LECTURE

MICROBIOLOGY OF WATER AND WASTE WATER

Dr. Reeta Goel Professor& Head Department of Microbiology College of Basic Sciences and Humanities G.B. Pant Uni. of Agri. & Technology, Pantnagar
In urban areas, the household consumption of water is about 150 liters per day per person. Water is used for bathing, washing utensils, washing clothes etc. This domestic water consumption may vary with the lifestyle of community and the availability of water. Most of the water taken into the house may be returned as wastewater through drainage system. Moreover, industries also consume large quantities of water and contribute to the discharged effluent. WATER AND HEALTH Water which is fit for human consumption is called drinking water or potable water. Sometimes the term safe water is applied to potable water of a lower quality threshold (i.e., it is used effectively for nutrition in humans that have weak access to water cleaning processes, and does more good than harm).Sometimes microorganisms that cause health problems can be found in drinking water. However, as drinking water is thoroughly disinfected today, disease caused by microorganisms is rarely caused by drinking water. There are various bacteria and protozoa that can cause disease when they are present in surface water (Table 1). Water Purity Tests Explain how water is tested for bacteriological quality Historically, most of our concern about water purity has been related to the transmission of diseases. Therefore, tests have been developed to determine the safety of water; many of these tests are also applicable to foods. It is not practical, however, to look only for pathogens in water supplies. Because, if we were to find the pathogen causing typhoid or cholera in the water system, the discovery would already be too late to prevent an outbreak of the disease. Moreover, such pathogens would probably be present only in small numbers and might not be included in tested samples.

Table 1. various bacteria that can be found in surface water, and the diseases caused by them . Bacteria Aeromonas Campylobacter jejuni Escherichia coli Disease/ infection Enteritis Symptoms Very thin, blood- and mucus-containing diarrhoea Campilobacteriose Flue, diarrhoea, headand stomachaches, fever, cramps and nausea Watery diarrhoea, Urinary tract infections, headaches, fever, neonatal meningitis, homiletic uraemia, kidney intestinal disease damage Plesiomonas-infection Nausea, stomachaches and watery diarrhoea, sometimes fevers, headaches and vomiting Typhoid fever Fevers Salmonellosis Sickness, intestinal cramps, vomiting, diarrhoea and sometimes light fevers (Gastro) intestinal disease Stomachaches, diarrhoea and fevers, sometimes vomiting (Light form of) Cholera Heavy diarrhoea

Plesiomonas shigelloides

Salmonella

Streptococcus Vibrio El Tor (freshwater)

The tests for water purity in use today are aimed for detection particular indicator organisms. There are several criteria for an indicator organism, the most important being that the microbe is consistently present in human feces in substantial numbers so that its detection is a good indication that human wastes are entering the water. The indicator organisms should also survive in the water at least as well as the pathogens would. The indicator organisms must also be detectable by simple tests that can be carried out by people with relatively little training in microbiology. Coliforms are defined as aerobic or facultatively anaerobic, gram-negative, non-endospore-forming, rod-shaped bacteria that ferment lactose to form gas within 48 hours of being placed in lactose broth at 35C. Because some coliforms are not solely enteric bacteria but are more commonly found in plant and soil samples, many standards for food and water specify the identification of fecal

coliforms. The predominant fecal coliform is E. coli, which constitutes a large proportion of the human intestinal population. There are specialized tests to distinguish between fecal coliforms and nonfecal coliforms. Further, coliforms are not themselves pathogenic under normal conditions, although certain strains can cause diarrhea and opportunistic urinary tract infections. The common methods for determining the presence of coliforms in water are largely based on the lactose-fermenting ability of coliform bacteria. The multipletube method can be used to estimate coliform numbers by the most probable number (MPN) method. The membrane filtration method is a more direct method of determining the presence and numbers of coliforms. A more convenient method of detecting coliforms, specifically the coliform E. coli, makes use of media containing the two substrates o-nitrophenyl--Dgalactopyranoside (ONPG) and 4-methylumbelliferyl--D-glucuronide (MUG). Coliforms produce the enzyme -galactosidase, which acts on ONPG and forms a yellow color, indicating their presence in the sample. E. coli is unique among coliforms in almost always producing the enzyme -glucurom'dase, which acts on MUG to form a fluorescent compound that glows blue when illuminated by longwave ultraviolet light. These simple tests, or variants of them, can detect the presence or absence of coliforms or E. coli and can be combined with the multiple-tube method to enumerate them. It can also be applied to solid media, such as in the membrane filtration method. The colonies fluoresce under UV light. Coliforms have been very useful as indicator organisms in water sanitation, but they have limitations. One problem is the growth of coliform bacteria embedded in layers of biological slime (or biofilms, discussed in detail shortly) on the inner surfaces of water pipes. These coliforms do not, then, represent external fecal contamination of the water, and they are not considered a threat to public health. Wastewater Wastewater is liquid effluent derived from domestic sewage of industrial sources that cannot be discarded in untreated form into lakes or streams due to public health, economic and aesthetic considerations. Sewage is liquid effluent contaminated with human or animal fecal materials. For technical purpose can be divided into urban and industrial wastewater. The composition of the former usually conforms to a general typology.

Most industrial processes emit wastewater during one or more stages of production. The composition of this type of water can vary dramatically as it is determined both by the products themselves and processes of production. All these waste waters contain organic and inorganic wastes as suspended or dissolved matter. In addition they may also contain microorganisms, including those of faecal origin and pathogenic nature. The solids content of an urban wastewater may be physically classified approximately as shown in fig. 1 (Metcalf and Eddy, 1987)

Fig.1 Classification of solids found in urban wastewater (Metcalf and Eddy, 1987)

In a typical urban wastewater, about 75 percent of the suspended solids and more than 50 percent of the filterable solids are organic in nature. These solids are derived from both the animal and plant kingdoms and the activities of these as related to the synthesis of organic compounds. The principal groups of organic substances found in wastewater are proteins (40 to 60 %), carbohydrates (25 to 50 %), fats and oils(10 % (Metcalf and Eddy, 1987). Beyond these substances, wastewater contains small quantities of a large number of different synthetic organic molecules. Surfactants, phenols and pesticides are typical compounds.

Various forms of nitrogen in urban wastewater (Ekama et al., 1984) Industrial waste generally has a strong odour. The substances responsible for causing odour and taste are phenol compounds, sulphur compounds, iron, manganese, sodium chloride, calcium chloride, magnesium salts, acids, hydrocarbons, often present in wastes from gas and wood industries, refineries and various chemical industries (Mendia, 1962). Microbiological Characteristics of Sewage The sewage composition varies depending upon the source of wastewater. This also causes variation in the microbial flora of sewage. Almost all groups of microorganisms, algae, fungi, protozoa, bacteria and viruses are present. The bacterial group comprises mainly the soil borne organisms, Bacillus subtilis, B. megaterium, B. mycoides, Pseudomonas fluorescens, Achromobacter spp. and Micrococcus spp. Bacteria of intestinal origin also occur in sewage in large numbers. Mostly these are pathogens. Examples of this type are Escherichia coli, and other coliforms, Proteus and Serratia species. Potential pathogens include enterococci (Streptococcus faecalis) and Clostridium perfringens. Pathogenic bacteria which cause serious illness like Vibrio cholerae, Salmonella typhi, S. paratyphi and Shigella dysenteriae may also occur in sewage. Viruses (released in the faeces from infected host) are also occasionally found in sewage, for example, poliomyelitis virus, infectious hepatitis virus and Coxsackies virus. Bacteriophages also occur in comparatively large numbers. During treatment process the microbial flora may be dominated by the corresponding physiological groups.

WASTEWATER AND SEWAGE TREATMENT Wastewater treatment refers to the process of removing pollutants from water previously employed for industrial, agricultural, or municipal uses. The main objectives of the sewage treatment are: To convert waste and wastewater into a readily reusable resource. To prevent pollution of any water body to which treated or reused water enters. To reduce the BOD (biochemical oxygen demand) of sewage from 30 mg/l to about 20 mg/l in the final effluent. To destroy the causative agents of waterborne diseases and sewage treatment involves a large-scale use of

Wastewater

microorganisms and can be considered a type of industrial-scale bioconversion. Wastewater enters a treatment plant and, following treatment, the effluent water is suitable for release into rivers and streams or to drinking water purification facilities. The techniques used to remove the pollutants present in wastewater can be broken into biological, chemical, physical, and energetic. These different techniques are applied through the many stages of wastewater treatment. Systems commonly used for treatment of urban wastewater are constituted of primary treatment by settling, a biological second stage, and a tertiary treatment by disinfection, in some cases following a filtration process. Primary sedimentation is most efficient in removing coarse solids. Biological processes are used to convert the finely dissolved organic matter in wastewater into flocculant settleable solids that can be removed in sedimentation tanks. These processes are employed in conjuction with physical and chemical processes and they are most efficient in removing organic sub-stances that are either soluble or in the colloidal size range. Disinfection is generally operated by chlorination with Cl2 or NaOCl. The main systems for removal of solids, organic matter and pathogens are the activated sludge process, trickling filters, aerated lagoons, high-rate oxidation ponds, stabilization ponds. Stabilization ponds or aerated lagoons are most often used for small installations. The activated sludge process, or one of its many

modifications, is most often used for larger installations. In some cases trickling filters are applied. Several processes have been used for activated sludge. The most important are (Metcalf and Eddy, 1987): tapered aeration process; modified aeration process; continuous-flow stirred tank; step aeration process; contact stabilization process; extended aeration process; oxidation ditch; carrousel system; high-rate aeration process. Wastewater treatment and biochemical oxygen demand The goal of a wastewater treatment facility is to reduce organic and inorganic materials in wastewater to a level that no longer supports microbial growth and to eliminate other potentially toxic materials. The efficiency of treatment is expressed in terms of a reduction in the biochemical oxygen demand (BOD), the relative amount of dissolved oxygen consumed by microorganisms to completely oxidize all organic and inorganic matter in a water sample. Higher levels of utilizable organic and inorganic materials in the wastewater result in a higher BOD. Typical values for domestic wastewater, including sewage are approximately 200 BOD units. For industrial wastewater for example from sources such as dairy plants, the values can be as high as 1500 BOD units. An efficient wastewater treatment facility reduces levels to less than 5 BOD units in the water released from the treatment plant. A typical wastewater facility must treat both sewage and industrial wastes. Treatment is a multistep operation employing a number of independent physical and biological process. Primary, secondary(Fig.3) and sometimes tertiary treatments are employed to reduce fecal and chemical contamination in the incoming water. Each level of treatment employs more complex and more expensive technologies. Primary treatment Primary treatment usually includes the removal of large solids from the wastewater via physical settling or filtration. The first step in primary treatment is screening. Wastewater entering the treatment plant is passed through a series of grates and screens that remove large objects. The effluent is left to settle down for a number of hours to allow suspended solids to sediment. Municipalities that provide only primary treatment suffer from extremely polluted water when the

effluent is discharged into adjacent waterways because high levels of organic matter and other nutrients remain in water following primary treatment. Therefore, most treatment plants employ secondary treatment to reduce the organic content of the wastewater before release to natural waterways. Secondary treatment is intimately tied to microbiological processes. Secondary Treatment Secondary treatment typically removes the smaller solids and particles remaining in the wastewater through fine filtration aided by the use of membranes or through the use of microbes, which utilize organics as an energy source. Energetic techniques may also be employed in tandem with biological techniques in the secondary phase to break up the size of particles thus increasing their surface area and rate of consumption by the microbes present. A common first step in the secondary treatment process is to send the waste to an aeration tank. Anoxic secondary wastewater treatment Anoxic wastewater treatment involves a series of digestive and fermentative reactions carried out by a number of bacterial species and is usually employed to treat materials that have large amounts of insoluble organic matter (and hence very high BOD), such as fiber and cellulose waste from food-and dairy processing plants. The anoxic degradation process itself is carried out in large enclosed tanks called sludge digesters or bioreactors and requires the collective activities of many different types of microorganisms.Through the action of the resident anoxic microorganisms, the macromoleculare waste components are first digested by polysaccharases, proteases and lipases into soluble components. These soluble components are then fermented to yield a mixture of fatty acids, H2 and CO2 and the fatty acids are further fermented to acetate, CO2 and H2. These products are then used as substrates by methanogenic bacteria, which are capable of carrying out the reactions CH3COOH CH4+CO2 and 4H2O + CO2 CH4 + 2H2O. Thus major products of anoxic sewage treatment are CH4 (methane) and CO2. The methane can be collected and either burned off or used as fuel to heat and power the treatment plant. Aerobic secondary treatment In general, nonindustrial wastewater can be treated efficiently using only aerobic secondary treatment. Several kinds of aerobic decomposition processes are used

for wastewater treatment, but the trickling filter and activated sludge methods (Fig.2) are the most common. A trickling filter is a bed of crushed rocks, about 2 m thick, on tip of which the wastewater is sprayed. The liquid slowly passes through the bed, the organic matter adsorbs to the rocks and microbial growth occurs on the rocks. The complete mineralization of organic matter to carbon dioxide, ammonia, nitrate, sulfate and phosphate takes place in the microbial biofilms on the rocks. The most common aerobic treatment systems is the activated sludge process. Here, the wastewater to be treated is mixed and aerated in a large tank. Slime-forming bacteria, including Zooglea ramigera, among others, grow and form flocs (large, aggregated masses) and these flocs form the substratum to which protozoa and small animals attach. Occasionally, filamentous bacteria and fungi is pumped into a holding tank or clarifier where the flocs settle. Some of the floc material (called activated sludge) is then returned to the aerator to serve as inoculum and the rest is sent to the anoxic sludge digestor or is removed, dried and burned or used for fertilizer.

