagic Zone of the Pacific Ocean." Nature 409 (January 2001): 507510. Ruiz, G.M., T.K. Rawlings, F.C. Dobbs, et al. "Global Spread of Microorganisms by Ships." Nature 406 (November 2000): 49.

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Review of literature

1. The united states has large number of impoundments which provide flood control and serve as water supply and recreational sites for million of inducers. Each of these impoundment possesses unique and complex hydrologic and water quality characteristics. Impoundments

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are subject to contamination from multiple sources including industrial and municipal discharges, agricultural, rural and urban run off,septic tanks,recreational user discharges and natural processes. These sources introduce chemicals, fertilizers, and fecal wastes which add microorganism and alter physicochemistry of impoundment, there by altering the natural microbial makeup of aquatic system. 2. Many microorganisms fond in run of, discharges, and impoundments are pathogenic to human, fish and wild life. During the period from 1971 to 1978, 2to 4 water born diseases outbreaks were reported in the united state, resulting in to deaths and 48,193 illnesses. Of the illnesses reported 11,435 occurred in 1978(Craun 1980 and Haley et al. 1980). In most of the outbreaks the waters were contaminated with chemicals or pathogenic microorganisms with drinking water being epidemiologically implicated as the source of illness. Table 1 lists the etiological agents identified in outbreaks from 1946 through 1978. All reported water born illnesses are not linked to injection of contaminated water and many cases are not reported at all, thus the true number of illnesses due to water born pathogens is provably underestimated. Most reported out breaks occur during December month (Craun 1978). 3. A summary of water born infectious diseases which may occur in north American impoundments is given in Table 2 . The list include agent swhich produce disease from water contact activities (eg. Swimming, boating, fishing) as well as drinking water , it serves as a general guide to potential water bordisease transmission in reservoir . 4. The constant potential for outbreaks of water borne disease from water supplies makes routine monitoring for contamination necessary. However , attempting to assess the occurance of microbial pathogen in aquatic systems is very difficult due to a
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multitude of factor. Because low level of pathogen However,attempting to assess the occurrence of microbial pathogens in aquaticsystems is very difficult due to a multitude of factors. Because low levels of pathogens may be present, it is necessary to monitor water supplies using indicator" organisms. Indicator organisms are easier to identify than pathogens because they occur in higher numbers. The basic assumption of this approach is that the presence of indicator organisms is Associated with the presenceof pathogens. Criteria (McFeters et a1. 1978) for an ideal indicator are as follows: a) An indicator should be applicable to all types of waters subject `to investigation. b) Microorganisms used as indicators should be present in greater numbers than the pathogen in all cases where the latter is found. c) Numbers of any indicator microorganism should not increase significantly in the absence of a health hazard. d) Indicator microorganisms should be more resistant to the physiologica stress within aquatic environments, hence exhibit greater survival, than pathogens. e) Indicator reaction or test data should be unique and characteristic of that microorganism or determination. f) Indicator methodology should be of minimal complexity, rapid, and inexpensive . g) Indicator microorganisms should be harmless to man under usual conditions. h) The indicator or test should be proportional to the health hazard that is present.

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Table 1 Waterborne Illness in the United States (from Haley et al. 1980,Craun 1978,Center for Disease Control 1977,1980, Pipes 1978, Craun and McCabe 1973)
1946-1970 Outbreak
Gastroenteritis** Salmonella typhi Other Salmonella Infectious Hepatitis-A Shigella Entamoeba Hystolytica Enterotoxigenic Escherichia coli Giardia lamblia Leptospira Tularemia Naegleria fowleri Pseudomonas Aeruginosa Schistosome Poliovirus Campylobacter fetus 937 Chemical poisoning

1971-1975 Cases 45255 610 16730 1833 7400 75 88 176 9 6 16 0 60 Outbreak 63 4 2 14 14 1 13 0 12 Cases 17252 222 37 368 2803 1000 5136 0 511

1976 Outbreak 25 0 1 0 2 0 3 0 3 Cases 1469 0 750 1 2175 0 639 0 35

1977 Outbreak 19 0 2 0 1 0 4 0 6 Cases 0 0 0 0 -

1978* Outbreak 16 0 2 0 4 1 4 3 4 1 1 3 2 Cases 2014 0 89 0 178 5171 3 256 30 3000 937 46

178 53 13 53 33 5 4 3 1 2 1 0 12

*In 1978, 55% of the outbreaks were of known atiology, 11% were caused by Giardia, 10% by chemicals,9 % by shigella,8% by virus, and 5 % by salmonella. **Agent Unknown

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Table 2 Agents of Waterborne Diseases (from Pipes 1978)

Bacterial Shigellosis Diarrhea Salmonelliosis Yersiniosis Leptospirosis Typhoid fever Tularemia Melioidosis Otitis externa Pustular dermatitis Folliculitis (dermatitis) Wound infections Legionelliosis* Viral Gastroenteritis . Shigella spp Enterotoxigenic E. coli Campvlobacter fetus spP. Jejuni Salmonella spp. Yersinia enterocolitica Leptospira spp. S. typhi Francisella tularensis Pseudomonas pseudomallei

P. aeruginosa P. aeruginosa
P. foliculitis Aeromonas hydrophila Legionella pneumophila Parvovirus-like agents (e.g. Norwalk) Enteroviruses (e.g. Coxsackie A and B, Polio, Echo) Unknown Hepatitis A Poliovirus Entamoeba hystolytica Giardi a 1ambli a Naegleria fowleri &Acanthamoeba Ascaris lumbricoides Trichuris trichura Balantidium coli Isopora spp. Schistosomes

Hepatitis Poliomyletis Parasitic Amebic dysentery Giardiasis Primary Amebic Meningoencephalitis Ascariosis Trichuriosis Balantidial dysentery Coccidiosis Swimmer sitch
I

*No reported cases reported case of waterborne infection.

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5. The traditional indicator group for microbial pathogens has been the coliform bacteria. As a result of the incorporation of coliform standards into Federal criteria and state water quality standards, continued use of coliform tests is required, despite the numerous shortcomings of these tests as indicators of microbial pathogens. 6. Not only are coliform bacteria inadequate as indicators, there are problems monitoring their presence in aquatic systems. Isolated "grab" samples from impoundments frequently result in data on coliform numbers which are essentially meaningless. In recent years many of the problems and shortcomings associated with using coliform bacteria as indicators for microbial pathogens in aquatic systems have been identified and alternatives suggested. These problems, alternate indicators, and appropriate sampling methods for impoundments will be discussed in the following sections. 7. As a result of the widespread presence of "total coliform" bacteria in nature, the use of the coliform group as an indicator is generally discouraged, except in finished drinking water Presence of coliform bacteria in drinking water has greater meaning since it indicates inadequate treatment. Fecal coliforms are those bacteria which are gram negative, asporogenous rods and produce gas from lactose at an incubation temperature of 44.50C (Dufour 1978). The presence of fecal coliforms in water suggests either animal or human wastes have contaminated the system and their associated pathogens may also be present (McKee and Wolf 1963, Moore 1959). Estimates of the percentages of microbial flora in human feces are listed in Table 3 and the percentages of warmblooded animals excreting enteric pathogens in Table 4. 8. Development of indicator standards, prediction of risk of waterborne disease, and assessment of pathogen levels requires knowledge of an indicator-pathogen relationship. For a given

