Wastewater Analysis and Study of Soil Microorganisms of Koparkhairane Nullah.

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Dissertation-Bachelors of Technology In Biotechnology

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  • 1. Wastewater Analysis and Study of Soil Microorganisms of Koparkhairane Nullah. The Project Dissertation submitted to Padmashree Dr D.Y.Patil University Department of Biotechnology and Bioinformatics For the degree of Bachelors of Technology In Biotechnology By PRIYESH V. WAGHMARE (BBT-2-06055) Research Guide: Mr. Manish Bhat Padmashree Dr.D.Y.Patil University Department of Biotechnology and Bioinformatics C.B.D. Belapur, Navi Mumbai 400 614 2010 1
  • 2. DECLARATION I, Mr Priyesh Vijay Waghmare, student of Dr. D. Y. Patil Institute forBiotechnology and Bioinformatics, Padmashree Dr. D. Y. Patil University, Navi Mumbaihereby state that to the best of my knowledge and belief, the project report entitled“Wastewater Analysis and Study of Soil Microorganisms of Koparkhairane Nullah.”was carried out under the guidance of Mr. Manish R.Bhat, Assistant Professor and beingsubmitted for partial fulfillment of Bachelor’s of Technology In Biotechnology (2007- 2010).This research project is an original work and has not been submitted in part/ full for any otherdegree/ diploma to any other University to the best of my knowledge.Place: Navi MumbaiDate: 30th June, 2010Mr Priyesh Vijay WaghmareBBT-2-06055Research StudentPadmashree Dr.D.Y.Patil UniversityDepartment of Biotechnology and BioinformaticsC.B.D BelapurNavi Mumbai 2
  • 3. CERTIFICATEThis is to certify that the project entitled “Wastewater Analysis and Study of SoilMicroorganisms of Koparkhairane Nullah.” has been carried out by Mr Priyesh VijayWaghmare in partial fulfillment of his degree of Bachelor of Technology in Biotechnology(B.Tech Biotechnology) at the Dr D. Y. Patil Institute for Biotechnology andBioinformatics, CBD Belapur, Navi Mumbai-400614. This work has been carried outunder my supervision and has not been submitted in part/ full for any other degree/ diplomato any other University.Date: 30th June, 2010Place: Navi Mumbai(Mr. Manish R.Bhat)Asst ProfessorResearch Guide 3
  • 4. ACKNOWLEDGEMENTSIn the first place, I thank God Almighty for His grace and all the blessings that have beenshowered upon me.I am deeply indebted to a number of people who enabled the completion of my project, all ofwhom especially need to be acknowledged for all the support and assistance redeemed to methrough each stage of this project.I owe my thanks to Dr. D.A. Bhiwgade, Dean of the Department of Biotechnology andBioinformatics, Padmashree Dr. D.Y. Patil University, for providing us the opportunity totake our first steps in exploring the field of biotechnology through this project.I avail this opportunity to express sincere reverence and deep sense of gratitude to my guideand mentor, Prof. Manish Bhat for his valuable and indispensable guidance throughout myproject tenure. I am much obliged to him for charting out the course of my work and for hisconstant words of encouragement and inordinate patience. His supervision over thelaboratory practicals and invaluable contribution to the writing of this thesis is greatlyacknowledged.I wish to thank Leena, Hrishikesh, Akshay, Jay, Abhishek, Manish, Vikas, Amit, Sid and toall my peers for their kind co-operation. My thanks are due to all teaching and non-teachingstaff and especially to the laboratory staff for their timely technical support.I am very much obliged to my family, my pillars of strength, for their unwavering faith andwhole hearted support for giving me a choice to pursue a wonderful career. Priyesh Vijay Waghmare. 4
  • 5. ABSTRACT:Physiochemical analysis of waste water was carried out from the effluent sample collectedfrom koparkhairane nullah near MIDC industrial area besides Furnace fabrica Ltd and Alokindustry-Navi Mumbai. Analysis of effluent showed that effluent samples were highlycoloured like dark black and reddish brown, foul smelling, alkaline and having temperatureapproximately 300C with high BOD and COD values. Traces of heavy metal contaminationwas found in textile wastewater Then the isolation and characterization of soilmicroorganisms from koparkhairane nullah and heavy metal tolerance studies of soil isolateswas carried out. The organisms isolated from the soil could belong to Pseudomonas spp,Bacillus spp, Micrococcus spp or Arthobacter spp. Optimization of various parameters likepH, Temperature, growth curve studies and evaluation of heavy metal tolerance of soilisolates was carried out for bioremediation.Key Words:Textile wastewater; BOD; Bioremediation; 5