Fig.2; Simple Activated Sludge with Trickling filter Wastewater normally stays in an activated sludge tank for 5 to 10h, a time too short for complete oxidation of all organic matter. However, during this time much of the soluble organic matter is adsorbed to the floc and is incorporated into microbial cells. The BOD of the liquid effluent is considerably reduced (by up to 95%) by this process, with most of the BOD now contained in the settled flocs and the goal of BOD reduction in the water is achieved. Nearly complete BOD reduction can occur if the flocs are then transferred to the anoxic sludge digestor.

Most treatment plants now chlorinate the effluent (to further reduce the possibility of biological contamination) and discharge the treated water to streams or lakes. A few plants, however, process wastewater through a tertiary stage. Tertiary treatment Tertiary treatment is the most complete method of treating sewage but has not been widely adopted because it is very expensive. Tertiary treatment is a physicochemical process employing precipitation, filtration and chlorination procedures similar to those employed for drinking water purification to sharply reduce the levels of inorganic nutrients, especially phosphate and nitrate, from the final effluent.

Fig.3: Primary and secondary treatment of raw water Physiochemical purification A typical drinking water treatment installation for a small city is shown in figure 4. Raw water is first pumped from the source, in this case a lake, to a sedimentation basin where anionic polymers, alum (aluminum sulfate), and chlorine are added. Sand, gravel and other large particles settle out. This pretreated water is then pumped to a clarifier or coagulation basin, a large holding tank where coagulation takes place. The alum and anionic polymers form larger suspended particles from the much smaller suspended colloidal particles. After mixing, the particles continue to interact, forming large, aggregated masses, a

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process known as flocculation. The large, aggregated particles, called floc, settle out by gravity, trapping any remaining microorganisms and absorbing organic matter and sediment. After coagulation and flocculation, the clarified water undergoes filtration. The water is passed through a series of filters designed to remove the remaining suspended particles and microorganisms. The filters usually consist of thick layers of sand and ionic filtration media. When combined with previous purification steps, the filtered water is free of all particulate matter, most organic and inorganic chemicals, and all microorganisms. Disinfection Clarified, filtered water must then be disinfected before it is released to the supply system as pure, potable finished water. Chlorination is the most common method of disinfection. In sufficient doses, chlorine kills microorganism within 30 minutes (certain pathogenic protozoa such as Cryptosporidium are not easily killed by chlorine treatment and thus can be important waterborne pathogens. In addition to killing microorganisms, chlorine reacts with organic compounds, oxidizing and effectively neutralizing them. Therefore, since most taste and odor-producing compounds are organic in nature, chlorine treatment also improves water taste and smell. Chlorine is added to water either from a concentrated solution of sodium or calcium hypochlorite or as a gas from pressurized tanks. The latter method is used most commonly in large water treatment plants because it is most amenable to automatic control.

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Fig. 4: Water treatment system

Further Reading Ekama G.A., G.v.R. Marais and I.P. Siebritz., (1984). Biological Excess Phosphorus Removal. In Theory, Design and Operation of Nutrient Removal Activated Sludge Processes, information document prepared for the Water Research Commission by the University of Cape Town, City of Council of Johannesburg and the National Institute for Water Research of the CSIR, Pretoria. Mendia L., (1962). Aspetti tecnici del problema degli scarichi industriali. Ingegneria Sanitaria, N. 1. Metcalf and Eddy, Inc., (1987). Wastewater Engineering: Treatment, Disposal, Reuse. Tata McGraw-Hill Publishing Company Ltd., New Delhi, second edition, 6th reprint.

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*Basic Terminology of Microbiology Autoclave Bacteria Biofilms Bioremediation Biotechnology Coccus Coliforms Colonization Colony Complex media Consortium Culture Culture medium Disease Eukarya Extremophile Fungi Gene Gram-negative cell : : ; : : : : : : : : : : : : : : : : A sterilizer that destroys microorganisms by high temperature using steam under pressure All prokaryotes that are not members of the domain Archaea Microbial colonies encased in an adhesive, usually polysaccharide material, and attached to a surface Use of microorganisms to remove or detoxify toxic of unwanted chemicals in an environment The use of living organisms to carry out defined chemical processes for industrial application a spherical bacterium Gram-negative, nonsporing, facultative rods that ferment lactose with gas formation within 48 hr at 35o C Multiplication of a microorganism after it has attached to host tissues or other surfaces A macroscopically visible population of cells growing on host tissues or other surfaces Culture media whose precise chemical composition is unknown. Also called underfined media A two-(or more) membered bacterial culture (or natural assemblage) in which each organism benefits from the others A particular strain or kind of organism growing in a laboratory medium An aqueous solution of various nutrients suitable for the growth of microorganisms Injury to the host that impairs host function The Phylogenetic domain containing all eukaryotic organism An organism that grows optimally under one or more chemical or physical extremes, such as high or low temperature or pH Nonphototrophic eukaryotic microorganisms that contain rigid cell walls A unit of heredity; a segment of DNA specifying a particular protein or polypeptide chain, a tRNA or an rRNA A prokaryotic cell whose cell wall contains relatively little petidoglycan but has an outer membrane composed of lipopolysaccharides, lipoprotein and other complex macromolecules A prokaryotic cell whose cell wall contains relatively little peptidoglycan and lacks the outer membrane or gram-negative cells

Gram-positive cell

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Growth Growth rate Guild Microorganisms Nutrient Parasite Pasteurization

: : : : : : :

Pathogen Pure culture Stationary phase Sterilization Strain Virus Water activity (aw) Xenobiotic Yeasts

: : : : : : : : :

In microbiology, an increase in cell number The rate at which growth occurs, usually expressed as the generation time A group of metabolically related organisms A microscopic organism consisting of a single cell or cell cluster, also including the viruses A substance taken by a cell from its environment and used in catabolic or anabolic reactions An organism able to live on and cause damage to another organism Destruction, usually by heat treatment, of all diseases-producing, microorganisms along with a reduction in the number of spoilage microorganism An organism able to inflict damage on a host it infects A culture containing a single kind of microorganism The period during the growth cycle of a microbial population in which growth ceases The killing or removal of all living organism and their viruses from a growth medium A population of cell of a single species all descended from a single cell; a clone A genetic element containing either DNA or RNA that replicates in cells but is characterized by haing an extracellular state An expression of the relative availability of water in a substance. Pure water has an aw of 1.000 A completely synthetic chemical compound not naturally occurring on Earth Unicellular fungi

* Source: Brock Biology of Microorganism 2003, 10th Edition

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Water Purification and Public Health

S.P. Singh Prof. & Head Department of Veterinary Public Health College of Veterinary and Animal Sciences, G. B. Pant University of Agri. & Tech., Pantnagar-263145
Water and Health The worlds population is expected to increase every year by 74.8 million. To meet requirement of water for such a huge population, sincere efforts are being made at international level. The United Nations have declared 2005-2015 as the International Decade for Water for life and World agenda has been set focusing the water related issues. This issue is of great consequence since approximately 1.8 million people die every year from diarrhoeal diseases (including cholera); mostly in developing countries where 88% of diarrhoeal disease is attributed to unsafe water supply, inadequate sanitation and hygiene. Improvements in drinking-water quality through household water treatment, such as chlorination at point of use can lead to a significant reduction of diarrheal episodes. There is a need to undertake an integrated water resources management so as to provide safe and clean water to all. Waterborne diseases occur not only as an endemic but also often appear as an epidemic. In the context of zoonoses, the waterborne diseases have significance in both developed as well as developing countries alike. The associated pathogens are transmitted predominantly by faecal-oral and occasionally by faecal-droplet routes. Consequent to the dynamics in the population as well as its resultant effect on the environment, many pathogens are taking newer and virulent forms resulting in the emergence and re-emergence of the waterborne disease. Such changes are not free from the adverse consequences on the public health and these include (i) changing patterns of water use (ii) population growth/migration/variation (iii) increased population of the immunocompromised (consequent to the malnourishment as well as immunodeficiency diseases such

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as AIDS and its deadly combinations with tuberculosis/toxoplasmosis etc) (iv) increased use of water due to the changed lifestyle and access to recreational activities (v) water scarcity, climate changes, disasters, & the emergencies (vi) war and bioterrorism (vii) increased population in the urban and periurban areas (viii) use of non-conventional alternatives to meet the never ending human demands (ix) increased use of agro-chemicals, antibiotics, growth promoters, and other veterinary drugs for the production and protection of plants, animals and human (x) altered ecological rhythm and (xi) the global trade, its regulations and their related consequences. Zoonotic pathogens have been encountered in water as a cause of gastroenteric infections with the symptoms of diarrhea in many countries. But, still there exist many diseases that just escape the diagnosis process. Leptospirosis, E. coli O157:H7, Cryptosporidiosis, Campylobacter, Toxoplasmosis and Giardiasis, occur regularly in some countries. It is also important to mention more than 75% of all the emerging pathogens are zoonotic on nature. Further, animals and other lower vertebrate or non-vertebrates play an equal role as that of human in the maintenance or transmission of such infections that ultimately threaten the human life by the way of vehicles, particularly water. A variety of bacteria, parasites, fungi, viruses can be acquired by the way of water. The transmission routes involve drinking, contact, water use (food preparation, agriculture) exposure to wastewater, faeces, urine, and abattoir waste. The viruses / prion particles possess considerable host specificity yet can infect the related species. There are 1.5 million cases of clinical hepatitis reported every year.Many of the bacterial pathogens are well established water borne pathogens such as Salmonella, E. coli O157:H7, Campylobacter, Yersinia, Mycobacterium avium (ssp. paratuberculosis) and Leptospira. They can be transmitted by improperly purified water and can put the end users of water at risk. The waterborne zoonotic bacteria are principally those shed in faeces by warmblooded animals (birds and mammals), although some are also harbored by reptiles.

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Although fungi are transmitted directly by contact but at times, water can act an agent of transmission and the infections such as Trichophyton spp., Cryptococcus, and Coccid odes may enter by such route. Protozoan pathogens originating from animal and human waste have been recorded from water sources throughout the world. A number of well documented waterborne zoonotic protozoa exist, including Giardia intestinalis, Cryptosporidium, Toxoplasma gondii, and Entamoeba histolytica. There are other potential candidates including Cyclospora, where waterborne transmission has been demonstrated but a zoonotic route remains to be established. Protozoan pathogens, including microsporidia, amoebae, ciliates, flagellates, and apicomplexans, originating in human or animal faeces have been found in surface waters worldwide The zoonotic protozoa that are emerging or are of renewed interest consequent to their spread associated with water include several species of microsporidia, the amoeba Entamoeba histolytica, Giardia duodenalis (G. lamblia), Toxoplasma gondii, and Cryptosporidium spp. Although Cyclospora cayetanensis is known to be a waterborne threat and has been detected in washings from vegetables contaminated with irrigation water, humans are the only confirmed hosts for this species. Major helminthic zoonoses include nematodes such as ascarids, pinworms, hookworms, strongylids, angiostrongylids, capillarids, and guinea worms, flukes such as schistosomes and liver flukes, and tapeworms such as the beef, pork, and fish tapeworms, as well as cystic and alveolar hydatid tapeworms. Poor sanitation and poor water quality facilitate transmission among animals and humans. Water purification The ultimate purpose of water purification is inactivation and removal of pathogens such as bacteria, viruses, parasites, microbial toxins and other miscellaneous pathogens, as well as elimination of contaminants that arise into it by the way of its pollution at various levels of the distribution system. Hence routine analysis of water is a mandate to assess the number of pathogens in the water, to select a suitable treatment facility to assure the consumers about wholesomeness of the water. Since decades, a composite system known as multiple barrier concept have been in use for the purification of water, which

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holds good till today. This includes protection of the source of water, coagulation, flocculation, sedimentation, filtration, disinfection, and finally protection of the water distribution systems. But the recent epidemiological data indicate lacunae in such traditional systems and there is a need to modify them by adding multistage filtration and disinfection especially to remove the pathogens and still stringent treatment so to remove the environmental pollutants, contaminants and other miscellaneous substances that have health implications. There is a need to implement HACCP in the water related industries so as to keep the contaminants at the lowest possible limits. Water safety plan has also to set and followed strictly as per the recommendations of the competent authorities. Frameworks need to be strengthened to so as to maintain minimum residual concentrations of the disinfectants in the distribution systems even taking care health implications arising from such chemicals. Keeping in view the public health significance of water, it must pass through the various stages of water purification system. The various water purification processes include are described as follows: Boiling for one minute can kill harmful organisms and thus can be considered as a reliable method. Various halogens such as iodine and chlorine preparations can also serve the function. Iodination is a very effective and convenient method for water purification as it destroys bacteria, viruses and protozoan cysts in concentration temperature and duration dependent destruction of such pathogens (8 mg/liter at 20 0C for 10 minutes). Preparations of iodine such as tincture of iodine (4 drops in a 1 litre of water or one drop for a glass), iodine crystals and tablets can be used for the purpose; but all the halogens are not effective against Cryptosporidium. While using iodine preparations for water purification, proper care must be taken for pregnant women, very young individuals and the persons suffering from thyroid disease or iodine allergy. After the iodine application, the taste due to remaining iodine residues can be eliminated by the use of vit-C tablets, lime or lemon juice. The various processes employed for removal of microbes from the water include (i) pre-treatment by using any process that modifies microbial water quality before, or at the entry to, a treatment plant; (ii) coagulation, flocculation and sedimentation by which small particles interact to form larger particles and settle