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concentration of indicator organisms, there should be a related concentration of pathogens under a known set of conditions. This hypothesis is based on the assumption that there are relatively constant levels of pathogens present in sewage which usually seems to be the case in large municipal sewage systems; but as the number of individuals who contribute to fecal wastes becomes smaller, the indicatorto-pathogen ratio variance increases. So a waste discharge into a lake from a healthy recreational user may be completely free of pathogens, or it may contain a high density of virulent pathogens if the user is infected (Cabelli 1979). Nearly 40 years ago it was suggested that a ratio of 3 to 120 of Salmonella ~ per million coliforms existed for a typhoid rate of 0.01/1000 to 30/1000 population per year (Kehr &Butterfield 1943). If one assumes one person per 100,000 individuals excretes Salmonella typhi, there would be roughly one pathogen per litre of sewage (Pipes 1978). Correlation between fecal coliform (FC) densities and the presence of Salmonella in recreational waters has been reported (Geldreich 1970, Bonde 1977, Smith et al.1973, Smith and Twedt 1971, Dutka 1973). Geldreich (1970) reported isolating Salmonella in approximately 30% of samples with less than 200 FC/100 mL but in more than 98% when the density was greater than 2000 FC/100 mL. Bonde (1977) reported Salmonella usually occurred at concentrations of I. coli greater than 1000/100 mL. Many times, however, when the actual density of Salmonella is examined, no correlation exists (Cabelli 1976). Several studies have discounted the coliformpathogen relationship (Smith et al. 1973, Smith and Twedt 1971, Dutka 1973, Gallagher and Spino 1968). Enteric pathogens have been found when low levels of indicator bacteria were present (Dutka 1973, Muller 1964, Colberg et al. 1974, Kraus 1977). Moreover, no studies have shown FC to indicate the presence of fecal pathogens such as Yersinia enterocolitica and Campylobacter fetus spp. JeJuni.

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Table 3 Estimated Percentages of Microbial Flora in human Feces (from geldrich et. Al 1978 and cabelli 1979)

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enterocolitica and other srecies have been implicated in disease from waters which have been found to be relatively free from FC

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(Schieman 1978, Ghirelli and Marker 1977). Small mammals, snails, poultry, and migratory waterfowl have been shown to be reservoirs of Yersinia and Campylobacter and are partially responsible for contaminating water (Botzler et al. 1976, Hacking and Sileo 1974, Kapperud 1975, Luechtefeld et al. 1980, Center for Disease Control 1979a). 9. Fecal coliforms do not serve as indicators of bacterial pathogens which are ubiquitous in aquatic environments. Pseudomonas eruginosa, Aeromonas hydrophila, and Klebsiella spp. are naturally occurring aquatic bacteria which have been implicated in disease. These organisms usually do not pose a health threat unless present in high numbers, yet they are frequently present in conjunction with fecal pollution (Cabelli 1980b). Protozoa and Viruses 10. Fecal coliform indicator validity is especially tenuous in predicting health hazards resulting from presence of pathogenic protozoa and viruses. Recent studies have demonstrated the existence of enteric viruses in waters containing low levels of fecal coliforms (Gerba 1980). No normal viral flora exists in humans, and any relationship between indicators and virus density is hampered by the following: (a) only a small percentage of the population may harbor and excrete enteric viruses, (b) intestinal infection and excretion of viruses is transient, (c) most cases are subclinical, (d) excretion of viruses is subject to seasonal variation, (e)no methods are available for the in vitro cultivation and quantitation of many viral pathogens, and (f) techniques for isolation and quantitation of the hundreds of viruses are primitive with poor recovery rates (Pipes 1978).

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Virus-to-coliform ratios have been established in one study (U.S.Environmental Protection Agency 1975) as follows: feces 1:65,000 polluted water 1:50,000 sewage 1:92,000 Bonde (1977) reported higher ratios of f. coli:virus when f. coli densities were greater than 1000 organisms per 100 mL. Large variations in the numbers of coliphage in sewage and in the coliphage:coliform bacteria ratio have been reported (Pretorius 1962). These ratios obviously change with environments and time; furthermore, improved isolation techniques for viruses In recent years have raised doubt about previously reported ratios (Mechalas et al. 1972). Several studies have demonstrated that no consistent virus: coliform ratio exists (Gerba 1980, Goyal et al. 1977,1979, Berg 1976a, Duma 1980); for example enteroviruses were detected 44% of the time in recreational waters considered acceptable as judged by FC criteria (Gerba 1980). 11. In the South, Naegleria and Acanthamoeba have been found to bubiquitous in aquatic systems. Relatively high densities have been reported in warm water such as thermal discharges from power plants and hot springs (Duma 1980, Stevens et al. 1977, Wellings 1977, O'Dell 1979). These pathogens have not been associated with fecal pollution; therefore, use of the FC as an indicator of their presence is inaccurate. 12. Another protozoal pathogen, Giardia lamblia, has been reported with increasing frequency as an etiologic agent in waterborne disease (Jakubowski and Hoff 1979). It is found in waters which are relatively free of FC (Craun 1979); moreover, its ability to encyst allows extended survival, thus preventing the use of FC as an

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indicator (Craun and McCabe 1973, Craun 1976, Rendtorff and Holt 1954, Rendtorff 1954). Presence of Sediments 13. The relationship of FC densities in the water column to pathogen densities in the sediments is also unclear. Sediment has been shown to harbor much higher numbers of bacteria, viruses, and protozoa, both pathogenic and nonpathogenic (Goyal et al. 1977, Grimes 1975 &1980, Hendricks 1971, LaBelle et al. 1980, Rittenberg et al. 1958, Van Donsel &Geldreich 1971, Gerba &Goyal 1978, Smith et al. 1978, U.S. Public Health Service 1965, Goyal et al. 1978, Metcalf &Stiles 1968, Slanetz et al. 1965, DeFlora et al. 1975, MatsGn et al. 1978, LaBelle &Gerba 1979, Farmer 1980, Gerba et al. 1977,Winslow 1976). When large numbers of enteric organisms are present in the sediment, a relationship exists between their numbers and the degree of contamination of the overlying water (Allen et al. 1953). Nearshore sediments at reservoir swimming areas have shown Fe concentrations as high as 48,000/100 cc (Winslow 1976). Van Donsel and Geldreich (1971) reported that a minimum of 150 FC/100 mL in the water was required for Salmonella to be present in the Sediment. In their studies, they sampled various streams and lakes and found 100-to 1000-fold more FC in the sediments than in overlying waters. They recovered Salmonella spp. from 46% of their sediment samples, whereas only 8% of the overlying waters contained this pathogen (Van Donsel and Geldreich 1971). Similar ratios of FC in sediment and water were found in the Mississippi River (Grimes 1980). Goyal et al. (1977) reported 47% of the sediment samples compared to 3% of the overlying water samples to be positive for Salmonella spp. Hendricks (1971) recovered 90% more Salmonella spp. from sediments, than from overlying waters. As a consequence of the great variability of sediments, it usually is not

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possible to establish a correlation between FC levels in the sediment and overlying waters (Winslow 1976). 14. There is no doubt that the present indicator system works to some degree. Fecal coliforms do indicate fecal pollution, and the present acceptable levels are so low that infection by fecal bacterial pathogens is unlikely. However, there are still insufficient data to support a direct,consistent relationship between indicator bacteria, pathogen concentration,and infection. It may be concluded that as indicator density increases there is a deterioration of water quality, but not necessarily an increase in health hazards (Pipes 1978). Die-Off Rates 15. Because of the tenuous relationship between indicator organisms and pathogens, it may be feasible to develop an indicator index based on survival as a function of exposure time in water. Such an index could relate a numerical survival ratio for a pathogen to an indicator survival value once pathogen survival data for different aquatic environments were known. If infective dose levels were known for each pathogen, then quantitative risk could be assigned to recreational activities. At the present,the data for such a system are inadequate (Mechalas et al. 1972, Andre et al. 1967, Geldreich et al. 1968, Klock 1971, Gordon 1972, Moore 1971,Rudolphs et al. 1950, Gyllenberg et al. 1960). 16. Numerous studies have measured the survival rates of fecal coliformand pathogenic bacteria in water (Carter et al. 1967). Enteric organismsare physiologically adapted to the nutrient-rich environment of warmblooded animal intestines. When placed in dilute, nutrient-poor, aquatic systems they become stressed and die (Bissonnette 1975). Factors affecting their rates of survival are many; with sunlight, nutrient availability, pH, and temperature, presence of protozoa, phage, and toxins, and other physicochemical