  • 6. INDEX:Sr. No. Topic Page No. 1 Introduction 11 2 Aims and Objectives 16 3 Literature Review 18 4 Materials and Methods 39 5 Results 66 6 Discussion 85 7 Conclusion 89 8 Future prospects 91 9 Bibliography 93 10 Appendix 98 6
  • 7. LIST OF TABLES AND GRAPHS:1) Table 1. Properties of Waste Water from Textile Chemical Processing. (Page no 20)2) Table 2. Composite textile industry wastewater characteristics. (Page no 20)3) Table 3.Effluent Characteristics From Textile Industry. (Page no 23)4) Table 4. Substances present in industrial effluents. (Page no 24)5) Table 5. Heavy Metals Found in Major Industries. (Page no 30)6) Table 6. Comparative strengths of wastewater from industry. (Page no 30)7) Table 7. Environmental conditions affecting degradation. (Page no 34)8) Table 8: Bioremediation strategies. (Page no 35)9) Table 9: Nutrient Agar Composition. (Page no 49)10) Table 10: Simmon’s Citrate Agar Composition. (Page no 53)11) Table 11: Starch agar medium. (Page no 59)12) Table 12: Milk agar medium. (Page no 60)13) Table 13: Macconkey agar medium. (Page no 61)14) Table 14: Gelatin Agar medium. (Page no 62)15) Table 15: Peptone Agar medium. (Page no 63)16) Table 16: Waste water analysis. (Page no 67)17) Table 17: Soil microorganisms. (Page no 68)18) Table 18: Wastewater microorganisms. (Page no 68)19) Table 19: Gram staining. (Page no 70)20) Table 20: Biochemical Tests. (Page no 70)21) Table 21: Acid production. (Page no 74)22) Table 22: Enzyme assay. (Page no 74)23) Table 23: ZnSO4 Tolerance. (Page no 76)24) Table 24: KCrO4 Tolerance. (Page no 77) 7
  • 8. 25) Table 25: MnSO4Tolerance. (Page no 78)26) Table 26: HgCl2 Tolerance. (Page no 80)27) Table 27: AlCl3 Tolerance. (Page no 81)28) Table 28: Growth at different pH. (Page no 82)29) Table 29: Growth at different Temperatures. (Page no 82)30) Table 30: Effect of various concentration of phenol on growth curve of the organism. (Page no 83)31) Graph 1: Effect of phenol on growth curve ( Page no 84) 8
  • 9. LIST OF FIGURES,DIAGRAMS AND PHOTOS:1) Diagram 1: Cotton Fabric Production & Associated Water Pollutants(Page no 22)2) Figure 1: Citrate Utilization test.(Page no. 69)3) Figure 2: Glucose Utlization Test( Page no. 69)4) Figure 3: Sucrose Utlization Test ( Page no. 71)5) Figure 4: Nitrate Utlization Test ( Page no. 71)6) Figure 5: Casein Hydrolysis Test (Page no. 72)7) Figure 6: Casein Hydrolysis Test (Page no. 72)8) Figure 7: Starch Hydrolysis Test (Page no. 73)9) Figure 8: ZnSO4Tolerance. (Page no. 75)10) Figure 9: KCrO4Tolerance. (Page no. 75)11) Figure 10: HgCl2Tolerance. (Page no. 79)12) Figure 11: HgCl2Tolerance. (Page no. 79) 9
  • 10. LIST OF ABBREVATIONS:1) pH- negative log of H- ion concentration2) ˚C- Degree celcius3) MRL- Maximum resistivity levels4) MIC- Minimum inhibitory concentration5) Mg/L- Milligram per liter6) COD- Chemical oxygen demand7) BOD- Biochemical oxygen demand8) As- Arsenic9) Mn- Manganese10) Ni- Nickel11) Cd- Cadmium12) Cu- Copper13) CO- Cobalt14) Cr-Chromium15) Pb- Lead16) Fe- Iron17) Zn- Zinc18) Hg- Mercury19) Al- Aluminium20) spp.- Species21) µ- Micro22) M- Molar 10
  • 12. INTRODUCTION:Our biosphere is under constant threat from continuing environmental pollution. Impact on itsatmosphere, hydrosphere and lithosphere by anthropogenic activities cannot be ignored. Manmade activities on water by domestic, industrial, agriculture, shipping, radio-active,aquaculture wastes; on air by industrial pollutants, mobile combustion, burning of fuels,agricultural activities, ionization radiation, cosmic radiation, suspended particulate matter;and on land by domestic wastes, industrial waste, agricultural chemicals and fertilizers, acidrain, animal waste have negative influence over biotic and abiotic components on differentnatural ecosystems. Some of the recent environmental issues include green house effect, lossin