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out by gravity; (iii) ion exchange used for the removal of calcium, magnesium and some radionuclide; (iv) Granular filtration, in which water passes through a bed of granular materials after coagulation pretreatment; (v) slow sand filtration, in which water is passed slowly through a sand filter by gravity, without the use of coagulation pretreatment. Pre-treatment Pre-treatment of water (roughing filters, microstrainers, off-stream storage and bank infiltration), help in the removal of algae, turbidity, viruses and protozoan cysts. During pretreatment a variety of treatment s are undertaken that vary in their complexity and may vary from disinfection to membrane filtration. Roughing employed for pretreatment are filters derived from rock or gravel that are used prior to filtration (slow sand) process to reduce turbidity (up to 6090%), coliform count (93-99.5%), algal cell (37%), total chlorophyll (53%). Further; color, organic carbon, and the turbidity can still be reduced by the use of alum coagulant. Micro strainers are made of fabric meshes woven of stainless steel or polyester wires and many large sized protozoa such as Balantidium coli, but smaller pathogens such as bacteria or viruses can not be removed and these also reduce turbidity (520%) which can even be enhanced by the use of coagulants (alum). The quality of water in the off-stream storage reservoirs that feed the potable water source directly or indirectly feeds a potable water intake is determined by the physical, biological and chemical processes taking place in it. The algal growth, influx of nitrogen, phosphorous and other contaminants and the faecal contamination at or near surroundings should be limited even attempts should be made to reduce birds. If properly stored at off-storage reservoirs there can be significant reduction in the counts of Cryptosporidium, E.coli, Giardia, and entero-viruses. Further, storage of water in divided reservoirs is better compared to single large reservoir. A process of surface water seeping from the bank or bed of a river or lake to the reduction wells of a water treatment plant is known as Bank infiltration which is used in some of the European countries. This process reduces Giardia,

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Cryptosporidium, Clostridia, bacteriophase and certain viruses such as Entero and Reoviruses. Coagulation, flocculation and sedimentation Coagulation, flocculation and sedimentation are used in conjunction with subsequent filtration. Coagulation promotes the interaction of small particles to form larger particles. In practice, the term refers to coagulant addition (i.e. addition of a substance that will form the hydrolysis products that cause coagulation), particle destabilization and inter-particle collisions. Flocculation is the physical process of producing inter-particle contacts that lead to the formation of large particles. Sedimentation is a solidliquid separation process, in which particles settle under the force of gravity. Most bacteria and protozoa can be considered as particles, and most viruses as colloidal organic particles that are eliminated by such processes. Conventionally, clarification refers to chemical addition, rapid mixing, flocculation and sedimentation. Here the chemical coagulation is critical for effective removal of microbial pathogens, in the absence of a chemical coagulant; removal of microbes is low because sedimentation velocities are low. When properly performed, coagulation, flocculation and sedimentation can result inconsiderable removals of bacteria, viruses and protozoa. However, Cryptosporidium and Giardia are found at very low levels, and methods for their detection have limitations use of coagulants further helps in the reduction of turbidity. Removal of bacteria (E. coli vegetative cells and Clostridium perfringens spores) and protozoa (Giardia cysts and Cryptosporidium oocysts) is possible but this can be achieved by the use of iron-based coagulants which are slightly more efficient than alum (aluminum hydroxide) or poly-aluminum chloride (PACl); however, coagulation conditions (i.e. dose, pH, temperature, alkalinity, turbidity and the level and type of natural organic matter) affect the efficiency of removal. High-rate clarification involves using smaller basins and higher surface loading rates than conventional clarifiers, and is therefore referred to as high rate clarification. Processes include floc-blanket sedimentation (also known as solidscontact clarification), ballasted-floc sedimentation, and adsorption or contact clarification. In floc-blanket sedimentation, a fluidized blanket increases the particle concentration, thus increasing the rate of flocculation and sedimentation.

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Ballasted-floc systems combine coagulation with sand, clay, magnetite or carbon to increase the particle sedimentation rate. Adsorption or contact clarification involves passing coagulated water through a bed where particles attach to previously adsorbed material. Such processes help in the removal of algae, Cryptosporidium and Giardia. In dissolved air flotation (DAF), bubbles are produced by reducing pressure in a water stream saturated with air. The rising bubbles attach to flocparticles, causing the agglomerate to float to the surface, where the material is skimmed off DAF can be particularly effective for removal of algal cells and Cryptosporidium oocysts. Precipitative lime softening is a process in which the pH of the water is increased (usually through the addition of lime or soda ash) to precipitate high concentrations of calcium and magnesium. Removal and reduction in the viability of disinfection efficiency of Giardia, viruses and coliform bacteria is achieved. In-line coagulation can be used with high-quality source waters (e.g. those where turbidity and other contaminant levels are low). The coagulants are added directly to the raw water pipeline before direct filtration. Ion exchange is a treatment process in which a solid phase pre-saturant ion is exchanged for an unwanted ion in the untreated water. The process is used for water softening (removal of calcium and magnesium), removal of some radionuclides (e.g. radium and barium) and removal of various other contaminants (e.g. nitrate, arsenate, chromate, selenate and dissolved organic carbon). The effectiveness of the process depends on the background water quality, and the levels of other competing ions and total dissolved solids. Filtration using a wide variety of filters removes sand, clay and other matter as well as organisms by means of small pore size membranes, adsorption, exchange resins and osmosis. They effectively remove bacteria and parasites but not viruses. Good filters are effective against Cryptosporidia and Giardia. Due to the inability to remove viruses, filtered water must also be chemically treated or boiled and hence many a times filtration is combined with other chemical sterililants such as iodine (or chlorine) hence, modern filters incorporate chemical disinfection, which is usually achieved by passing water through iodine exchange resins. When negatively charged contaminants contact the iodine resin, iodine is

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instantly released so killing the microorganisms without large quantities of iodine being in solution. Various filtration processes (diatomaceous earth; micro-filtration; nanofiltration; reverse osmosis; ultra-filtration) are used in drinking-water treatment. Filtration can act as a consistent and effective barrier for microbial pathogens. Granular high rate media filtration is the most widely used filtration process in drinking water treatment. Under optimal conditions, a combination of coagulation, flocculation, sedimentation and granular media filtration can result better removal of protozoan pathogens with chlorine-resistant cysts. The use of slow sand filtration to protect drinking-water consumers from microbial risk was well established more than 100 years ago. Numerous disease outbreaks due to chlorine-resistant protozoan pathogens in the past two decades have increased interest in slow sand filtration because of its ability to remove parasites. It can provide some degree of protection against microbial pathogens reducing bacteria, protozoa (Cryptosporidium, Giardia) and turbidity. Pre-coat filtration was developed by the US Army during World War II as a portable unit for the removal of Entamoeba histolytica (a protozoan parasite prevalent in the Pacific war zone) from drinking-water. The process involves forcing water under pressure or by vacuum through a uniformly thin layer of filtering material pre-coated onto a permeable, rigid, supporting structure (referred to as a septum). Diatomite grades used for drinking-water treatment have a mean pore diameter of 17 m. Pre-coat filtration can remove protozoan parasites such Giardia very effectively and the removal of Cryptosporidium can be significant, but because organism is smaller than Giardia, it is more difficult to remove. In membrane filtration, a thin semi-permeable film (membrane) is used as a selective barrier to remove contaminants from water. There are very few contaminants that cannot be removed by membrane processes. For the past two decades, the use of membrane filtration in drinking-water treatment (including pathogen removal) has been growing, due to increasingly stringent drinking-water regulations and decreasing costs of purchasing and operating membrane filters. The membrane processes most commonly used to remove microbes from drinking-water are micro-filtration (pore size 0.1 m or more), ultra-filtration ((pore

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size 0.01 m or more), nano-filtration (NF) and reverse osmosis (RO). Membrane filtration eliminates most of bacteria, virus, protozoa and algae. Bag, cartridge and fibrous filters are widely used in the recent past. A bag filter is one that has a non-rigid fabric medium for the filter. Water flow is usually pressure-driven from the inside of the filter bag to the outside. A cartridge filter is one that has a rigid fabric medium or membrane for the filter. In this type of filter, water flow is usually pressure-driven from the outside of the filter to the inside. Bag and cartridge filters are often developed for small systems and for point-ofuse filtration applications. They are also sometimes applied as a pretreatment process for membrane filtration. Bag filters and cartridge filters remove microorganisms by physical straining. The removal efficiency thus depends primarily on the pore size of the filter medium and on the size of the microbes. A typical pore size range is from 0.2 to 10 m. The pore size of the filter medium is usually designed to be small enough to remove protozoa such as Cryptosporidium and Giardia. Submicron particles, including viruses and most bacteria, can pass through the filters. As water passes through a bag or cartridge filter, pressure drop increases to a level impractical for operation. The bag or cartridge is then replaced by a clean one. Since the removal mechanism is physical straining, chemical pretreatment is usually not required for bag filters and cartridge filters. Straining of large compressible particles can blind the filters and reduce filter life. High turbidity and algae can also clog these filters. These processes are therefore only appropriate for high-quality waters. A pre-filtration process may be employed to remove large particles. Disinfection of water and public health Various disinfectants are used in the treatment of water used for drinking purposes. Water treatment to inactivate pathogenic microbes: The disinfection processes have strong bearing on the final quality of the water used for the drinking purpose viz., (i) pre-treatment oxidation (wherein oxidants are added to water early in the treatment process) (ii) primary disinfection which is a common component of primary treatment of drinking-water, and important because granular filter media do not remove all microbial pathogens from water and (iii) secondary disinfection which is employed to maintain the water quality achieved at the treatment plant throughout the distribution system up to the tap.

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The factors like disinfectant concentration, contact time, temperature and pH influence disinfection efficiency. Further, disinfection kinetics and CT of the disinfectant (CT = concentration x contact time) have practical implications. Increased resistance to disinfection may result from attachment or association of microorganisms to various particulate surfaces, including, (i) macro-invertebrates (Crustacea, Nematoda and Platyhelminthes); (ii) particles that cause turbidity; (iii) algae; (iv) carbon fines and other miscellaneous substances. Primary disinfection A disinfection barrier is a common component of primary treatment of water and is typically a chemical oxidation process, although ultraviolet (UV) irradiation and membrane treatment are gaining increased attention. Different types of disinfectant such as chlorine, monochlorine, chlorine dioxide, ozone, UV light and mixed oxidants can be used various pathogenic microorganisms. Chlorine and silver based preparations destroy most the bacteria (e.g. V.cholerae), but are less effective against viruses (hepatitis A) and cysts (Giardia, amoebic cysts, and Cryptosporidia). Chlorine alone is readily inactivated by organic matter and its action varies with pH. However if used in combination with Phosphoric acid it is more effective and this combination will destroy both Giardia and Cryptosporidia. Chlorine gas and water react to form HOCl and hydrochloric acid (HCl), further HOCl dissociates into the hypochlorite ion (OCl) and the hydrogen ion (H+) which act as a germicide by destroying microorganisms by combining with proteins to form N-chloro compounds and has effects on sulfhydryl groups and convert them to several alpha amino acids by oxidation into a mixture of corresponding nitriles and aldehydes. For nearly 100 years of chlorination of drinking-water has demonstrated the effectiveness of this process for inactivation of microbial pathogens, with the notable exception of Cryptosporidium. Even certain bacteria show a high level of resistance to free chlorine. Spore forming bacteria such as Bacillus or Clostridium are highly resistant when disseminated as spores. Acid-fast and partially acid-fast bacteria such as Mycobacterium and Nocardia can also be highly resistant to chlorine disinfection. Since Gram-positive bacteria have thicker walls than Gram-negative ones the pathogenic group that survives chlorination are gram positive as well as acid fast pathogens. Also,

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enteric viruses are generally more resistant to free chlorine than enteric bacteria due to the protective nature of the particle surface (Coxsackie A2). Protozoan cysts such as Entamoeba histolytica and Giardia lamblia are highly resistant to chlorine disinfection and may require prolonged contact times at high chlorine residuals (23 mg/l) to achieve 99.9% acceptable inactivation and chlorine-based disinfectants are generally not effective at inactivation of Cryptosporidium. Monochloramine interact with nucleic acids or free purine and pyrimidine bases by inducing single and double stranded breaks by transforming activity of DNA and enhanced the sensitivity of DNA to endonuclease cleavage, it also reacts to lesser extent with amino acids. Monochloramine is not recommended as a primary disinfectant because of its weak disinfecting power also it is not effective for inactivation of Cryptosporidium and hence in systems using monochloramine, free chlorine is usually applied for a short time before addition of ammonia, or an alternative primary disinfectant is used (e.g. ozone, chlorine dioxide). Treatment to produce a monochloramine residual poses the risk of nitrite formation in the distribution system, especially in low-flow stagnant areas, because bacteria on surfaces and in deposits may nitrify any slight excess of ammonia. Chlorine dioxide is a strong oxidant that can be used to control iron, manganese and taste and odour causing compounds. It is highly soluble in water (particularly at low temperatures), and is effective over a range of pH values (pH 510). Chlorine dioxide is thought to inactivate microorganisms through direct oxidation of tyrosine, methionyl, or cysteine containing proteins, which interferes with important structural regions of metabolic enzymes or membrane proteins. In water treatment, chlorine dioxide has the advantage of being a strong disinfectant, but not forming THMs or oxidizing bromide to bromate. Chlorine dioxide is roughly comparable to free chlorine for inactivation of bacteria and viruses at neutral pH but is more effective than free chlorine at pH 8.5. Chlorine dioxide is an effective disinfectant for control of Giardia lamblia and Cryptosporidium. Chlorine dioxide forms undesirable inorganic by-products (chlorite and chlorate ions) upon its reaction with constituents of water such as dissolved organic carbon, microbes and inorganic ions. Therefore, a water utility may need to provide additional treatment depending on the level of these inorganic by-products and their specific regulatory requirements.