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factors predominating (Faust et al. 1975, Gameson and Saxon 1967, Van Donsel et al. 1967, Mitchell 1967). Since such a multitude of environmental variables influences survival, die-off varies as much between aquatic systems as do the variables. In general enteric bacteria die in fresh water within three days (Rudolphs 1950). Attempts at determining whether certain types of bacteria such as f. coli survive as long as pathogens such as Salmonella have produced conflicting results. The majority of studies have shown f. coli to be a good indicator because it survives as long or longer than Salmonella spp. (Geldreich 1970, Geldreich et al. 1968, Rudolphs et al. 1950, Mitchell and Starzyk 1975, Orlob 1956), while others have reported the opposite (Gudding and Krogstad 1975, McFeters et al. 1974, Vasconcelos and Swartz 1976). The inconsistency of these findings can be attributed to varied strain characteristics and different methodologies and, more importantly, to unknown environmental variables. One study (McFeters et al. 1974) comparing die-offs of fecal indicator bacteria and enteric pathogens reported that 50% reductions in initial population required an average of 17 hours for coliforms and from 2.4 to 19.2 hours for Salmonella spp. These survival rates are somewhat lower than die-off in marine waters (Chamberlin and Mitchell 1978). Chamberlin and Mitchell (1978) compared numerous freshwater and seawater investigations of coliform survival and calculated average dieoff rates, assuming that first order decay followed the relationship known as Chick1s law; i.e., dB/dt = -KB, where B is the bacterial density at time t, K is the die-off rate, and d is the difference from beginning to end. In fresh water, rates of decrease averaged 0.015 to 0.02/hr, while a rate of l/hr was found in seawater. Their studies indicated sunlight was the most important factor contributing to die-off. Thornton et al. (1970) measured coliform densities associated with turbidity when

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storm flows were tracked into a reservoir. The authors attributed die-off to be most closely associated with water temperature. The model developed predicted relatively well disappearance of coliforms associated with storm flow. 17. Die-off of other pathogens varies considerably from the FC survival.Survival of FC may not be representative of other pathogens because FC and Salmonella have the most rapid die-off of all microorganisms of health significance tested (McFeters and Stuart 1972). r. enterocolitica, a pathogenic bacterium, survives for long periods in cold waters which are low in nutrients (Shillinger and McFeters 1978). High densities of heterotrophic and coliform bacteria are inhibitory to Yersinia spp. survival, as they are also to survival of Shigella, Leptospira, and enteric viruses (Schieman 1978, Highsmith et al. 1977, Geldreich 1972). Aeromonas hydrophila, ~.aeruginosa, h. pneumophila, and ~. fowleri are not only pathogens, but are also naturally occurring aquatic organisms with indefinite survival. Giardia, parasitic ova, and enteric viruses survive adverse conditions such as water treatment much better than the coliform indicators (Craun and McCabe 1973, Craun 1976, Rendtorff and Holt 1954, Rendtorff 1954, Berg 1973, Malina 1976, lin et al. 1971, Mack et al. 1972, Petrilli et al. 1974, Nestor and Coston 1976, Berg 1973). This characteristic of greater persistence and the fact that, in contrast to bacterial pathogens, very low numbers of organisms may cause infection when ingested makes the occurrence of these organisms critical (Pipes 1978, Mechalas et al. 1972, Rendtorff 1979, Center for Disease Control 1979b, Melnick 1976). Viruses may persist for several weeks to months in coldwater environments (Hill et al. 1971, Katzenelson 1978). One study showed survival of viruses was closely related to temperature: fewer than 5 days at 370 C, 2.5 to 9 days at 22-250 C, and 40 to 90 days at 3-50C (Bitto~ 1978).

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These survival periods are much longer than the periods for coliform indicators.Kott (1981) reported that enteroviruses survive twice as long as FC in secondary wastewater; however, I. coli B die-off was similar to that of the viruses. Viruses, like bacteria, vary in their ability to survive in waters, therefore adding another complicating factor in determining water quality (Colwell and Foster 1980, Metcalf 1971). 18. Sediments greatly extend the survival of most microbial organisms of health significance, as indicated by the much higher organism numbers reported (Winslow 1976, Horak 1974). Studies of survival in sediments are few (Van Donsel and Geldreich 1971, Gerba and McLeod 1976, LaBelle and Gerba 1982, Laliberte and Grimes 1982, Schaub et al. 1974, Roper and Marshall 1978). Van Donsel and Geldreich (1971) reported a 90% die-off in seven days of both FC and Salmonella spp. in various sediments. Studies by the authors comparing survival of ~. newport, I. coli, ~. aeruginosa, and ~. Pneumoniae in five different sediments showed I. coli and Salmonella to have comparable die-off rates of 2 to 5 logarithms in 2 weeks, whereas ~. aeruginosa and ~.pneumoniae decreased only 1 to 2 orders of magnitude. At initial concentrations of 108 colony-forming units per milliliter (CFU/mL) such as are found in sewage, these pathogens could survive in the sediments for months. This increased survival partially accounts for the high numbers of indicators and pathogens in sediments.Other Indicator Organisms 19. Several other bacteria have been proposed as indicator organisms for microbial pathogens. These include I. coli, fecal streptococci, enterococci, total bacteria, ~. perfringens, ~. pneumoniae, Aeromonas, and Pseudomonas (McFeters et al. 1978, Cabelli 1979). 20. I. coli has been used in many European countries as the primary fecal indicator organism. It has the advantage of being specific for

27

Bacterial Diversity

warmblooded animals and is not found in nature as are some fecal coliforms such as Klebsiella, Enterobacter, and Citrobacter. For this reason it serves as a better indicator of recent fecal pollution. In a 3-year epidemiological study, the occurrence of I. coli was shown to correlate better to incidences of waterborne illness than FC, fecal streptococci, total coliforms, Aeromonas, ~.aeruginosa, Klebsiella, and Enterobacter-Arthrobacter (Geldreich et al. 1978, Ktsanes et al. 1981, Cabell i et al. 1976). 21. There was originally no scientific justification for using fecal coliforms rather than -E. -co-li as indicators other than that facile (easily accomplished) methods for enumerating I. coli were not available. However, now accurate, facile methods do exist which are specific for I. coli (Cabelli 1979, Dufour et al. 1981). 22. Another widely used indicator has been the fecal streptococci (FS). This includes ~. faecal is, ~. faecium, ~. bovis, ~. eguinus, and ~. avium (Bordner and Winter 1978). However, some species of Streptococcus are capable of reproducing outside of warmblooded animals and are widespread in nature (Mundt 1962a, 1962b), which violates one essential requirement of indicators. When FS information is combined with FC data to provide a factor known as the FC/FS ratio, sources of contaminants are more clearly identified (Galvani 1974, Geldreich and Kenner 1969). FC/FS ratios of 4 or greater indicate human feces, whereas ratios of less than 0.7 are indicative of animal wastes (Geldreich and Kenner 1969, Geldreich et al. 1980). However, due to differing die-off rates, dilution, and the occurrence of background coliforms levels, these ratios are often useless in determining animal or human origin unless derived from the immediate source within a few hours of pollution (Cabelli 1980b, Geldreich and Kenner 1969). Some studies have shown FC to survive longer than FS, but most report longer survival of FS (Pipes 1978, McFeters et al. 1974, Geldreich et al. 1980). This would cause the