bio-diversity, rising of sea level, abnormal climatic change and ozone layer depletion etc.In recent years, different approaches have been discussed to tackle man made environmentalhazards. Clean technology, eco-mark and green chemistry are some of the most highlightedpractices in preventing and or reducing the adverse effect on our surroundings.Among many engineering disciplines – Civil Engineering, Mechanical Engineering,Electrical Engineering etc., Textile Engineering has a direct connection with environmentalaspects to be explicitly and abundantly considered. The main reason is that the textileindustry plays an important role in the economy of the country like India and it accounts foraround one third of total export. Out of various activities in textile industry, chemicalprocessing contributes about 70% of pollution. It is well known that cotton mills consumelarge volume of water for various processes such as sizing, desizing, scouring, bleaching,mercerization, dyeing, printing, finishing and ultimately washing. Due to the nature ofvarious chemical processing of textiles, large volumes of waste water with numerouspollutants are discharged. Since these streams of water affect the aquatic eco-system innumber of ways such as depleting the dissolved oxygen content or settlement of suspendedsubstances in anaerobic condition, a special attention needs to be paid. Thus a study ondifferent measures which can be adopted to treat the waste water discharged from textilechemical processing industries to protect and safeguard our surroundings from possiblepollution problem has been the focus point of many recent investigations.[5] 12
  • 13. Sources of industrial wastewater:Iron and steel industry:The production of iron from its ores involves powerful reduction reactions in blast furnaces.Contamination of waste streams includes gasification products suchas benzene, naphthalene, anthracene, cyanide, ammonia, phenols, cresols together with arange of more complex organic compounds known collectively as polycyclic aromatichydrocarbons (PAH).Mines and quarries:Some specialized separation operations, such as coal washing to separate coal from nativerock using density gradients, can produce wastewater contaminated by fineparticulate haematite and surfactants. Oils and hydraulic oils are also common contaminants.Food industry:Wastewater generated from agricultural and food operations has distinctive characteristics, itis biodegradable and nontoxic, but that has high concentrations of biochemical oxygendemand (BOD) and suspended solids (SS).Complex organic chemicals industry:A range of industries manufacture or use complex organic chemicals. Theseinclude pesticides, pharmaceuticals, paints and dyes, petro-chemicals, detergents, plastics, paper pollution, etc. Waste waters can be contaminated byfeed-stock materials, by-products, product material in soluble or particulate form, washingand cleaning agents, solvents and added value products such as plasticisers.Water treatment:Water treatment produces organic and mineral sludges from filtration and sedimentation. Ionexchange using natural or synthetic resins removes calcium, magnesium and carbonate ionsfrom water, replacing them with hydrogen and hydroxyl ions. Regeneration of ion exchangecolumns with strong acids and alkalis produces a wastewater rich in hardness ions which arereadily precipitated out, especially when in admixture with other wastewaters. 13
  • 14. Agricultural waste:Pesticides are widely used by farmers to control plant pests and enhance production, butchemical pesticides can also cause water quality problems. Farms withlarge livestock and poultry operations, such as factory farms, can be a major source of pointsource wastewater.