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Water can also be purified by the use of Ozone, which is very effective in purifying water. Ozone use in water applications for treatment is now easier, more efficient and much less costly. Ozone systems can be applied safely to any home or business in water applications or effective air purification disinfectant. Ozone has been used for more than a century for water treatment, mostly in Europe, although its use is now spreading to okther countries. Ozone in aqueous solution may react with microbes either by direct reaction with molecular ozone or by indirect reaction with the radical species formed when ozone decomposes. Ozone is known to attack unsaturated bonds, forming aldehydes, ketones or carbonyl compounds. Additionally, ozone can participate in electrophilic reactions, particularly with aromatic compounds, and in nucleophilic reactions with many of the components of the microbial cell. Of the vegetative bacteria, Escherichia coli are one of the most sensitive, while Gram-positive cocci (Staphylococcus and Streptococcus), Gram-positive bacilli (Bacillus) and mycobacteria are the most resistant. Mycobacterium avium can be effectively controlled by low doses of ozone (CT=99.9 of 0.10.2 mg/min l1), whereas the organism is highly resistant to free chlorine (CT=99.9 of 5511552 mg/min l1 for water-grown isolates). Viruses are generally more resistant to ozone than vegetative bacteria, although phages appear to be more sensitive than human viruses. For the protozoa Giardia lamblia and Naegleria gruberi, ozone inactivation did not follow linear kinetics, due to an initial latent phase. Ozone is effective for removal of Cryptosporidium. Ozonation is an effective process for destruction of both intracellular and extracellular algal toxins. Essentially complete destruction of microcystins, nodularin and anatoxin-a can be achieved if the ozone demand of the water is satisfied. UV light can be categorized as UV-A, UV-B, UV-C or vacuum-UV, with wavelengths ranging from about 40 to 400 nm. The UV light in the UV-B and UVC ranges of the spectrum (200310 nm) is effective for inactivating microorganisms with maximum effectiveness at around 265 nm. Thymine bases on DNA and ribonucleic acid (RNA) are particularly reactive to UV light and form dimers (thyminethymine double bonds) that inhibit transcription and replication of nucleic acids, thus rendering the organism sterile. Thymine dimmers can be repaired in a process termed photo-reactivation in the presence of light, or dark

26

repair in the absence of light. As a result, the strategy in UV disinfection has been to provide a sufficiently high dosage to ensure that nucleic acid is damaged beyond repair. Adenoviruses are double-stranded DNA viruses and are very resistant to UV inactivation. Typical doses used for drinking-water disinfection would not be effective for treatment of adenoviruses. Similarly, protozoa are also sensitive to UV rays. The use of mixtures of oxidants for microbial inactivation has gained attention as a way to maximize the efficiency of current disinfectants. The chemistry of mixed oxidant production is complex, resulting in a solution of free chlorine, chlorine dioxide, ozone and various oxidation states of chlorine. Secondary Disinfection Secondary disinfection strategy is employed to maintain water quality in distribution systems. The purpose of a secondary disinfectant is to maintain the water quality achieved at the treatment plant throughout the distribution system up to the tap. Secondary disinfection provides a final partial barrier against microbial contamination and serves to control bacterial growth. The practice of residual disinfection has become controversial, with some opponents arguing that if biological stability is achieved and the system is well maintained, the disinfectant is unnecessary. Occasionally, corrosion of iron pipes can influence the effectiveness of chlorine-based disinfectants for inactivation of biofilm bacteria. Microbial quality of drinking-water cannot depend only on maintenance of a residual disinfectant. The extensive nature of the distribution system, with many kilometres of pipe, storage tanks, interconnections with industrial users and the potential for tampering and vandalism, provides opportunities for contamination. Cross-connections are a major risk to water quality. Although the risk can be reduced by vigilant control programs, complete control is difficult to achieve and water utilities worldwide face challenges in maintaining an effective cross-connection control program. Backflow devices to prevent the entry of contaminated water are important as a distribution system barrier. Because of high costs, backflow devices are installed mainly on service lines for facilities that use potentially hazardous substances (e.g. hospitals, mortuaries, dry cleaners and industrial users). Recent research is focusing on transient pressure waves that can result in hydraulic surges in the

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distribution system. These waves have both positive and negative amplitude, meaning that they can create transient negative pressures (lasting only a few seconds) in a distribution system, which may be missed by conventional pressure monitoring. Because these waves travel through the distribution system, any point where water is leaking out of the system is a potential entry point for microbes during the brief period of negative pressure. Conclusions The water used for drinking purpose should not only be visibly clean but also be wholesome and free from microbial as well as non-microbial contaminants. The water purification is never an accident, stringent exercises need to be undertaken so as to keep it away from these contaminants. Various processes used for the purification of water suffer from one or the other lacunae, thus, there is a need to use a composite system which can enhance safety. In the industries, where water is used directly or indirectly for the preparation of food, HACCP system needs to be implemented in order to reduce contamination of water. The source of water should be kept clean and suitable primary purification system should be employed as per the recommendations of the competent authority. In situations, where the secondary disinfection is required, a strategy needs to be first defined and then implemented to keep the pathogens away from the water distribution system. Assurance for the supply of safe water to the consumers should be the prime objective of public health administration in order to safeguard the health of the public from the water associated problems.

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WATER BORNE MICROBIAL DISEASES

V. D. P. RAO Department of Veterinary Microbiology College of Veterinary and Animal Sciences G.B. Pant University of Agriculture & Technology, Pantnagar 263145
Water-borne diseases are infectious diseases spread primarily through contaminated water. Many classes of pathogens excreted in the feces are able to initiate waterborne infections. These are bacterial pathogens, including enteric and aquatic bacteria, enteric viruses, and enteric protozoa. Though these diseases are spread either directly or through flies or filth, water is the chief medium for spread and hence they are termed as water-borne diseases. Water borne microbial diseases are one of the major health hazards mainly in the developing countries. Worldwide, in1995 contaminated water and food caused death of more than three million persons of which more than 80% were among children of 5 years of age (Mary and Ross, 1996). In India, more than 70% of the epidemic emergencies are either water borne or water related. Most intestinal (enteric) diseases are infectious and are transmitted through faecal waste. Pathogens which include virus, bacteria, protozoa, and parasitic worms are disease-producing agents found in the faeces of infected persons. These diseases are more prevalent in areas with poor sanitary conditions. These pathogens travel through water sources and interfuses directly through persons handling food and water. Since these diseases are highly infectious, people looking after an infected patient should maintain extreme care and hygiene. Hepatitis, cholera, dysentery, and typhoid are the more common water-borne diseases that affect large populations in the tropical regions. Water borne bacterial diseases: Clostridium: The bacteria are found in soil, fresh water or marine sediments. The Genus Clostridium is having many species that are pathogenic in animals and human beings that can be classified into neurotoxic, histotoxic, enteropathogenic and enterotoxemia producing Clostridia.

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Neurotoxic clostridia include C. tetani and C. botulinum. C. tetani causes tetanus in animals and humans and leads to synaptic inhibition and muscular spasms. C. botulinum inhibits neuromuscular transmission and leads to flaccid paralysis. Histotoxic group of clostridia includes many species of genus Clostridium causing variety of diseases in animals. C. chauvoie causes black leg in cattle and sheep. C. septicum causes malignant oedema in cattle, pig and sheep and abomasitis in sheep. C. novyi type A causes big head disease in young ram and type B causes infectious necrotic hepatitis (black disease) in sheep and occasionally in cattle. C. haemolyticum causes bacillary haemoglobiniuria in cattle and sheep. C. perfringens type A causes necrotic enteritis in chicken and necrotizing enterocolitis in pigs. Enteropathogenic and enterotoxemia producing Clostridia: This group includes type A to type E. Clostridium perfringens type A causes disease conditions viz., necrotic enteritis in chicken, necrotizing enterocolitis in pigs and canine haemorrhagic gastroenteritis. Type B causes lamb dysentery and haemorrhagic enteritis in calves and foals. Type C causes struck in adult sheep, necrotic enteritis in chickens and haemorrhagic enteritis in neonatal piglets. Type D causes pulpy kidney in sheep, enterotoxaemia in calves, adult goats and kids. Type E is responsible for haemorrhagic enteritis in calves and enteritis in rabbits (Quinn et al. 2002). Listeria: This bacterium can replicate in the environment and can be recovered from herbage, faeces of animals, sewage effluents and bodies of fresh water. L monocytogenes causes encephalitis, abortion, septicaemia or encephalomyelitis mainly in case of sheep, goat and cattle but some times dog, cat, horse and pigs may also get affected. Mycobacteria: Lipid rich wall of mycobacteria is hydrophobic and resistant to adverse environmental influences. The bacteria are found in soil, vegetation and water and are obligate pathogens, shed by infected animal, can survive in environment for long periods. The bacteria cause tuberculosis and J.D. in various species of animals and also in human beings. Legionella and Mycobacterium avium complex (MAC) are environmental pathogens and found have ecological micro in drinking and hot water supplies.

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Leptospira: It can survive in ponds, river surface water, and moist soil and in mud when environment temperature is warm. The bacteria causes abortion, still birth, agalactia, influenza like illness, nephritis in pups, chronic renal disease in dogs, septicaemia in calves, piglets and lambs. In dog and human it causes jaundice and hepatitis. Vibrio spp: Mostly found in brackish and salt water. The emergence in early 1992 of serotype O139 of Vibrio cholerae with epidemic potential in Southern Asia suggests that other than V. cholerae 01 could also getting on epidemic. Along with important human pathogen Vibrio cholerae, there are five more species, which cause enteric infections. Vibrio cholerae is the major pathogen of human beings causing cholera. Vibrio metschnikovii causes enteric disease in chickens. Vibrio anguillarum and some Vibrio spp are pathogens of fish. Escherichia coli: The bacteria have a worldwide distribution, inhabit the intestinal tract of human and animals and contaminate vegetation, soil and water. Such water becomes the most frequent source of infection. Colonization of the intestinal tract by E. coli from environmental sources occurs shortly after birth. These organisms persist as important members in the intestine as normal microflora throughout life. Most strains of E. coli are of low virulence but may cause opportunistic infection in extra intestinal locations such as the mammary gland and urinary tract. Pathogenic strains of E. coli possess virulence factors, which allow them to colonize mucosal surfaces and subsequently produce disease. The main categories of pathogenic strains of E. coli and their clinical effects are as followsEnteric disease Enterotoxigenic E. coli (ETEC): It produces heat labile (LT) and heat stable (ST) enterotoxins. LT induces hypersecretion in gut and ST reduces absorption leading to diarrhoea in neonatal piglets, calves and lambs; also causes post- weaning diarrhoea in pigs. Enteropathogenic E. coli (EPEC): Although nature of toxins of these organisms are uncertain but they are found to cause destruction of microvilli, atrophy and shedding of enterocytes leading to maldigestion, malabsorption and diarrhoea in piglets, lamb and pups.

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Vreotoxigenic E. coli (VTEC): It binds to enterocytes and produces verotoxins viz: VT1, VT2, VT2e leading to damage to vasculature in intestine and in other locations and causes oedema disease in pigs, haemorrhagic enterocolitis in calves, post- weaning diarrhoea in pigs and heamorrhagic colitis-haemolytic uraemic syndrome in man. Necrotoxigenic strains of E. coli: These organisms binds to enterocytes and produces cytotoxic necrotizing factors CNF1 and CNF2 leading to damage to enterocytes and blood vessels and ultimately causes haemorrhagic colitis in cattle, enteritis in piglets and calves, diarrhoea in rabbits and dysentery in horses. Septicaemia: Septicaemic strains of E. coli invade blood stream and causes colisepticaemia in calves, piglets, pups and chickens and watery mouth in lambs and arthritis and meningitis in many species. Non enteric localized disease caused by E. coli: Uropathogenic strains of E. coli: For these bacteria adhesion is required for colonization. Local reaction attributed to endotoxin and exotoxin and causes cystitis in many bitches. Invasion by opportunistic E. coli: They can cause coliform mastitis in cattle and sows, pyometra in bitches and omphelitis in calves, lambs and chicks if they get entry inside the organ. Salmonella: The serotypes occur worldwide and infect many mammals, birds, and reptiles and mainly excreted in faeces. Ingestion is the main route of infection. The organism may be present in water, soil, and raw meat, offal and in vegetable material. Source of environmental contamination is invariably faeces. Salmonellosis is of common occurrence in animals and human and the consequences of infection range from sub-clinical carrier state to acute fatal septicaemia. Salmonella serotypes of clinical importance are as follows: Serotype Salmonella Typhimurium Salmonella Dublin Species affected Humans Animals Cattle Sheep, horses and dogs Disease/ syndrome Food poisoning Enterocolitis and septicaemia Septicaemia, abortion, joint osteomylitis and dry gangrene Enterocolitis and septicaemia ill,

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Salmonella Choleraesuis Salmonella Pullorum Salmonella Gallinarum Salmonella Arizonae Salmonella Enteritidis

Pigs Chicks Adult birds Turkeys Poultry Mammals Humans Sheep

Enterocolitis and septicaemia Bacillary white diarrhoea Fowl typhoid Paracolon infection Sub-clinical infection Clinical infection Food poisioning Abortion