28

Bacterial Diversity

FC/FS ratio to decrease with time, thus attributing the pollution to animals. Some lower animals have an intestinal flora with high FC/FS ratios similar to humans, which adds to the problems of using such a ratio (Wheater et al. 1979, Thomas and Levin 1978). 23. Enterococci are a subgroup of the FS and are more specific for human wastes (Bordner and Winter 1978). Enterococci have been observed to survive longer than fecal streptococci (McFeters et al. 1974, Geldreich et al. 1980). They have been reported to be an adequate indicator for organisms that have a longer survival, such as viruses. Studies by Cabelli (1979, 1980b) and Cabelli et al. (1974) of lake beaches have shown numbers of enterococci to correlate highly with waterborne illness and be better indicators thanI. coli, FC, or others. Their use as an indicator does not account for possible input of pathogens from animals. 24. Clostridium perfringens has been proposed by some as an indicator organism (Bonde 1963). The occurrence has correlated well with other indicators.Due to its ability to form spores under stressful conditions,it is a good indicator when disinfectants (water treatment) are present and when water samples cannot be analyzed quickly. Its presence in water or sediments does not necessarily indicate recent pollution, due to this same spore-forming characteristic; however, information concerning the occurrence of past pollution is often desireable. On the other hand, for water quality maintenance and enforcement information, it is essential to know if pollution was recent. Two additional factors which limit the usefulness of this organism as an indicator are its requirement of anaerobic conditions for growth and its widespread occurrence in nature (McFeters et al. 1978, Bonde 1963). 25. In recent years, the use of K. pneumoniae as an indicator of sanitary significance has frequently been challenged. Although it is associated withI. coli in fecal material and is an opportunistic

29

Bacterial Diversity

pathogen, it has been isolated in many natural sources and is capable of proliferating in water (McFeters et al 1978, Menon and Bedford 1973, Seidler et al. 1975, Dufour and Cabelli 1976). Its presence as a constituent of the FC population contributes to the shortcomings of FC as an indicator (Cabelli 1980b). It is, however, a good indicator of organic pollution, being found in high numbers in textile, paper, and pulp mills, beet processing, and other wastes (Bordner and Winter 1978). 26. Two other bacteria have recently been proposed as indicators, Aeromonas and t. aeruginosa (McFeters et al. 1978). There has, however, been much controversy concerning their significance as indicator organisms (Colwell and Foster 1980). Both organisms are widespread in nature and are capable of reproducing in water, thus maintaining relatively stable populations (Carson et al. 1973, Fliermans et al. 1977, Nemedi and Layni 1971). ~. hydrophila is often a good indicator of nutrient loading (including sewage), thus its densities are associated with water quality and serve as an index of the trophic state of a water body (Cabelli 1980b, Cabelli et al. 1974, Rippey and Cabelli 1980). Other studies have shown good correlations with conductivity, redox potentials, and temperature but question use of this organism as a trophic-level indicator (Colwell and Foster 1980). It has a seasonal distribution, being present in high numbers only during warmer

30

Bacterial Diversity

months unless thermal discharges are present (Colwell and Foster 1980, Straskrabova 1974). As with many bacteria, densities in the sediment are elevated and remain relatively stable throughout the year. t. aeruginosa is also frequently present in high numbers in sewage (Wheater et al. 1979, Miescier 1977, Dutka 1979). Cabelli et al. (1976) suggest P. aeruginosatoFC ratios greater than 20 indicate the source is not of fecal origin. 27. The use of the yeast, Candida albicans, as an indicator has been gaining acceptance in recent years (Dutka 1979, Buck 1977). It is a known pathogen, and its occurrence is apparently directly related to man's activities. It can be isolated from the mouth, throat, skin, and feces of normal individuals; but it is not a good fecal indicator because only about 18% of the population has f. albicans present in their feces (Cabelli et al. 1976). Survival in fresh waters is relatively longer than most enteric bacteria, often lasting several weeks (Cabelli et al. 1976). Its presence in uncontaminated water is rare. Beaches with high FC counts produced samples containing ~. albicans counts of 20-25/1itre, while low FC areas had counts of 0-2/1itre (Buck 1977). 28. Two absolute indicators of fecal pollution which have potential

31

Bacterial Diversity

for use are numbers of bifidobacteria and the fecal sterol, coprostanol. Bifidobacteria are anaerobic organisms found only at high densities in feces of humans and higher animals and have survival characteristics similar toI. coli. Their use as water quality indicators has been suggested by a few authors, but further verification of enumeration procedures and natural sources is needed (Evison and James 1975, Levin 1977). Coprostanol is a fecal component that has several traits which make it an ideal chemical indicator of fecal pollution; however, it's identification requires complex laboratory methods which prevent its use at present (Colwell and Foster 1980, Smith and Gouron 1969, Dutka et al. 1974, Dutka and El-Shaarawi 1975, Murtaugh and Bunch 1967). 29. Perhaps the most important organisms of sanitary significance are the viruses, yet they are the most difficult to detect. No facile method has been developed for identifying virus indicators, and much disagreement and uncertainty exists regarding proper indicators for viruses of fecal origin (Carson et al. 1973). There is agreement, however, that a significant

32

Bacterial Diversity

number of waterborne cases of gastroenteritis are probably caused by enteroviruses (Pipes 1978). Relatively involved isolation methods have implicated viruses directly and indirectly in waterborne outbreaks of illness (Pipes 1978). There has been much discussion on the use of coliphage as a virus indicator (Kott 1981, Colwell and Foster 1980, Leahy et al. 1980). It has been suggested by Kott et al. (1976) that the f2 coliphage was a good indicator for human enteroviruses and this was supported by studies on the frequency of these viruses in sewage treatment. There are several characteristics of coliphage which qualify its use as an indicator: (a) it is prevalent in sewage at ratios to enterovirusus of 103:1, (b) it can be enumerated within 24 hrs , (c) it is more resistant to chlorine than the enteroviruses tested, (d) it survives as long or longer than enteroviruses in water, and (e) reliable isolation methods exist which are less expensive than most virus isolation methods (Colwell and Foster 1980). However, coliphage does have the following shortcomings as a fecal indicator.

33

Bacterial Diversity

MATERIAL AND METHODS

Material and Method

1. StudySite:-The study site is situated in northern

Rajasthan and Himachal pradesh. The total human population of

34

Bacterial Diversity

northen Rajasthan is about 17.89 million and out of this nearly 72.6 percent in rural areas. Thefirst sample was taken from Sri Ganganagardistrict ,which is under the influence of Great Thar desert .It is bounded on the north by Punjab state ,on east by Hanumangarh district of Rajasthan, on the south by Bikaner district of Rajasthan, and on the west by Pakistan. The annual rainfall of this area is ranged between 250-300mm. The climate of this region is semi arid with extreme temperature condition in summer (up to 45 c) and winter (up to 1c) .The site is influenced by the Indian southern-west or summer monsoon (Juneand during winter (December-February) by September)

anticyclones. The main rainy season is during July and August when almost 80% of the annual rainfall. The annual rainfall was recorded as 253.00mm.Himachal Pradesh is a state in Northern