[25]Hazards:Water pollution has number of effects. The effects could be classified as:1] Effects on ecosystem2] Effects on animal health3] Effects on human healthVirtually all types of water pollution are harmful to the health of humans and animals. Waterpollution may not damage our health immediately but can be harmful after long termexposure. Different forms of pollutants affect the health of animals in different ways: Heavymetals from industrial processes can accumulate in nearby lakes and rivers. These are toxic tomarine life such as fish and shellfish, and subsequently to the humans who eat them. Heavymetals can slow development; result in birth defects and some are carcinogenic. Industrialwaste often contains many toxic compounds that damage the health of aquatic animals andthose who eat them. Some of the toxins in industrial waste may only have a mild effectwhereas other can be fatal. They can cause immune suppression, reproductive failure or acutepoisoning. Microbial pollutants from sewage often result in infectious diseases that infectaquatic life and terrestrial life through drinking water. Microbial water pollution is a majorproblem in the developing world, with diseases such as cholera and typhoid fever being theprimary cause of infant mortality. Organic matter and nutrients causes an increase in aerobicalgae and depletes oxygen from the water column. This causes the suffocation of fish andother aquatic organisms. Sulfate particles from acid rain can cause harm the health of marinelife in the rivers and lakes it contaminates, and can result in mortality. Suspended particles infreshwater reduces the quality of drinking water for humans and the aquatic environment formarine life. Suspended particles can often reduce the amount of sunlight penetrating thewater, disrupting the growth of photosynthetic plants and micro-organisms. Someorganochlorine pesticides (like DDT, BHC, Endrin) are known for bioaccumulative andbiomagnifiable characters. [21] 14
  • 15. Bioremediation:By definition, bioremediation is the use of living organisms, primarily microorganisms, todegrade the environmental contaminants into less toxic forms. It uses naturally occurringbacteria and fungi or plants to degrade or detoxify substances hazardous to human healthand/or the environment. The microorganisms may be indigenous to a contaminated area orthey may be isolated from elsewhere and brought to the contaminated site. Contaminantcompounds are transformed by living organisms through reactions that take place as a part oftheir metabolic processes. Biodegradation of a compound is often a result of the actions ofmultiple organisms.[10]Types of Bioremediation: Biostimulation Bioaugmentation Intrinsic BioremediationAdvantages of bioremediation Bioremediation is a natural process and is therefore perceived by the public as an acceptable waste treatment process for contaminated material such as soil. Microbes able to degrade the contaminant increase in numbers when the contaminant is present; when the contaminant is degraded, the biodegradative population declines. Theoretically, bioremediation is useful for the complete destruction of a wide variety of contaminants. Many compounds that are legally considered to be hazardous can be transformed to harmless products. This eliminates the chance of future liability associated with treatment and disposal of contaminated material. Instead of transferring contaminants from one environmental medium to another, for example, from land to water or air, the complete destruction of target pollutants is possible. Bioremediation can often be carried out on site, often without causing a major disruption of normal activities. This also eliminates the need to transport quantities of waste off site and the potential threats to human health and the environment that can arise during transportation. Bioremediation can prove less expensive than other technologies that are used for clean-up of hazardous waste.[10] 15
  • 17. AIMS & OBJECTIVES:1) Collection of wastewater & soil sample from KoparKhairane Nullah.2) Physiochemical Analysis of wastewater.3) Isolation and characterization of soil microorganisms.4) Study of heavy metal tolerance of soil isolates.5) Optimization of various parameters for bioremediation. 17