Salmonella Brandenburg

Corynebacterium: It is a gram-positive pleomorphic bacterium that can survive for months in the environment. C. pseudotuberculosis causes caseous lymphadenitis in sheep and goat and ulcerative lymphadenitis in horse and cattle. It is prevalent in Australia, New Zealand, Middle East, Asia, Africa and North and South America. Erysipelothrix rhusiopathiae: Soil and surface water become contaminated with the organism mainly with pig faeces. Bacteria are often present in the slime layer of fish, a potential source of human and animal infection. In sheep the bacteria causes polyarthitis, post-dipping lameness, pneumonia and valvular endocarditis. In case of human beings the organism affects mainly workers of fish and poultry industry or agriculture based occupation. The organism enters through minor cuts and aberration in the skin and leads to local cellulitis known as erysipeloid. In rare cases disease extends to blood leading to joint and heart involvement. Bacillus: The bacteria are sporulated and thus persist in soil and water for a long time. B. anthracis causes anthrax in cattle, sheep, horse and pigs. In human beings the bacteria causes cutaneous, pulmonary and intestinal form of anthrax. Another species, B. cereus causes mastitis in cattle and food poisoning and eye infection in human. The significance of Aeromonas species in the drinking water to the occurrence of acute gastroenteritis need to be evaluated by further epidemiological studies. Shigella: The organism essentially S.sonnei causes bacillary dysentery; stool containing blood and mucus along with heavy inflammation of colonic mucosa, in human, chimpanzees and monkeys. The organism is transmitted by oral-faecal route. Shigella can be found in surface water and drinking water and is highly

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significant mode of transmission in developing countries. Within clean water the organism may survive from 14 days to several weeks (Percival et al. 2004). In addition Giardia, Cryptosporidium, some species of genera Cyclospora, Isospora and of family Microsporidia are emerging as opportunistic pathogens and may have waterborne routes of transmission. Water borne viral diseases : A relatively small group of viruses have been incriminated as cause of acute gastroenteritis in humans and fewer have proven to be true etiologic agents, including rotavirus, calcivirus, astrovirus, and some adenoviruses. More than 15 different groups of viruses, encompassing more than 140 distinct types can be found in the human get. Avian Influenza viruses: Avian Influenza virus has caught attention of the whole world in recent times. These viruses belong to family Orthomyxoviridae and infect domestic birds, wild waterfowls, humans, sea mammals, horses, felines and pigs (Webster, 1997). Wild waterfowls are the known carriers, which carry these viruses in their gut across the continents. They are usually asymptomatic carriers but mortality has been observed in recent outbreaks among these birds also. This virus replicates in gut, which is in contrast to human influenza viruses (Gupta, 2005). The virus is excreted in large quantity from nasal and oral secretions and cloaca of affected birds. Infected waterfowl may be able to excrete up to 3 109 EID50 of virus per gram of faeces. Infection to domestic birds occurs by mixing of these wild birds with local population or droppings of these birds may contaminate the water sources. Avian influenza viruses do not affect human population directly. Infection to humans is spread only after gene assortment with human influenza viruses in swine (Webster, 1997). But in recent outbreaks with H5N1 subtype direct transmission to human beings has been recorded. Therefore, it is possible that humans may get this infection directly from contaminated water bodies. Virus is also excreted in the faeces of affected human beings. Picornaviruses: These viruses belong to Picornaviridae family (Murphy et al. 1999 e). Following Genera of this family are transmitted through water:

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1. Apthovirus: Foot and mouth disease virus (FMD) affects cloven-hoofed animals. Besides other routes of transmission, it is also transmitted by contaminated waters (Schijven et al., 2005). Equine rhinovirus I causes disease similar to FMD virus in equines. 2. Enterovirus: Human poliomyelitis: Polio is a highly contagious disease. Poliovirus can survive in the body, and in raw sewage or freshwater systems; polio is frequently found in areas where raw sewage directly enters a water source without treatment. Transmission of the virus occurs either by direct person-to-person contact, or by indirect contact with infectious saliva or faeces, or with contaminated sewage or water (Cliver, 1997; Thapliyal, 1999). Porcine polioencephalomyelitis: causative agent is porcine enterovirus I. Avian encephalomyelitis causes high morbidity and mortality in affected flock. Mode of transmission is by faecal-oral route (Murphy et al. 1999 e). VIRAL GASTROENTERITIS: Rotaviruses: Rotaviruses belong to RNA virus family Reoviridae (Murphy et al. 1999a). Infection has been reported all over the world. These are classified into seven groups; from A to G. Transmission is through faecal-oral route. Virus is excreted in the faeces of infected animals in high titers. Virus can survive in the faeces for several months. Therefore, contaminated water and poor sanitary conditions are responsible for its transmission. Group A viruses affect multiple species of mammals and birds. Group B viruses show species specificity. They may infect cattle, sheep, swine and man. Group C viruses are present in swine and man, group E viruses in swine whereas group D, E and F affect chickens (Thapliyal, 1999; Hill-king, 2005). The virus affects villi of proximal part of small intestine resulting in malabsorption and severe diarrhoea. In animals, disease is referred as white scours or milky scours and mainly affects young ones. Faeces of affected animals are voluminous soft or liquid. Young animals may die as a result of dehydration or secondary bacterial infection (Murphy et al., 1999 a).

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In human beings it accounts for significant proportion of diarrhoea cases in children. It affects children during Ist four weeks of life. Death may occur due to dehydration (Hill-king, 2005). Caliciviruses: Water borne caliciviruses have been incriminated in the cases of diarrhoea in adults and older children. These RNA viruses are the members of the family Caliciviridae (Murphy et al. 1999 b). Calciviruses and some protozoan agents such as Cryptosporidium, are the best candidates to reach the highest levels of endemic transmission, because they are ubiquitous in water intended for drinking, being highly resistant to chemical disinfecting procedures. Norovirus: These were earlier known as Norwalk virus and Norwalk like viruses and are recognized as major causes of water-borne illnesses world-wide. Main feature of these infections is severe vomiting. Aerosol infection may also occur (Hederberg and Osterholm, 1993). Sappovirus: Main feature of infection is persistent watery diarrhoea. It can infect young children also. Virus is excreted in the faeces of affected persons. Mortality is usually less. Adenoviruses: These are the members of family Adenoviridae (Murphy et al., 1999c). Enteric adenoviruses of human beings are second most common cause of viral diarrhoea. These enteric viruses are usually non- cultivable and cause severe watery diarrhoea in children of one to two years of age. Infection is usually faecooral but nosocomial infection may also occur through contaminated fomites. Virus is excreted in faeces and urine (Hill-king, 2005). In animals and birds these are associated with respiratory and gastrointestinal tract infections (Murphy et al., 1999c). Infectious canine hepatitis: Fever, vomiting, diarrhoea, petechial haemorrhages and jaundice in pups mark canine adenovirus-1 infection. Virus is excreted in high concentration in faeces. Fowl adenoviral infections: fowl adenoviruses have been categorized in three serogroups.

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Group I adenoviruses are associated with fowl, geese, ducks and turkeys. In fowls, 12 serotypes of this virus are associated with inclusion body hepatitishydropericardium syndrome. Virus has natural tropism for liver of poultry. The disease is characterized by hydropericardium, severe hepatitis, anaemia and sometimes-yellowish diarrhoea. Mortality is usually high in broiler birds. Group II adenoviruses are associated with haemorrhagic enteritis of turkeys and marble spleen disease of pheasants. The disease is usually acute and there is sudden onset of bloody diarrhoea. Group III adenoviruses are associated with egg-drop syndrome in poultry. Virus infects the pouch shell gland of oviduct resulting in decreased thickness of eggs. Equine adenoviruses: sometimes cause mild diarrhoea in horses. Astroviruses: These RNA viruses are the members of Astroviridae family. These have wide host range and are present in gastrointestinal tract of young ones of almost every mammalian species and young ducklings. Affected animal may develop mild diarrhoea which is not life threatening. But in ducklings of less than 6 week of age it may cause severe hepatitis (Murphy et al., 1999d). In human beings, it usually infects children and diarrhoea is of mild nature (Hill-King, 2005). VIRAL HEPATITIS: Hepatitis A, Hepatitis E and Hepatitis F viruses are transmitted by contaminated water (Cliver, 1997). Hepatitis A virus (HAV) and Hepatitis E virus (HEV) known to cause illness unrelated to the get epitheliums. Numerous large outbreaks have been documented in the U.S. between 1950 and 1970, and incidence rate has strongly declined in the developing countries since the 1970s. Hepatitis E is mostly confined to tropical and subtropical areas, but recent reports indicate that it can occur at a low level in Europe. These cause mild form of hepatitis in adult humans. Hepatitis E virus may sometimes cause fatal disease in pregnant ladies. Infection in children is usually asymptomatic. Virus is excreted in the faeces. Water-borne epidemics and health hazards in the aquatic environment are mainly due to improper management of water resources. Proper management of

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water resources has become the need of the hour as this would ultimately lead to a cleaner and healthier environment (Mara and Huran, 2003). In order to prevent the spread of water-borne diseases, people should take adequate precautions. The city water supply should be properly checked and necessary steps be taken to disinfect it. Water pipes should be regularly checked for leaks and cracks. At home, the water should be boiled; filtered or other necessary steps should be taken to ensure that it is free from infectious agents (Environmental protection agency 1975). References: Cliver, D.A. (1997). Viral transmission via food. Food tech. 51 (4): 71-78. Gupta, S. and Arvind Nath (2005). Human disease due to an avian influenza virus: The influenza (H5N1) virus. ICMR bulletin 34(2-3): 13-18. Environmental Protection Agency. 40 CFR Part 141. Water programs: national interim primary drinking water regulations. Federal Register 1975;40:5956674. Hederberg, C.W. and Osterholm, M.T. (1993). Outbreak of food-borne and water borne viral gastroenteritis. Clinic. Microbial. Rev. 6(3). 199-210. Hill-king, L. (2005). Viral diarrhea. The biomedical scientist 5: 462-466. Leclerc, H. Schwartzbrod, L. and Dei-Cas, E. (2002). Microbial agents associated with Waterborne diseases. Crit Rev Microbial. 28(4): 371-409 Mary, A and Ross, M.A. (1996) Microbiological water pollution. Health effect review 1(7). Pp 1-2 Mara D. and Huran N. (2003). Faecal indicator organism. In: Handbook of water and water born disease. Academic press. Pp193-208 Murphy, F.A.; Gibbs, E.P.J.; Horzinek, M.C. and Studdert, M.J. (1999a). Reoviridae. In: Veterinary Virology. 3rd edn. Academic press. Pp 391-404. Murphy, F.A.; Gibbs, E.P.J.; Horzinek, M.C. and Studdert, M.J. (1999b). Caliciviridae. In: Veterinary Virology. 3rd edn. Academic press. Pp 533-542. Murphy, F.A.; Gibbs, E.P.J.; Horzinek, M.C. and Studdert, M.J. (1999c). Adenoviridae. In: Veterinary Virology. 3rd edn. Academic press. Pp 327334.

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Murphy, F.A.; Gibbs, E.P.J.; Horzinek, M.C. and Studdert, M.J. (1999d). Astroviridae. In: Veterinary Virology. 3rd edn. Academic press. Pp 543-545. Murphy, F.A.; Gibbs, E.P.J.; Horzinek, M.C. and Studdert, M.J. (1999e). Picornaviridae. In: Veterinary Virology. 3rd edn. Academic press. Pp 391404. Percival S.L., Chalmers R.M., Embrey M. Hunter P.R., Sellwood and WynJones P. (2004). Shigella species. In: Microbiology of water born disease. Academic press. Pp 185-196 Pontius F.W., Roberson J.A. (1994) The current regulatory agenda: an update. Journal of the American Water Works Association.86:54-63. Quinn P.J., Markey B.K., Carter M.E., Donnelly W.J.C. and Leonard F.C..(2002) Clostridium species. In: Veterinary Microbiology and Microbial Disease.Pp63-106 Schijven, J., Rijs, G. B. J. and De Roda Husman A. M. (2005). Quantitative Risk Assessment of FMD virus transmission via water. Risk analysis 25 (1). 1321. Thapliyal, D.C. (1999). Diseases caused by viruses. In: diseases of animals transmissible to man. 1st edn. International book distributing company, Lucknow. Pp. 57-71. Webster, R.G. (1997). Influenza virus: transmission between species and relevance to emergence of the next human pandemic. Arch. virol. suppl. 13. 105-113.

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Microbiological Analysis of Narmada River: A Case Study

Anjana Sharma Bacteriology Lab, Department of Biosciences, R.D. University, Jabalpur (M.P.) India
River Narmada (21o23i to 24o46i N latitude, 72o32i to 81o 46i E Longitude) is the largest west flowing and the fifth largest river in Peninsula. The total length of the river from the head to its outfall into the sea is 1312 km. The first 1077 km is in M.P, the next 35 km forms boundary between the states of Madhya Pradesh and Maharashtra further 39 km from the boundary between Maharashtra and Gujarat and the rest of the 161 km lies in Gujarat. The basin had an elongated shape almost like a thin ribbon with a maximum length of 953 km east to west and a maximum width of 234 km north to south. River was divided into 11 different stations for the complete study from its origin to end viz. Amarkantak, Dindori , Mandala, Jabalpur, Narsinghpur, Hoshangabad , Omkareshwar, Koral, Neelkantheshwar, Ankleshwar and Dahez was investigated for its Physicochemical and Bacteriological status. We screened the river for seven very important genera of pathogenic potential belonging to the family Aeromonadaceae, Enterobacteriaceae and Vibrionaceae viz. Aeromonas, Enterobacter, Serratia, Shigella, Salmonella, Klebsiella and Vibrioand were identified on the basis of biochemical serological & molecular techniques.These clinically significant microorganisms were investigated for their enzymatic reactions, antibiotic sensitivity/resistance,

hemolytic activity, complement sensitivity/resistance, virulent genes. All the species showed multidrug resistance and the plasmid detected among the isolates were diverse.We screened several virulent genes viz. ipaH, ipaBCD and STX 1 in Shigella species, ctx A, tcp A (classical), tcp A(ESTor) and ompW in Vibrio species, STX 2 gene in Enterobacter isolates, act, hly A and hemolysin was found in Aeromonas isolates. Aeromonas, Enterobacter and Serratia isolates showed complement sensitivity. Phylogenetic diversity of all the three families was investigated using 16S rRNA analysis. This demonstrated the dynamic nature of the population structure and the high level of inter and intra specific diversity of microorganism in river Narmada.