India. It is spread over 21,495 sq mi (55,670 km2),[4] and is bordered by the Indian states of Jammu and Kashmir on the north, Punjab on the west and south-west, Haryana and Uttar Pradesh on the south,Uttarakhand on the south-east and by the Tibet Autonomous Region on the east. The literal meaning of Himachal Pradesh is In the lap of Himalayas.[5] Himachal Pradesh is known to be abundant in natural beauty[6] After the Anglo Gorkha War, the British colonial government came into power.In 1950 Himachal was declared as a union territory but after the State of Himachal Pradesh Act 1971, Himachal emerged as the 18th state of the Republic of India. Himachal has many prestigious boarding schools. Hima means snow in Sanskrit. It was named by one of the great Sanskrit scholars of Himachal Pradesh, Acharya Diwakar Datt Sharma. Himachal Pradesh has one of the highest per capita incomes of any state in India. Due to the abundance of perennial rivers,

35

Bacterial Diversity

Himachal also sells hydro electricity to other states such as Delhi, Punjab and Rajasthan.[7] The economy of the state is highly dependent on three sources: hydroelectric power, tourism and agriculture.[8][9] Hindus make up 95% of the state population, making it the most Hindu ranked state the (proportionally), second-least in India. state According in the to a 2005 Transparency Internationalsurvey, Himachal Pradesh is corrupt country after Kerala.[10]

A summer view of Khajjiar. Climate Temperature Avg. Winter Summer Precipitation 1,469 mm (57.8 in) Himachal is situated in the western Himalayas. Covering an area of 55,673 kilometres (34,594 mi),[4] Himachal Pradesh is a mountainous state with elevation ranging from about 350 metres (1,148 ft) to 7,000 metres (22,966 ft) above the sea level.[15] The drainage system of Himachal is composed both of rivers and glaciers. Himalayan rivers criss-cross the entire mountain chain. In fact the rivers are older than the mountain system.[16] Himachal Pradesh provides water to both the Indus and Ganges basins.[17] The drainage systems of the region are the Chandra Bhaga or theChenab, the Ravi, the Beas,
36
[citation needed]

7 C (45 F) 28 C (82 F)

Avg.

Bacterial Diversity

the Sutlej and the Yamuna. These rivers are perennial and are fed by snow and rainfall. They are protected by an extensive cover of natural vegetation.[17] There is great variation in the climatic conditions of Himachal due to extreme variation in elevation. The climate varies from hot and sub-humid tropical in the southern tracts to cold, alpine and glacial in the northern and eastern mountain ranges with more elevation.[18] The state has areas like Dharamsala that receive very heavy rainfall, as well as those like Lahaul and Spiti that are cold and almost rainless. Broadly Himachal experience three seasons; hot weather season, cold weather season and rainy season. Summer lasts from mid April till the end of June and most parts become very hot (except in alpine zone which experience mild summer) with the average temperature ranging from 28 C(82 F) to 32 C (90 F). Winter lasts from late November till mid March. Snowfall is common in alpine tracts (generally above 2,200 metres (7,218 ft) i.e. in the Higher and TransHimalayan region).We done the sampling from beas river and satluj river. 2. Collection of water sample:The bacteria were isolated from the sample of water in northern Rajasthan, Srigangangar zone and Himachal Pradesh(Kangra and Hamirpur) . We collect the 3 sample , one from northen Rajasthan(gang canal) , two from Himachal (Beas, satluj river) in the sterile bottles carefully with the standard methods . Collected samples were transported to Department of Microbiology M.D. (P.G.) College, Srigangangar. In laboratory, samples bottles were stored at 4c. The collected samples were used for further microbiological analysis. All bacteriological

37

Bacterial Diversity

analysis of soil sample was done during of September 2011 to 2012 January, by using standard methods. 3. Methodology: 3.1 Enumeration Enumeration of microbial populations was accomplished by Total Viable Count (TVC). It was performed on nutrient agar media by means of serial dilution agar plating method, which is based on the principle that when sample containing microorganisms are plated, each viable microorganism will develop into a colony. 10-6, 10-7 and 10-8 dilutions were used from all the samples. 0.1ml of appropriate diluted suspension was transferred to eachagar petridishes in replicates. Plates were incubated at 370C for 24 hrs.

3.2

Isolation: Isolation was completed by serial dilution method, spreading and pours plate method on agar media. Cultivation was performed with appropriate incubation, temperature and time required for growth. Isolation of pure cultures was completed by the streak plate method on various agar media such as nutrient agar (NA), macConkeys agar and eosin methylene blue. . Further cultivation and biochemical tests were performed and the optimum conditions were maintained as required for the particular reaction. The Gram staining was used on each step after the transfer of a single colony to check the purity of culture.

3.3

Sub culturing:

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Bacterial Diversity

Bacterial isolates were sub cultured in nutrientat regular intervals to maintain viability and metabolic activities. 4. Preservation and Maintenance: Nutrient broth tubes were stored at a temperature of 4 0C which, protects from damage due to evaporation of medium and preserves the cultures. Isolates were maintained in replicates one as the working culture to be used as a source for identification tests and other as stock culture from which new working cultures were prepared whenever required. 5. Primary identification: Primary identification of the bacteria was carried out on the basis of their cultural characteristics on agar plates and microscopic observations, which formed the basis for the selection of biochemical tests to be, performed . 5.1 Colony characteristics: 5.1.1 Filamentous. 5.1.2 Colour: Cream, Pink, Purple, Blue, Yellow. 5.1.3 Margin: Entire, Lobate, Undulate, Erose, Curled. 5.1.4 Elevation: Flat, Convex, Raised, Pulvinate, Umbonate. 6. Secondary identification: Secondary Identification of the bacteria was carried out on the basis of their biochemical characteristics, the detection of which aid in the identification and classification of bacteria those were found morphological identical. Many distinctive enzymatic activities were tested by observing the resulted byproducts or the action of enzymes on specific substrates supplemented in media. Forms: Circular, Punctiform, Irregular, Rhizoid,

39

Bacterial Diversity

6.1

Amylase production test/Starch hydrolysis: Amylase is an exo enzyme that hydrolyses a polysaccharide. The ability to degrade starch is used as a criterion for the determination of amylase production by a microbe. Amylase production is known in some bacteria while well-known in fungi.

6.2

Fermentation of carbohydrates: Fermentative degradation of carbohydrates is carried out in a fermentation tube (a culture tube that contains a Durham tube) under facultative conditions. The fermentation broth contains ingredients of nutrient broth, a specific carbohydrate (lactose) and a pit indicator (phenol red) which is red at neutral pH and turns yellow at or below a pH of 6.8 due to the production of an organic acid.

6.3

Indole production test: Indole is a component of the amino acid tryptophan. Some bacteria have the ability to break down tryptophan for nutritional needs using the enzyme tryptophase into indole, pyruvic acid and ammonia. Tryptophan
tryptophanase

Indole + Pyruvic acid + NH3

The pyruvic acid can be further metabolized to produce large amounts of energy. This ammonia is available for use in synthesis of new amino acid. Indole can be detected by reaction with kovacs reagent (para-dimetheylaminonesaldenude in alcohol) to produce a cherry red compound. Indole + Kovacs 6.4 Citrate utilization test:
HCL

Rosidole + H2O (Butanol)(Cherry-red compound)

40

Bacterial Diversity

The ability to metabolize citrate is useful for differentiating among Enterobacteriaceae. Simmons Citrate Agar is a medium containing citrate as the sole carbon source and ammonium salts as the some nitrogen source. The utilization of citrate depends on the presence of an enzyme citrase produced by the organism that breaks down the citrate to oxaloacetic acid and acetic acid. These products are later converted to pyruvic acid and Co2 enzymatic ally. Citric acid
CITRASE

Oxaloacetic acid + Acetic acid Pyruvic acid + Co2

decarboxylase Co2 generated combines with sodium and water to form sodium carbonate an alkaline product, which changes the colors of the indicator from green to blue and this constitutes a positive test. Co2 + 2 Na+ + H2O Na2CO3 (alkaline pH)(blue colour) Bromothymol blue is used as an indicator which is green when acidic (pH 6.8 and below) and blue when alkaline (pH 7.6 and higher). 6.5 Catalase test: Catalase is an enzyme that splits hydrogen peroxide (H2O2) into water and oxygen. H2O2 is produced as a byproduct of respiration and is lethal if it accumulates in the cell. positive test. 2H2O2CATALASE 6.6 2H2O + O2 The evolution of gas causes bubbles to form and is indicative of a

Methyl-red and voges proskauer test (MR-VP)

41

Bacterial Diversity

The Methyl Red (MR) and the Voges Proskauer (VP) tests are used to differentiate two major types of facultative anaerobic enteric bacteria that produce large amounts of acid and those that produce the neutral product acetoin as end product. Both these are performed simultaneously because they are physiologically related and are performed on the same medium MR-VP broth. The MR-VP broth contains dextrose as the Some bacteria well ferment the After carbohydrate source. pH 5.

dextrose to acid products that will cause the pH to drop below This is called a mixed acid fermentation. incubation, the addition of methyl red, a dye which turns red below pH 4.4, will indicate whether such fermentation has occurred or not. Other bacteria will convert dextrose to less acidic products such as ethanol. These bacteria are negative in the methyl red test. Ethanol fermentation is demonstrated by VP test which measures the presence of acetoin (acetyl methyl carbinol, a precursor to butanedio). This test uses the same medium as the methyl red test. Barritts reagents, alpha-napthol and potassium hydroxide, are added to a 24-48 hour culture and the tube is shaken to aerate the solution. The development of a pink or red colour after agitation is a positive reaction for the production of acetoin.

42

Bacterial Diversity

6.7 Nitrate reduction test:This test is done for the presence of the enzyme nitrate reductase , which causes the reduction of nitrate in the presence of suitable electrone donor to nitrate or free nitrogen gas . A number of bacteria respires under anaerobic conditions by using nitrates , sulphates as a terminal electron acceptor . so there is reduction of nitrate to nitrite with help of reductase enzyme . the end product possibilities of nitrate rediuction is not only nitrite but may be ammonia and nitrogen gas. It depends on the bacterial species .the most common end product from nitrate reduction to nitrite is nitrogen gas .this reduction to end product is called denitrification and can be cheked in a durham tube as gas bubble. NO3+2H++2eNitrate
nitrate reductase

NO2- +H2O Nitrite Water

Hydrogen electron

6.8 Urease test:This is done to determine the ability of organism to split urea, forming ammonia by action of the enzyme urease .The urease is a hydrolytic enzyme which attack the carbon and nitrogen bond amide compound (urea) with the liberation of ammonia . NH2CO.NH2 + H2O UREA
UREASE

2NH3 + CO2 AMMONIA

The presence of this enzyme can be tested by growing the organism in the presence of urea and testing for alkali production by means of suitable pH indicator .The production of ammonia raises the pH of the medium which changes the color of indicator from a yellow color to red or deep pink color. The optimum pH of urease activity is 7.

43

Bacterial Diversity

6.9 Gelatin Liquefaction test:This test is done to determine the ability of an organism to produce proteolytic like enzyme which liquefy gelatin. Naturally occurring proteins are too large to enter a bacterial cell ,so they must be first catabolized into smaller components . The exocellular proteolytic like enzyme gelatinases are secreted by certain bacteria to breakdown certain protein and this ability aids in bacterial identification. 7.0 Tripple sugar iron agar test:TSIA medium is composed of three sugars lactose, sucrose and very small amount of glucose(1%) ,iron and phenol red as a indicator .This indicator is employed for the detection of fermentation of the sugar indicated by change in color of the medium due to production of organic acid and hydrogen sulfide .If organism ferment any of three sugar or any combination of them , the medium will become yellow due to the production of acid as end product of fermentation.

44

Bacterial Diversity

Result & Discussion

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Bacterial Diversity

Result & Discussion

In present study total 30 Isolates were Isolated in which the both type of Gram Negative and Positive bacteria are present (table III & IV). The soil samples of both crops showed the relative higher bacterial count. Total 11 bacterial strain Pseudomonas, E.coli, Bacillus, Klebsiella, Streptococcus, Azospirillum, Azotobacter, Proteus, Rhizobium, Staphylococcus, Enterobacter was most frequently distributed spp. in collected samples. The occurrence of bacterial strains was recorded in mustard and wheat crops shown in table III & IV. The habitat of identified bacterial strain was also shown in table VI. In present studies Table 5 shows that the percentage of bacterial species mustard crop field soil is described below in descending order : Pseudomonas>E.coli>Bacillus>Klebsiella>Streptococcus>Azospirillum> Azotobacter>Proteus>Rhizobium>Staphylococcus>Enterobacterspp. In present studies Table 5 shows that the percentage of bacterial species wheat crop field soil is described below in descending order : E.coli = Bacillus > Pseudomonas = Aztobacter > Azospirillum > Klebsiella > Streptococcus > Proteus > Staphylococcus >Rhizobium >Enterobacter. In mustard and wheat fields the highest amount of identified bacteria was Pseudomonas, Bacillus, E.coli, Azospirillum.The main reason for the presence of highest amount of Pseudomonas, Bacillus, E.coli because these genera responsible for organic matter degradation.Klebsiella, Azotobacter, Rhizobium, Azospirillum, are mainly present due to the nitrogen fixation
46

Bacterial Diversity

mainly in two modes that are symbiotic and asymbiotic. Next bacteria population found in studies Proteus spp., Staphylococcus spp., Enterobacter spp., Streptococcus spp. Because these are mainly Pathogenic and nonpathogenic in terms.

47

Bacterial Diversity

Table- I : Total viable count on nutrient agar of Wheat soil sample

Sr. No. 1 Sample code W1 Media NA Dilution 10 Count 140 67 42 170 51 34 160 72 42 185 75 44 148 52 30 165 77 56 182 61 42 167 56 39 141 47 30 179 86 59 197 91 57 199 91 57 199 89 61 172 64 55 181 77 44

W2

NA

W3

NA

W4

NA

W5

NA

W6

NA

W7

NA

W8

NA

W9

NA

10

W10

NA

11

W11

NA

12

W12

NA

13

W13

NA

14

W14

NA

15

W15

NA

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Bacterial Diversity

Table-II : Total viable count on nutrient agar of mustard soil sample

Sr.no. 1 Sample code M1 Media NA Dilution Count 147 56 38 142 59 31 152 78 54 162 61 31 178 79 38 151 86 37 170 69 42 142 75 39 186 42 30 130 52 33 201 98 65 130 52 33 177 44 36 190 69 42 172 65 44

M2

NA

M3

NA

M4

NA

M5

NA

M6

NA

M7

NA

M8

NA

M9

NA

10

M10

NA

11

M11

NA

12

M12

NA

13

M13

NA

14

M14

NA

15

M15

NA

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Bacterial Diversity

Table- III : Biochemical Activities of Unidentified Bacteria In Wheat Field

SAMPLE CODE W1 W2 Gram stain Rod + Rod Rod + Rod + Rod Rod + Rod Rod + Rod + Rod + Rod Rod + Rod Coccus + Rod + Colony characteristics opaque,white, waxy growth Slimy,white translucent, raised growth opaque,white, waxy growth opaque,white, waxy growth Abundant,thin, white media turns green opaque,white, waxy growth Thin blue,grey, spreading growth, opaque,white, waxy growth Thin white, glistening, opaque Opaque ,white, Raised Slimy,white Translucent opaque,white, waxy growth Thin white, glistening, white,spreading viscous growth opaque,white, waxy growth Starch hydrolysis +ve -ve +ve +ve -ve +ve -ve +ve +ve -ve +ve +ve +ve -ve +ve Fermentation
Lactose Dextrose Sucrose