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The microbiological standards for fresh water have been the cornerstone of one of the oldest and the most effective programme for the control of the infectious diseases. Since the pathogenic organisms studied, are autochthonous inhabitants of the aquatic environment, the traditional population indicators cannot access the pollution status of the river water.

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Modern Molecular Tools & Techniques for Detection of Water Borne Pathogens
Anil Kumar Professor & Head Dept of Molecular Biology & Genetic Engg., College of Basic Sciences & Humanities, G.B. Pant Univ. of Agri. & Technology, Pantnagar
"Today we saw our first public water tap. It is now rather run down, but looked like it was beautiful at one time. The spouts were decorated with dragon heads. While we were there a woman with an aluminum jar came to collect some water to take home with her" Annette Dietz Introduction: Increases in population over the past century have placed tremendous pressures on water resources of both the developed and developing world. These pressures include direct contamination from domestic, industrial, and agricultural wastes and less direct effects caused by climate change and other ecological disturbances. Population projections for the next century suggest that these pressures can only increase without appropriate intervention. Development, implementation, and maintenance of low cost, low technology water treatment systems are critical for reduction of global mortality associated with waterborne diseases. Waterborne diseases cannot be eradicated because of the variety of disease agents transmitted by water. Waterborne diseases must be made reportable with active surveillance implemented as well as improved risk assessment methodology used. Waterborne diseases: A status Paul R. Hunter, consultant medical microbiologist and director of the Chester Public Health Laboratory and honorary professor of epidemiology and public health at the University of Central Lancashire, presented World Health Organization data that showed high morbidity and death rates worldwide due to consumption of unsafe drinking water. Currently, about 20% of the worlds population lacks access to safe drinking water, and more than 5 million people die annually from illnesses associated with unsafe drinking water or inadequate sanitation. If everyone had safe drinking water and adequate sanitation services,

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there would be 200 million fewer cases of diarrhea and 2.1 million fewer deaths caused by diarrheal illness each year. The wide variety of microbes recognized since 1980 as waterborne disease agents, including Cryptosporidium, Cyclospora, Escherichia coli O157:H7, Legionella, Helicobacter pylori, hepatitis E virus, Toxoplasma, and others in developed country. Water-borne diseases are among the most recent emerging and reemerging infectious diseases throughout the world and have recently proven to be the biggest health threat worldwide and they contribute between 70- 80% of health problems in developing countries. The most well known water-borne diseases such as cholera, dysentery, and typhoid are the leading causes of morbidity and mortality. The causative agents of water-borne diseases may be bacterial, viral and protozoal in nature, and this is true during both epidemic and endemic periods. The burden of these diseases is most felt in almost all African countries, especially in the tropical areas of the region, including Kenya. The bulk of these have been reported from the other countries in the tropical rain forests, e.g., Tanzania, Uganda and the Central African Republic, Rwanda and Burundi, however, the extent of their problem had not been clearly defined within the health facilities. Accessibility of safe drinking water, particularly among the low-income communities is still a problem in developing countries. Water supply falls short of demand, resulting in many residents using less than what is considered sanitary. Poor waste disposal mechanisms in both urban and rural areas are below satisfactory requirements, and this contributes in the pollution of water sources in the district. Disease causing waterborne pathogens: The following is a list of some of the disease-causing micro-organisms that have been reported or known to cause infection in people after exposure to contaminated water or food. Bacteria The following is a list of the 8 main genera of waterborne bacteria that are harmful to humans. Symptoms include dysentery, vomiting and anorexia. These genera are as given below: Leptospira Mycobacterium avium complex

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Pseudomonas E. coli Vibrio Salmonella Campylobacter Francis Ella Tulare sis

Viruses The following is a list of the 6 groups of viruses which cause gastroenteritis. These have all been found and isolated in contaminated drinking water and beaches. Symptoms include acute vomiting and mild to severe dysentery. Rotaviruses Norwalk-like agents Caliciviruses Astroviruses Small Round Structured Virus (SRSV) Enteric Adenoviruses The following is a list of the 3 main genera of waterborne protozoa that are harmful to humans. They exist independently of the host and are spread through fecal contact. Symptoms include dysentary, vomiting and anorexia. Giardia Entamoeba Cryptospordium This is a list of diseases that do not cause dysentery, but do pose a threat to humans and can be present in contaminated drinking water. Symptoms include lung, liver damage and Weil's disease. Legionellosis Leptospirosis Hepatitis A

Protozoa

Non Dysentery Diseases

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Assessment of water quality For over a century we have been making measurements of microorganisms in water to help lower the incidence of water borne diseases. In some cases this is due to limited water surveillance and water treatment necessary to ensure the microbiological safety of water, but in other cases it is the result of the inadequacy of the methodologies that are used to monitor water quality or failures of water treatment systems. Current methods tend to rely upon indicators, which offer a good margin of safety against most bacterial pathogens but are not effective protectors against some other bacteria (Campylobacter jejuni, Salmonella typhimurium, E. coli, Giardia, Cryptosporidium) viruses, and protozoan parasites. Thus, there is a need to examine newer approaches to monitor the microbiological quality of water that will lead to a reduction of waterborne disease transmission. To be useful, any methodologies for assaying microbial water quality must fulfill the needs of public health and environmental regulators who are charged with oversight of drinking water safety. It is essential when developing molecular methodologies for assessing microbial quality of water that they meet real world needs. The ultimate measure of the value of molecular methodologies is whether they represent improvements over conventional methodologies in terms of achieving a reduction of waterborne diseases and an improvement of human health. Molecular technologies present powerful new tools for assessing the microbial quality of water but their current application to water is very limited. There is a need to define the differing requirements for testing of microorganisms inclusive of the detection of appropriate indicator or specific microorganisms in source waters, the application of detecting microorganisms for the assessment of treatment performance. Limitations of conventional methods: Monitoring and regulation of the microbiology relevant to public health in warm-water recreational pools and other environments has relied primarily on culture-based analyses that specifically target classical indicator organisms. Culture can be successful for assessment of some microbes. Traditional microbiological techniques for the isolation and identification of bacteria from water have depended on obtaining pure cultures. Enrichment and selection step

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are often time consuming and the biochemical identification of a particular species may add several days to the procedure. Such methods are insufficient and inadequate when outbreaks of water borne disease are occurring. A large body of gene sequence-based studies shows that standard enrichment techniques significantly underestimate the actual quantity and diversity of microorganisms in a wide variety of environments. DNA hybridization methods first developed for research use in the molecular biology, such as DNA gene probes used for colony hybridization and the polymerase chain reaction have shorten the time required for analysis, by obviating the need for pure cultures. Molecular method for detection: Detection and identification of micro-organisms are disciplines that complement each other in order to present a classification system that serves both applied and general microbiologists. For detection of waterborne pathogens some basic steps are necessary which are shown as flow chart: Sample Collection

Sample Storage

Sample Preparation

Detection and Analysis

Result Interpretation The foremost element to be taken into account in the development and application of analytical methods in molecular diagnostics is the sample preparation.

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Discussion of some of these methods is relevant at this stage because many of them are distinguished by the relative lack of sample preparation required before the analysis can be performed. However, some methods do require the sample preparation for biological amplification. The biological amplification comprises of extraction stage and culture of pathogen on an enriched medium. The recent and rapid pace of developments in molecular biology has provided new opportunities in diagnostic areas. Modern diagnostic methods are based on high affinity biomolecular interaction between ligand and binder. These include the nucleic acid hybridization (DNA based) and antibody based techniques involving complementary interaction of DNA-DNA, DNA-RNA, RNA-RNA and antigen antibody (Ag Ab) interactions that have been applied to diagnostics of water borne pathogens. 1. Membrane filter detection: The most common indicator of potential pathogen contamination is still the coliform test in its various guises. The Total coliform test has many forms; the most common so far has been the membrane filter method in which a known volume of water is filtered through a 0.45 or 0.22 filter and the filter incubated on M-endo or LES-Endo agar. Red colonies with a metallic sheen are considered coliforms. A variant of this test (the Colilert method) uses ortho-nitrophenyl-betaD-galactosidase (ONPG) and 4-methyl umbelliferyl-beta-D-glucuronide (MUG) for detecting total coliforms and Escherichia coli in a single solution. The coliforms break down ONPG with their beta-galactosidase enzymes releasing the yellow coloured indicator portion of the molecule. If E. coli is also present, the enzyme glucuronidase hydrolyzes the MUG to glucuronide and the indicator portion 4methyl-umbelliferone that fluoresces under ultraviolet light. This permits separate and independent estimates of total coliform and E. coli counts in the same sample. 2. Immunological detection: Immunomagnetic separation: Magnetic beads are coated with either antibody or gene probe. These are then incubated with sample so that binding of specific target take place to the probe .Bound target can then be removed by magnetic separation of the beads. The bacterial or bacterial genome can then be detected by other means. This has

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been applied to the separation of bacteria from water or liquefied samples and will almost certainly, becomes a standard method for processing of water samples. The technique encompasses separation, concentration and specificity of diagnosis.

Fig. Immunomagnetic separation of mycobacterium Immunoassays: Many immunoassays are now available for detecting for water borne pathogens. These are dependent on inherent ability of living systems to produce antibodies against foreign substances (antigens) which are specific for that antigen. There are three main kinds of antibody preparations: polyclonal antibodies, monoclonal antibodies (MAb) and recombinant antibodies. It is the specificity of this interaction that makes it a useful diagnostic tool. These antigens can be proteins, lipid constituents or nucleic acid. Immunoassays can be in a number of method formats: Enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunodiffusion, immunoblotting, latexagglutination and countercurrent immunoelectrophoresis(CIE). Immunofluorescence and immunogold labelling methods rely on visualization of the fluorochrome or gold-

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labelled antibody-antigen interactions by microscopy. These methods can provide useful information on the nature of the antigen in the test sample. Immunoassays offer ease of use, relative low cost and the feasibility of testing large number of specimens rapidly. 3. Nucleic acid based detection: Oligonucleotide-Based Microarrays Microarrays represent an important advance in molecular detection technology, allowing the simultaneous detection of specifically labeled DNAs from many different pathogenic organisms on a small glass slide containing thousands of surface-immobilized DNA probes. Both basic types of microarrays, i.e., immobilized oligonucleotide probes and PCR amplicons, have been used successfully to detect and/or characterize pathogens. As the sensitivity of microarrays hybridized with total genomic DNA from complex mixtures is usually inadequate to provide detection of low pathogen concentrations, the hybridized DNA (target) usually consists of PCR amplicons. This mode of pathogen detection necessitates the combination of many PCRs prior to their hybridization on microarrays. Target DNA amplification with universal primers to ubiquitous genes prior to microarray hybridization can circumvent this limitation. However, within the Enterobacteriaceae, 16S rRNA and cpn60 sequences may share sufficient similarity to generate cross-hybridization reactions, even when short oligonucleotides are used as probes. Polymerase chain reaction (PCR): A detection method has been developed for strict and opportunistic pathogenic bacteria (Salmonella, enterohemorragic Escherichia coli and Aeromonas hydrophila) in raw and treated water. This method is composed of a bacterial DNA purification step followed by PCR detection. Compared to the traditional culture techniques, this method has an enhanced specificity and sensitivity. Furthermore, the simple and rapid protocol of the proposed technique provides results at a fraction of the time required by the traditional culture techniques (24 hours compared with two to six days). However, unlike the culture methods, detection by PCR does not provide information related to the viability of the bacteria, since the detected bacteria can be viable and cultivable, viable but non-cultivable, or dead. The viability concept is very important for interpreting the

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detection of pathogenic bacteria in relation to public health issues. To overcome this limitation, an indirect approach has been developed for assessing the viability of PCR-detected bacteria from water samples. This method is based on the analysis of each sample before and after a 20-hour culture step in a non-selective medium: an increase in the PCR response after cultivation indicates the occurrence of bacterial multiplication and thus demonstrates the viability of the detected bacteria.