H2s production -ve -ve -ve -ve -ve -ve +VE -ve +ve -ve -ve -ve -ve -ve -ve

Indole production +ve -ve +ve +ve -ve +ve +ve +ve +ve +ve +ve +ve +ve +ve +ve

MR

VP

Citrate utiliazation +ve +ve +ve +ve +ve +ve -ve +ve -ve -ve +ve +ve +ve +ve +ve

Catalase

-ve AG -ve -ve -ve -ve -ve -ve -ve +ve -ve -ve -ve -ve -ve

-ve AG -ve -ve -ve -ve AG -ve -ve +ve -ve -ve -ve -ve -ve

-ve AG -ve -ve -ve -ve AG -ve -ve +ve -ve -ve -ve -ve -ve

+ve -ve +ve +ve -ve +ve +ve +ve +ve +ve +ve +ve +ve +ve +ve

-ve +ve -ve -ve -ve -ve -ve -ve +ve +ve -ve -ve -ve -ve -ve

+ve +ve +ve +ve +ve +ve +ve +ve +ve +ve +ve +ve +ve +ve +ve

W3 W4 W5

W6 W7 W8 W9

W10 W11 W12 W13 W14 W15

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Bacterial Diversity

Table- IV

Biochemical Activities of Unidentified Bacteria In Mustard Field

SAMPLE CODE M1 Gram stain Rod + Rod Rod Rod Rod Cocco bacilli Coccus + Coccus + Rod + Rod + Rod Rod + Rod Coccus + Rod + Colony Characteristics Abundant, opaque,white,waxy growth White,moist ,glistening Slimy,white translucent,raised growth Thin blue,grey,spreading growth, Abundant,thin,white media turns green Thin white,spreading,viscous growth Abundant oaque,golden growth Abundant thin,even growth Thin white,glistening, opaque Opaque ,white,raised Slimy,white translucent Thin white,glistening, opaque Thin white,glistening, white,spreading,viscous growth opaque,white,waxy growth Starch hydrolysis +ve -ve Fermentation Lactose A Dextrose A Sucrose H2s production -ve Indole production -ve MR VP Citrate utiliazation -ve Catalase -ve +ve -ve

M2 M3 M4

-ve -ve -ve

AG AG -ve

AG AG AG

A+ AG AG

-ve -ve +VE

+ve -ve +ve

+ve -ve +ve

-ve +ve -ve

-ve +ve -ve

+ve +ve +ve

M5 M6

-ve -ve

-ve -ve

-ve -ve

-ve -ve

-ve -ve

-ve -ve

-ve -ve

-ve -ve

+ve -ve

+ve +ve

M7 M8 M9 M10 M11 M12 M13 M14 M15

-ve -ve +ve -ve +ve +ve +ve -ve +ve

A A -ve +ve -ve -ve -ve -ve -ve

A A -ve +ve -ve -ve -ve -ve -ve

A A -ve +ve -ve -ve -ve -ve -ve

-ve -ve +ve -ve -ve +ve -ve -ve -ve

-ve -ve +ve +ve +ve +ve +ve +ve +ve

+ve +ve +ve +ve +ve +ve +ve +ve +ve

+ -

-ve -ve -ve -ve +ve -ve +ve +ve +ve

+ve -ve +ve +ve +ve +ve +ve +ve +ve

-ve +ve +ve -ve +ve -ve -ve -ve

- Negative

+ - Moderate 51

Bacterial Diversity

+ Positive A Acid Production

AG Acid + Gas Production

Table-V Isolates

S.No.

Isolates

Percentage of mustard

Percentage of wheat 12.0 08.4 04.2 12.7 10.0 07.5 13.0 13.0 07.8 03.3 06.0

:Identified Percentage

1 2 3 4 5 6 7 8 9 10 11

Pseudomonas spp. Klebsiella spp. Rhizobium spp. Azotobacter spp. Azospirilum spp. Proteus spp. E.coli Bacillus spp. Streptocoocusspp. Enterobacter spp. Staphylococcus spp.

17.2 12.8 06.3 08.1 08.1 07.4 15.0 13.0 09.0 04.0 05.0

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Table-VI :Normal Habitat of identified Bacteria

Sr.No. 1 2 3 4 5 6 7 8 9 10 11 Organism Pseudomonas spp. Klebsiella spp. Rhizobium spp. Azotobacterspp. Azospirillumspp. Proteus spp. E.coli Bacillus spp. Streptococcusspp. Enterobacterspp. Growth Facultative aerobes Aerobic Facultative aerobe Facultative aerobe Facultative aerobe Facultative Facultative Facultative aerobe Facultative Facultative Human nnasopharynx, Soil Normal intestinal flora, Soil Endogenus human flora, Soil Pneumonia , meningenitis, others Skin and tissue Sterp throat:toxic shock , flesh eating Phage lysine has potential to eradicate spp. Opportunistic:nosocomial :debilitated patients Staphylococcusspp. Facultative Rheumatic fever and glomerulonephrities streptococci Water & oil Normal intestine flora, Soil Soil, water, food UTIs and wound infection Gastroentritis,UTIs,Septicemia Nonpathogenic Swarming motility biodilms,meat Fast grower:disease at in appropriate site Form spores(all bacilli) Free living bacteria in soil Non pathogenic Non symbiotic nitrogen fixer Free living bacteria in soil Non pathogenic Human trophical/subtropical, Soil Soil plant nodules Nonpathogenic Granuloma inguinate Habitat Soil,water,humans,plants Pathogenesis Lungs and many other sites Comments Opportunist harmful for cystic fibrosis others STD,forms intracellular Donovan bodies Major terrestrial nitrogen;fixing plant symbionts Non symbiotic nitrogen fixer

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CONCLUSION

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CONCLUSION Soil is living resource that provides numerous essential services, releasing nutrients in forms that can be used by organisms. When this recycling function is impaired agriculture, forestry and ultimately all life on earth is threatened. The microorganisms contained in soil contribute to water purification and help remove pollution and pathogen. The loss of this service would reduce the quality and quantity of ground and surface waters. Increasing the risk of erosion and landslides in mountain areas and flooding in lowland. Soil also contains the second largest carbon pool on the planet. The loss of soil biodiversity reduces the ability of soils to regulate the composition of the atmosphere diminishing their role in counteracting global warming Soil organisms constitute a major source of chemical and genetic resources. Antibiotic resistance develops fast, so the demand of new pharmaceutical products is almost unending and soil biodiversity can be an important source. In present studies showed that in this region bacterial diversities is very rich and it is very good for the crops of this region. The complex ecological interactions between soil organisms and agriculture are not fully understood. There is a need to continued research. However, there are many tools and management techniques already available for the sustainable management of soil although uptake of these tools and techniques could be improved. Soil requires protection and careful management by farmers, the public and policy makers. This is essential if we are to conserve the medium that supports our life and help us grow our future.