Single colony selected

Total DNA extraction

PCR with specific primers PCR+ Salmonella PCRNot Salmonella

Fig. Flow chart for the detection of salmonella direct DNA detection. IS restriction fragment length polymorphism IS are short stretches of DNA that have the ability to copy themselves in a random or semi-random fashion. This means that they are often present in multiple copies in a bacterial chromosome and the pattern of IS distribution can vary significantly from one strain to the next. By using restriction enzymes to specifically cut the bacterial chromosome into fragments, size-separating those fragments by gel electrophoresis, and then probing the fragments with a labelled copy of the IS, it is possible to obtain an IS banding pattern or fingerprint for that

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strain. This technique is used for several environmental mycobacteria. Normally, MAC isolates can be readily discriminated by RFLP Analytical methods 71 analysis using IS1245 and IS1311 (Guerrero et al. 1995), Inter-insertion sequence polymerase chain reaction Different strains have varying distances between the IS copies, depending on the pattern of distribution of each IS within that strain. Inter-IS PCR uses PCR to amplify between adjacent copies of different IS. Outward facing primers are designed to each IS type and a PCR is performed on genomic DNA extracted from the isolate. The resulting amplified DNA fragments are separated and visualized by gel electrophoresis. This method is rapid and simple to perform. It is used for genotype analysis of MAC by targeting IS1245 and IS1311 (Picardeau & Vincent 1996). It also has the advantage of not requiring a high concentration or high quality DNA. Using this technique, it has been possible to genotype strains of 72 Pathogenic Mycobacteria in Water. Random amplified polymorphic DNA (RAPD) This technique uses short oligonucleotides of random sequence in a low stringency PCR reaction to produce a strain-specific pattern of PCR fragments after gel electrophoresis. It is a rapid test that, like PFGE, requires no prior knowledge of the strain. RAPD has been used relatively widely and has shown utility in outbreak investigations (Zhang et al. 2002). There are issues surrounding the reproducibility of this method but attempts have been made to try and standardize the procedure (Ramasoota et al. 2001). Multi-locus sequence typing MLST is a recently developed technique, widely used now for bacterial molecular epidemiological and population genetics studies (Clarke 2002). The technique is analogous to Multi-Locus Enzyme Electrophoresis except that the nucleotide sequences for the genes of housekeeping enzymes are determined rather than looking for differences in the electrophoretic mobility of the enzymes themselves. This technique identifies unique combinations of alleles. A strain displaying a unique allele combination is assigned a sequence type (analogous to a genotype). The method is quite straightforward. As described for 16SrRNA and hsp65 sequencing, DNA is extracted from a strain and then PCR is used to amplify specific gene sequences of approximately 500 bp. It is usual to select

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seven or more distinct loci. The more loci that are analyzed, the greater the level of discrimination. The products are then subjected to nucleotide sequencing and then sequence comparisons are made using combinations of alignment and phylogenetic software. Adaptation of methods to natural waters: In addition to this taxonomic ignorance, several unnamed species cannot grow on presently available culture media, and a number of bacterial cells of culturable species are in a viable and nonculturable state. Thus, detection of bacterial species by culture on agar media gives a grossly distorted view of the bacterial diversity in the environment (including water). Molecular methods targeting nucleic acids are the necessary tools for unveiling bacterial diversity. Ribosomal ribonucleic acids are universally present in bacteria, have diversely conserved portions of their sequences, and occur in about 30 000 copies per cell. Fluorescent oligonucleotide probes can be devised for in situ hybridization (FISH). Such probes can react with all bacteria, a given phylogenetic branch, a genus, or a single species. Different fluorescent labels can be used enabling multicolor reactions. Conclusion: The identification and control of threats posed by waterborne pathogens will require effective molecular technologies, such as those developed in study, present potentially new tools for assessing microbial quality of water, their widespread application to water may depend on several factors. For example, the detection costs must be low and the benefits must outweigh the continued use of conventional methods; the molecular methods must be specific for the microorganisms of concern, which means specifically being able to detect live organisms capable of causing disease; and the sensitivity must be adequate to provide protection against water-borne disease, which means being able to concentrate targets for detection from large volumes of water and to overcome interfering factors that may be present so as to detect very low numbers of microorganisms. The studies have shown that it is possible to detect different types of pathogenic bacteria from water samples within 24 h. The combination of membrane filtration, an enrichment procedure and PCR provided a sensitive, specific and easy method for the detection of pathogens in environmental water samples. Inhibiting substances hampered PCR detection only in a very limited number of samples and these consisted mainly of drinking water and heavily contaminated effluents. The inhibitory substances, however, may be removed by extracting the DNA prior to analysis or by further dilution of the samples.

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Further readings: Alm, E. W., D. B. Oerther, N. Larsen, D. A. Stahl, and L. Raskin. 1996. The oligonucleotide probe database. Appl. Environ. Microbiol. 62:3557-3559. Bayardelle, P., and M. Zafarullah. 2002. Development of oligonucleotide primers for the specific PCR-based detection of the most frequent Enterobacteriaceae species DNA using wec gene templates. Can. J. Microbiol. 48:113-122. Bej, A. K. 2003. Molecular based methods for the detection of microbial pathogens in the environment. J. Microbiol. Methods 53:139-140. Bej, A. K., J. L. DiCesare, L. Haff, and R. M. Atlas. 1991. Detection of Escherichia coli and Shigella spp. in water by using the polymerase chain reaction and gene probes for uid. Appl. Environ. Microbiol. 57:1013-1017. Bekal, S., R. Brousseau, L. Masson, G. Prefontaine, J. Fairbrother, and J. Harel. 2003. Rapid identification of Escherichia coli pathotypes by virulence gene detection with DNA microarrays. J. Clin. Microbiol. 41:2113-2125. Berthelet, M., L. G. Whyte, and C. W. Greer. 1996. Rapid, direct extraction of DNA from soils for PCR analysis using polyvinylpolypyrrolidone spin columns. FEMS Microbiol. Lett. 138:17-22. Brewster, D. H., M. I. Brown, D. Robertson, G. L. Houghton, J. Bimson, and J. C. Sharp. 1994. An outbreak of Escherichia coli O157 associated with a children's paddling pool. Epidemiol. Infect. 112:441-447. Brousseau, R., J. E. Hill, G. Prefontaine, S. H. Goh, J. Harel, and S. M. Hemmingsen. 2001. Streptococcus suis serotypes characterized by analysis of chaperonin 60 gene sequences. Appl. Environ. Microbiol. 67:4828-4833. Burge, H. (1990) J. Allergy Clin. Immunol. 86, 687-701. Call, D. R., M. K. Borucki, and F. J. Loge. 2003. Detection of bacterial pathogens in environmental samples using DNA microarrays. J. Microbiol. Methods 53:235-243. Colwell, R. R., Brayton, P. B. & Al., E. (1985) BioTechnology 3, 817-820. Falkinham, J. O., III. (2003) Emerg. Infect. Dis. 9, 763-767. Flannigan, B., McCabe, E. M. & McGarry, F. (1991) J. Appl. Bacteriol. Symp. Suppl. 70, 61S-73S. Hussong, D., Colwell, R. R., O'Brien, M., Weiss, E. & Pearson, A. D. (1987) BioTechnology 5, 947-950.

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Kujundzic, E., Angenent, L. T., Zander, D. A., Henderson, D. E., Miller, S. L. & Hernandez, M. T. (2005) Air Waste 55, 210-218. Lemarchand, K., L. Masson, and R. Brousseau. 2004. Molecular biology and DNA microarray technology for microbial quality monitoring of water. Crit. Rev. Microbiol. 30:145-172. Leoni, E., Legnani, P. P., Bucci Sabattini, M. A. & Righi, F. (2001) Water Res. 35, 3749-3753. McCabe, K. M., Y. H. Zhang, B. L. Huang, E. A. Wagar, and E. R. McCabe. 1999. Bacterial species identification after DNA amplification with a universal primer pair. Mol. Genet. Metab. 66:205-211. Milton, D. K. (1999) in Bioaerosols: Assessment and Control, eds. Macher, J., Ammann, H. A., Burge, H. A., Milton, D. K. & Morey, P. R. (American Conference of Governmental Industrial Hygienists (ACGIH), Cincinnati, OH). Mukoda, T. J., Todd, L. A. & Sobsey, M. D. (1994) J. Aerosol Res. 25, 15231532. Pace, N. R. (1997) Science 276, 734-740. Rose, C. S., Martyny, J. W., Newman, L. S., Milton, D. K., King, T. E., Jr., Beebe, J. L., McCammon, J. B., Hoffman, R. E. & Kreiss, K. (1998) Am. J. Public Health 88, 1795-1800. Shafer, M. P., Fernback, J. E. & Jensen, P. A. (1998) AIHA J. 59, 540-546. Sherwood, R. L. (2000) in Pulmonary Immunotoxicology, eds. Cohen, M. D., Zelikoff, J. T. & Schlesinger, R. B. (Kluwer, Boston), pp. 181-197. Swerdlow, D. L., B. A. Woodruff, R. C. Brady, P. M. Griffin, S. Tippen, H. D. Donnell, Jr., E. Geldreich, B. J. Payne, A. Meyer, Jr., J. G. Wells, et al. 1992. A waterborne outbreak in Missouri of Escherichia coli O157:H7 associated with bloody diarrhea and death. Ann. Intern. Med. 117:812-819. Van der Giessen, J. W., A. Eger, J. Haagsma, R. M. Haring, W. Gaastra, and B. A. van der Zeijst. 1992. Amplification of 16S rRNA sequences to detect Mycobacterium paratuberculosis. J. Med. Microbiol. 36:255-263. Vora, G. J., C. E. Meador, D. A. Stenger, and J. D. Andreadis. 2004. Nucleic acid amplification strategies for DNA microarray-based pathogen detection. Appl. Environ. Microbiol. 70:3047-3054.

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Tools and Techniques for Purification of Water and Waste Water

Sanjeev Agrawal Professor and Head Department of Biochemistry College of Basic Sciences & Humanities G. B. Pant University of Agri. & Tech., Pantnagar-263145
What is pure water? We know that all life is dependent on water. Water exists in nature in many forms- clouds, rain, snow, ice and fog. Chemically pure water does not exist for any appreciable length of time in nature. When water falls as rain, it picks up small amount of gases, ions, dust and particulate matter from the atmosphere. Then, as it flows over or through the surface layers of the earth, it dissolves and carries with it some or almost everything it touches, including that which is dumped into it by man. These added substances may be arbitrarily classified as biological, chemical (both inorganic and organic), physical and radiological impurities. They include industrial and commercial solvents, metal and acid salts, sediments, pesticides, herbicides, plant nutrients, radioactive materials, decaying animal and vegetable matter etc. and cause hardness, corrosiveness, staining or frothing. They may damage growing plants and transmit disease. Many of these impurities are removed or rendered harmless in municipal drinking water treatment plants. The meaning of pure water varies from person to person. Homeowners are primarily concerned with domestic water problems related to color, odor, taste and safety to family health. Chemist and engineers working for industry are concerned with the purity of water as it relates to scale deposition and pipe corrosion. Regulatory agencies are concerned with setting standards to protect public health. Farmers are interested in the effect of irrigation water on soil, particularly as they influence crop production; hence, they are concerned with the waters total mineral content, proportion of sodium, or content of ions toxic to plant growth. Chemicals in drinking water which are toxic may cause either acute or chronic health effect. An acute effect usually follows a large dose of a chemical

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and occurs almost immediately. Example of acute health effect are nausea, lung irritation, skin rash, vomiting, dizziness and in the extreme, death. The levels of chemicals in drinking water however are seldom high enough to cause acute health effects. They are more likely to cause chronic effects, that occur after exposure to small amount of a chemical over a long period. Example of chronic health effects includes cancer, birth defects, organ damage, disorders of the nervous system and damage to the immune system. Waste water On the other hand, waste water is sewage, storm water and water that has been used for various purposes around the community. Unless properly treated, waste water can harm public health and environment. Chemically, waste water is mostly water by weight. Other materials make up only a small portion of waste water but can be present in large enough quantities to endanger public health and the environment. Because practically any things that can be flushed down a toilet, drain or sewer can be found in sewage water. Even house hold sewage contains many potential pollutants. The waste water components that are most concerned to the community are those that have the potential to cause diseases or detrimental environmental effect. Many different types of organism live in waste water and some are essential contributors to treatment. A variety of bacteria, protozoa and worms work to break down certain carbon based (organic) pollutants in waste water by consuming them. Through this process, organisms turn waste into carbon di oxide, water or new cell growth. Many disease causing viruses, parasites and bacteria also are present in wastewater and enter from almost any where in the community. These pathogens often originate from people and animals, who are infected with or are carriers of a diseases. Gray water or black water from typical home contains enough pathogens to pose a risk to public health. Gastroenteritis can result from a variety of pathogens in waste water. The cases of illnesses caused by the parasitic protozoa Giardia lambia and Cryptosporidium are not usual. The other important waste water related diseases include hepatitis A, typhoid, polio, cholera & dysentery. Outbreaks of these diseases can occur due to drinking water from wells gets polluted by waste water, eating contaminated fish or recreational

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activities in polluted water. Some illness can be spread by animals and insets that come in contact with waste water. Even municipal drinking water source are not completely immune to health risks from waste water pathogens. The water we use never really goes away. In fact, there never will be any more or any less water on earth than there is right now. Which means that all of the waste water generated by our communities each day from houses, farms, business and factories, eventually returns back to the environment to be used again. So when waste water receives inadequate treatment, the overall quality of the worlds water supply suffers. The waste water treatment is a relatively recent practice? Prior to the mid 1800s human and other wastes usually were just dumped or conveyed to the nearest body of water without treatment. As a result, ground water and other sources of drinking and bathing were regularly contaminated with sewage. Epidemics of cholera, typhoid, dysentery and other water born disease killed thousands and out breaks were especially devastating in densely populated areas. After 1854, when the connection between a cholera outbreak and sewage contaminated water was first discovered, better attempts were made to treat and dispose of sewage separately from drinking water. . For this reason, waste water treatment is as important to public health as drinking water treatment. Need of water analysis Clean, fresh drinking water is rapidly becoming a scarce and valuable resource. There are a long list of common ways in which surface water can become contaminated and unsafe, such as through the addition of organic wastes from livestock or even human settlements, through chemical runoff from agriculture lands or through industrial effluents entering a water source. Point sources of pollution, such as pipes discharging contaminated water from a factory into a stream are relatively easy to identify. Non-point pollution sources, such as agriculture runoff that may have many pathways by which it enters a surface water supply, are vary hard to manage. It is becoming necessary to test fresh water streams and reservoirs regularly in order to determine their quality and detect any new contamination from point or non-point sources.