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References

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References
Allen, O.N. 1957Experiments in Soil Bacteriolog, 3rd Edition, Burgen Publi.. Co.Minneapclis ,Minn; USA Alexander, M. & Clark, F.E., 1965. Nitrifying bacteria. In method of Soil analysis, Part 2 Chemi. & Microbiological Properties PP. 1477-1483 Eds. C.A. Black, American Society of Agronomy, Inc., Madison, Wisconsin, USA Alexander, M. 1961. Introduction to Soil Microbiology John Wiley & Sons, Inc., New York&London. Bear, F.E. 1968. Chemistry of the soil pp 346-367. Oxford & IBH publishing Co. Calcutta, Bombay, New Delhi. Baker, K.F. & Cook, R. J. 1974. Biological control of plant pathogens. W.N. freeman& co., Sanfrancisco. Black, C.A. 1968.Soil Plant Relationship John wiley & sons, Inc., New York, London Burns, R.G.Ed. 1978. Soil Enzymes. Academic Press, London Donehue RL Shicklauna, JC & Robertson LS 1971 :Soil introduction to soil & plant Growth. Hardy, R.M.F, &Holstein, R.D. 1977. Methods for measurement of dinitrogen fixation. In A Treatix on Dinitrogen fixation Sect, Agronomy & Ecology, PP. 451-486 John Wiley & Sons, Inc. New York Harris R.f. Chesters G. & Allen, On 1966 dynamics of soil aggregation, Adv. Agyon PP 107-169 Layer, F., Ghebremedhin, B., Moder, K.A., Konig, W., and Koing, B. Comparative study using various methods for identification of Staphylococcus species in Clinical specimens, J. Clin. Microbiol., 44, 2824, 2006 Macfaddin, J.F, Biochemical Tests for the identification of Medical Bacteria, 3rd ed. Lippincott, Williams & Wilkings Co., Philadelphia, PA, 2000

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Martin, W.P. Talor G.S. engibous, J.C. & Brunett E. 1952. Soil of crop responses from field application of soil condition as soil science 455-471 Okon, 4. Albrecht, S.L. & Burris, R.N. 1977Method for Growing spirillum lipoferuim & for counting it in pure culture & in association with plants J. Appl. Environ. Microbiol., 33, 85-88 OHara, C.M., Weinstein, M.P., and Miller, J.M., Manual and automated systems for detection and identification of Microorganisms, in Manual of Clinical Microbiology, 8th ed. Murray. P.R. et al., Eds. ASM Press, Washington, DC, 2003, Chap. 14. Paul, P. & Clark, F.E. 1989. Soil Microbiology & Bio Chemistry Academic Press, New York Rai, B & Srivastava, A. 1982Decomposition of leaf litter in relation to microbial population & their activity in tropical dry mix deciduous forest, Pedobiol 24 :151159. Russel J.R. 1962Soil conditions & plant growth longmasns, Green & Co. Ltd., London pp. 239-246. S Offerman, R. Sc. & Morris, M. 1963Algal Virus-isolation Science N. 4, 140, 679-680 Singh, B.N. 1946, A Method for estimating the number of Soil protozoa especially amoeba Bared on Their differential feeding of bacteria Ann. Appl. Biol, 33, 112119 Sundara Rao, WVB & Sinha M.K. 1963, Phosphate dissolving organism in the soil & rhizosphere Ind. J. Agric. Science. 33, 272-278 Waksman SA 1959, the actinomycetes Vol. 1 nature, occurrence & activates. YORK, M.K. Traylor, M.M., Hardy, J., and Henry, M.,Biochemical tests for the identification of aerobic bacteria, in clinical Microbiology procedures Handbook, 2nd ed., Isenberg H.D., Ed., ASM Press, Washington, DC, 2004, Vol. 1 Section 3.17.

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APPENDIX

APPENDIX - I
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Composition of media and their function Composition of nutrient broth medium (pH 7.0) S.No. 1 2 Components Peptone Beef extract Qty.
(gm/1000ml)

Functions Carbon source Source of nitrogen & vitamin

5.0 3.0

NaCl

5.0

Ions transport

Composition of nutrient agar medium (pH 7.0) S. No. Components Qty. (gm/1000 ml) 1 2 Peptone Beef extract 5.0 3.0 Carbon source Source of nitrogen & vitamin 3 4 NaCl Agar 5.0 15.0 Ions transport Solidifying agent Functions

Composition of starch agar medium (pH 7.0) S. No. Components Qty. (gm/1000 ml) 1 2 Peptone Beef extract 5.0 3.0 Carbon source Source of nitrogen & vitamin 3 4 Starch Agar 20.0 15.0 Energy source Solidifying agent Functions

Composition of MR-VP Broth (pH 6.9)

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S. No.

Components

Qty. (gm/1000 ml)

Functions

1 2 3

Peptone Glucose Potassium phosphate

7.0 5.0 5.0 Methyl red pH indicator.

Carbon source Energy source Ions transport

VP regent I (alpha naphthol solution) VP regent II (405KOH-40%) Composition of tryptone broth (pH 6.9) S. No. Components Qty. (gm/1000 ml) 1 2 3 Tryptone NaCl CaCl2 10.0 5.0 .01 - .03M Amino acids Ions transport Ionization, buffering capacity Kovacs Reagent - dimethylaminobenzaldehyde CaCl2 addition after autoclave. Composition of fermentation broth (pH 7.3) S. No. Components Qty. (gm/1000 ml) 1 2 3 4 Peptone Carbohydrates NaCl Phenol red 10.0 5.0 5.0 0.018 Carbon source Carbon source Ions transport Indicator Functions Functions

(indicator)

Composition of Simmons citrate agar medium (pH 6.9)

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S. No.

Components

Qty. (gm/1000 ml)

Functions

Ammonium-di hydrogen phosphate

1.0

Ions transport

2 3 4 5 6 7

K2HPO4 Sodium citrate NaCl MgSO4 Bromothymol blue Agar

1.0 2.0 5.0 0.2 0.08 15.0

Ions transport Carbon source Ions transport Ions transport Indicator Solidifying agent

Composition of eosin methylene blue agar medium (pH 7.2) S. No. Components Qty. (gm/1000 ml) 1 2 3 4 5 6 Peptone Lactose Eosin K2HPO4 Methylene blue Agar 10.0 5.0 0.4 2.0 0.065 15.0 Source: (Aneja, 2003) Carbon source Energy source Bacteriostatic Ions transport Bacteriostatic Solidifying agent Functions

Composition of yeast extract mannitol agar medium(pH 6.8)

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S.No . 1 2 3 4 5 6

Components

Qty.(gm/100 0ml)

Functions

Mannitol K2HPO4 MgSO4.7H2O NaCl Yeast Extract Agar

10 0.5 0.2 0.1 0.5 15.0

Element Element Element Ions transport mineral Solidifying agent

Composition of NFB(nitrogen free bromothymol blue)pH 6.5

S.No. Components Qty. (gm/1000ml) 1 2 3 4 5 6 7 8 9 10 11 Malic acid KOH K2HSO4 MgSO4.H2O FeSO4.7 H2O NaCl CaCl2 Na2Mo4 MnSO4.7H2O Bromothymol blue Agar 5.0 4.0 0.5 0.1 0.05 .01 .02 .01 .002 2.0 1.75 Carbon source Element Element Element Element Ion transport Ionization Ion transport Element Indicator Solidifying agent Functions

Composition of Kings medium (pH 7.2)

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S.No.

Components

Qty. (gm/1000ml)

Function

1 2 3 4 5

Protease Peptone Glycerine Agar K2HPO4 MgSO4.7H2O

20.0 15.0 20 2.5 6.0

Carbon source Stuff Solidifying agents Element Element

Composition of Jensens medium (pH6.5)

Sr.No. Components Qty. (gm/1000ml) 1 2 3 4 5 6 7 MgSO4.7H2O NaCl FeSO4.7H2O CaCO3 Sucrose K2HSO4 Agar 0.5 0.5 0.1 2.0 20.0 1.0 15 Element Ion transport Element Element Carbon source Element Solidifying agent Function

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