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Water quality assessment Chemical attributes of a waterway can be important indicators of water quality. Chemical attributes of water can affect aesthetic qualities such as how water looks, smells and tastes. Chemical attributes of water can also affect its toxicity and whether or not it is safe to use. Since the chemical quality of water is important to the health of humans as well as the plants and animals that live in and around streams, it is necessary to assess the chemical attributes of water. Assessment of water quality by its chemistry includes measures of many elements of molecules dissolved or suspended in the water. Chemical measures can be used to directly detect pollutants such as lead or mercury. Chemical measures can also be used to detect imbalances within the ecosystem. Such imbalances may indicate the presence of certain pollutants. For example, elevated acidity levels may indicate the presence of acid mine drainage. Commonly measured chemical parameters include pH, alkalinity, hardness, nitrates, nitrites and ammonia, ortho- and total phosphates and dissolved oxygen and biochemical oxygen demand. The presence of fecal coliform, bacteria is also determined using a chemical test. This microscopic organism is too small to detect during the biological assesment.of macroinvertebrate populations. In addition, some chemical measurements actually indicate the physical presence of pollutants in water. These include measurement such as conductivity and density. Wastewater quality indicators Any oxidizable material present in a natural waterway or in an industrial wastewater will be oxidized both by biochemical (bacterial) or chemical processes. The result is that the oxygen content of the water will be decreased. Basically, the reaction for biochemical oxidation may be written as: Oxidizable material + bacteria + nutrient + O2 CO2 + H2O + oxidized inorganics such as NO3 or SO4 Oxygen consumption by reducing chemicals such as sulfides and nitrites is typified as follows: S-- + 2 O2 SO4-NO2- + O2 NO3Since all natural waterways contain bacteria and nutrient, almost any waste compounds introduced into such waterways will initiate biochemical reactions

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(such as shown above). Those biochemical reactions create what is measured in the laboratory as the Biochemical oxygen demand (BOD). Oxidizable chemicals (such as reducing chemicals) introduced into a natural water will similarly initiate chemical reactions (such as shown above). Those chemical reactions create what is measured in the laboratory as the Chemical oxygen demand (COD). Both the BOD and COD tests are a measure of the relative oxygendepletion effect of a waste contaminant. Both have been widely adopted as a measure of pollution effect. The BOD test measures the oxygen demand of biodegradable pollutants whereas the COD test measures the oxygen demand of biogradable pollutants plus the oxygen demand of non-biodegradable oxidizable pollutants. The so-called 5-day BOD measures the amount of oxygen consumed by biochemical oxidation of waste contaminants in a 5-day period. The total amount of oxygen consumed when the biochemical reaction is allowed to proceed to completion is called the Ultimate BOD. The Ultimate BOD is too time consuming, so the 5-day BOD has almost universally been adopted as a measure of relative pollution effect. There are also many different COD tests. Perhaps, the most common is the 4-hour COD. It should be emphasized that there is no generalized correlation between the 5-day BOD and the Ultimate BOD. Likewise, there is no generalized correlation between BOD and COD. It is possible to develop such correlations for a specific waste contaminant in a specific wastewater stream, but such correlations cannot be generalized for use with any other waste contaminants or wastewater streams. Water purification Water purification is the process of removing contaminants from a raw water source. The goal is to produce water for a specific purpose with a treatment profile designed to limit the inclusion of specific materials; most water is purified for drinking purpose. It may also be purified for a variety of other purposes, including to meet the requirements of medical, pharmacology, chemical and industrial applications. Methods include ultra violet light, filtration, water softening,

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reverse osmosis, ultrafiltration, molecular stripping, deionization, and carbon treatment. Water purification may remove: particulate sand; suspended particles of organic matter; Parasites, Giardia; Cryptosporidium; bacteria; algae; virus; fungi; etc. Minerals calcium, silica, magnesium, etc., and Toxic metals lead; copper; chromium; etc. Some purification may be elective in the purification process, including smell (hydrogen sulfide remediation), taste (mineral extraction), and appearance (iron incapsulation). Governments usually dictate the standards for drinking water quality. These standards will require minimum / maximum set points of contaminants and the inclusion of control elements that produce potable drinking water. Ground water (usually supplied as well water) is typically a more economical choice than surface water (from rivers, lakes and streams) as a source for drinking, as it is inherently pre-filtered by the aquifer from which it is extracted. Over large areas of the world, aquifers are recharged as part of the hydrologic cycle. It is not possible to tell whether water is safe to drink just by looking at it. Simple procedures such as boiling or the use of a household charcoal filter are not sufficient for treating all the possible contaminants that may be present in water from an unknown source. Widely varied techniques are available to remove the fine solids, micro-organisms and some dissolved inorganic and organic materials. The choice of method will depend on the quality of the water being treated, the cost of the treatment process and the quality standards expected of the processed water. Distilled water has an average pH of 7 (neither alkaline nor acidic) and sea water has an average pH of 8.3 (slightly alkaline). If the water is acidic (lower than 7), lime or soda ash is added to raise the pH. Lime is the more common of the two additives because it is cheaper, but it also adds to the resulting water hardness. Making the water slightly alkaline ensures that coagulation and flocculation processes work effectively and also helps to minimize the risk of lead being dissolved from lead pipes . Coagulation, flocculation and sedimentation are used in conjunction with subsequent filtration. Coagulation promotes the interaction of small particles to

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form larger particles. Flocculation is the physical process of producing interparticle contacts that lead to the formation of large particles. Sedimentation is a solid-liquid separation process, in which particles settle under the force of gravity. Most bacteria, viruses and protozoa are eliminated by such processes. Conventionally, clarification refers to chemical addition, rapid mixing, flocculation and sedimentation. Here the chemical coagulation is critical for effective removal of microbial pathogens. Removal of bacteria (E.coli vegetative cells and clostridium perfringens spores) and protozoa (Giardia cysts and Cryptosporidium oocysts) is possible but this can be achieved by the use of ironbased coagulants which are slightly more efficient than alum (aluminum hydroxide) or poly-aluminum chloride (PACI); however, coagulation conditions (i.e. dose, pH, Temperature, alkalinity, turbidity and the level and type of natural organic matter) affect the efficiency of removal. Precipitate lime softening is a process in which the pH of the water is increased (usually through the addition of lime or soda ash) to precipitate high concentrations of calcium and magnesium. Removal and reduction in the viability of Giardia, viruses and coliforms is achieved. Filtration After separating most floc, the water is filtered as the final step to remove remaining suspended particles and unsettled floc. The most common type of filter is a rapid sand filter. Water moves vertically through sand which often has a layer of activated carbon or anthracite coal above the sand. The top layer removes organic compounds, which contribute to taste and odour. The space between sand particles is larger than the smallest suspended particles, so simple filtration is not enough. Most particles pass through surface layers but are trapped in pore spaces or adhere to sand particles. Effective filtration extends into the depth of the filter. This property of the filter is key to its operation: if the top layer of sand were to block all the particles, the filter would quickly clog. To clean the filter, water is passed quickly upward through the filter, opposite the normal direction (called backflushing or backwashing) to remove embedded particles. Prior to this, compressed air may be blown up through the bottom of the filter to break up the compacted filter media to aid the backwashing process; this is known as air scouring. This contaminated water can be disposed of, along with the sludge from

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the sedimentation basin, or it can be recycled by mixing with the raw water entering the plant. Some water treatment plants employ pressure filters. These work on the same principle as rapid gravity filters, differing in that the filter medium is enclosed in a steel vessel and the water is forced through it under pressure. Membrane filtration is essentially a thin film of synthetic polymer through which there are pores of fairly uniform size. This filters water as it flows through. Membrane filters are widely used for filtering both drinking water and sewage for reuse. For drinking water, membrane filters can remove virtually all particles larger than 0.2 um--including Giardia and Cryptosporidium. Membrane filters are an effective form of tertiary treatment when it is desired to reuse the water for industry, for limited domestic purposes, or before discharging the water into a river that is used by towns further downstream. They are widely used in industry, particularly for beverage preparation (including bottled water). However no filtration can remove substances that are actually dissolved in the water such as phosphorus, nitrates and heavy metal ions. Ultrafiltration Ultrafiltration membranes are a relatively new development; they use polymer film with chemically formed microscopic pores that can be used in place of granular media to filter water effectively without coagulants. The type of membrane media determines how much pressure is needed to drive the water through and what sizes of micro-organisms can be filtered out. Common microorganisms and the filter size needed: Organism Protozoa Bacteria Examples Giardia, Cryptosporidium Cholera, E. coli, Salmonella Hepatitis A, Viruses rotavirus, Norwalk virus General Size 5 microns or larger 0.20.5 microns 0.004 microns Filter Type Water filter Microfilter Particle Size Rating 1.04.0 microns 0.21.0 microns to 0.004 microns

Water purifier

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Disinfection Disinfection is normally the last step in purifying drinking water. Water is disinfected to kill any pathogens which pass through the filters. Possible pathogens include viruses, bacteria, including Escherichia coli, Campylobacter and Shigella, and protozoans, including G. lamblia and other Cryptosporidia. In most developed countries, public water supplies are required to maintain a residual disinfecting agent throughout the distribution system, in which water may remain for days before reaching the consumer. Following the introduction of any chemical disinfecting agent, the water is usually held in temporary storage tank to allow the disinfecting action to complete. This is done by adding gaseous dissloved chlorine in the water. Chlorine at a concentration of 1 or 2 ppm destroys bacteria and some viruses. Sufficient chlorine is added to the water (with careful monitoring) to ensure that the concentration stays slightly above 1ppm until the water reaches the end user. 1). Chlorination- The most common disinfection method is some form of chlorine or its compounds such as chloramine or chlorine dioxide. Chlorine is a strong oxidant that kills many micro-organisms. Because chlorine is a toxic gas, there is a danger of a release associated with its use. This problem is avoided by the use of sodium hypochlorite, which is either a relatively inexpensive solid that releases free chlorine when dissolved in water. The generation of liquid sodium hypochlorite is both inexpensive and safer than the use of gas or solid chlorine. Both disinfectants are widely used despite their respective drawbacks. One drawback to using chlorine gas or sodium hypochlorite is that they react with organic compounds in the water to form potentially harmful chemical by-products trihalomethanes (THMs) and haloacetic acids (HAAs), both of which are carcinogenic in large quantities. 2). 3). Chlorine dioxide is another fast-acting disinfectant. It is rarely used, because it may create excessive amounts of chlorate and chlorite. Chloramines are another chlorine-based disinfectant. Although chloramines are not as strong of an oxidant or provide a reliable residual, as compared to chlorine gas or sodium hypochlorite, they are less prone to form THMs or haloacetic acids.

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4).

Ozone (O3) is a powerful oxidising agent which is toxic to most water borne organisms. It is a very strong, broad spectrum disinfectant that is widely used in Europe. It is an effective method to inactivate harmful protozoans that form cysts. UV radiation (light) is very effective at inactivating cysts, as long as the water has a low level of colour so the UV can pass through without being absorbed. The main disadvantage to the use of UV radiation is that, like ozone treatment, it leaves no residual disinfectant in the water. Because neither ozone nor UV radiation leaves a residual disinfectant in the water, it is sometimes necessary to add a residual disinfectant after they are used. This is often done through the addition of chloramines.

Additional treatment options 1). 2). Fluoridation Addition of fluoride to water for the purpose of preventing tooth decay.is known as Water fluoridation Water conditioning: This is a method of reducing the effects of hard water. Hardness salts are deposited in water systems subject to heating because the decomposition of bicarbonate ions creates carbonate ions which crystalise out of the saturated solution of calcium or magnesium carbonate. Water with high concentrations of hardness salts can be treated with soda ash (sodium carbonate) which precipitates out the excess salts. 3). Plumbosolvency reduction: In areas of naturally acidic waters ,the water may be capable of dissolving lead from any lead pipes that it is carried in. The addition of small quantities of phosphate ion and increasing the pH slightly both assist in greatly reducing plumbo-solvency by creating insoluble lead salts on the inner surfaces of the pipes. 4). Fluoride Removal: In some areas of the world have excessive levels of natural fluoride is found in the source water. Excessive levels can be toxic or cause undesirable cosmetic effects such as staining of teeth. It can be reduced through treatment with activated alumina. Other water purification techniques Other popular methods for purifying water, especially for local private supplies are listed below. Particularly important are distillation (de-salination of seawater) and reverse osmosis.

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1).

Boiling: Water is heated hot enough and long enough to inactivate or kill micro-organisms that normally live in water at room temperature. Near sea level, a vigorous rolling boil for at least one minute is sufficient. At high altitudes (greater than two kilometers or 5000 feet) three minutes is recommended.

2).

Carbon filtering: Charcoal, a form of carbon with a high surface area, absorbs many compounds including some toxic compounds. Water passing through activated charcoal is common in household water filters and fish tanks. Household filters for drinking water sometimes contain silver to release silver ions which have an anti-bacterial effect.

3).

Distillation involves boiling the water to produce water vapour. The vapour contacts a cool surface where it condenses as a liquid. Because the solutes are not normally vaporised, they remain in the boiling solution. Even distillation does not completely purify water, because of contaminants with similar boiling points and droplets of unvaporised liquid carried with the steam. However, 99.9% pure water can be obtained by distillation.

4).

Reverse osmosis: Mechanical pressure is applied to an impure solution to force pure water through a semi-permeable membrane. Reverse osmosis is theoretically the most thorough method of large scale water purification available, although perfect semi-permeable membranes are difficult to create. Unless membranes are well-maintained, algae and other life forms can colonise the membranes.

5).

Ion exchange: Most common ion exchange systems use a zeolite resin bed to replace unwanted Ca2+ and Mg2+ ions with benign (soap friendly) Na+ or K+ ions. This is the common water softener.

6).

Electrodeionization: Water is passed between a positive electrode and a negative electrode. Ion selective membranes allow the positive ions to separate from the water toward the negative electrode and the negative ions toward the positive electrode. High purity deionized water results. The water is usually passed through a reverse osmosis unit first to remove nonionic organic contaminants. It may be concluded that water is an essential ,precious commodity. It

should be purified before supplying to the community.The water used for the

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purpose of drinking should not only be visibly clean but should also be free from microbes and other contaminants.The best way to ensure clean water is not to pollute it. Various processes used for the purification of water suffer from one or the other lacunae. So we need to use a composite system that can enhance safety.

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