  • 19. LITERATURE REVIEW:Textile Effluent:Sources and Causes of Generation of Textile Effluent:Textile industry involves wide range of raw materials, machineries and processes to engineerthe required shape and properties of the final product. Waste stream generated in this industryis essentially based on water-based effluent generated in the various activities of wetprocessing of textiles. The main cause of generation of this effluent is the use of huge volumeof water either in the actual chemical processing or during re-processing in preparatory,dyeing, printing and finishing. In fact, in a practical estimate, it has been found that 45%material in preparatory processing, 33% in dyeing and 22% are re-processed in finishing. Thefact is that the effluent generated in different steps is well beyond the standard and thus it ishighly polluted and dangerous.[5]Textile wastewater includes a large variety of dyes and chemicals additions that make theenvironmental challenge for textile industry not only as liquid waste but also in its chemicalcomposition (Venceslau et al., 1994). Main pollution in textile wastewater came from dyeingand finishing processes. These processes require the input of a wide range of chemicals anddyestuffs, which generally are organic compounds of complex structure. Because all of themare not contained in the final product, became waste and caused disposal problems. Majorpollutants in textile wastewaters are high suspended solids, chemical oxygen demand, heat,colour, acidity, and other soluble substances. The removal of colour from textile industry anddyestuff manufacturing industry wastewaters represents a major environmental concern. Inaddition, only47% of 87 of dyestuff are biodegradable (Pagga and Brown, 1986). It has been documentedthatresidual colour is usually due to insoluble dyes which have low biodegradability as reactiveblue21, direct blue 80 and vat violet with COD/BOD ratio of 59.0, 17.7, and 10.8, respectively.[32]The textile sector has a high water demand. Its biggest impact on the environment is relatedto primary water consumption (80–100 m3/ton of finished textile) and waste water discharge(115– 175 kg of COD/ton of finished textile, a large range of organic chemicals, low 19
  • 20. biodegradability, colour, salinity). Therefore, reuse of the effluents represents an economicaland ecological challenge for the overall sector (Li Rosi et al., 2007).[6] Table 1. Properties of Waste Water from Textile Chemical Processing:[5]Table 2. Composite textile industry wastewater characteristics:[2]Categorization of Waste Generated in Textile Industry:Textile waste is broadly classified into four categories, each of having characteristics thatdemand different pollution prevention and treatment approaches. Such categories arediscussed in the following sections:1. Hard to Treat WastesThis category of waste includes those that are persistent, resist treatment, or interfere with theoperation of waste treatment facilities. Non-biodegradable organic or inorganic materials arethe chief sources of wastes, which contain colour, metals, phenols, certain surfactants, toxicorganic compounds, pesticides and phosphates.The chief sources are: Colour & metal-dyeing operation 20
  • 21.  Phosphates- Non-biodegradable organic materials-Since these types of textile wastes are difficult to treat, the identification and elimination oftheir sources are the best possible ways to tackle the problem. Some of the methods ofprevention are chemical or process substitution, process control and optimization,recycle/reuse and better work practices2. Hazardous or Toxic WastesThese wastes are a subgroup of hard to treat wastes. But, owing to their substantial impact ontheenvironment, they are treated as a separate class. In textiles, hazardous or toxic wastesinclude metals, chlorinated solvents, non-biodegradable or volatile organic materials. Someof these materials often are used for non-process applications such as machine cleaning.3. High Volume WastesLarge volume of wastes is sometimes a problem for the textile processing units. Mostcommon large volume wastes include:• High volume of waste water• Wash water from preparation and continuous dyeing processes and alkaline wastes frompreparatory processes.• Batch dye waste containing large amounts of salt, acid or alkaliThese wastes sometimes can be reduced by recycle or reuse as well as by process andequipment modification.4. Dispersible Wastes:The following operations in textile industry generate highly dispersible waste:• Waste stream from continuous operation (e.g. preparatory, dyeing, printing and finishing)• Print paste (printing screen, squeeze and drum cleaning)• Lint (preparatory, dyeing and washing operations)• Foam from coating operations• Solvents from machine cleaning• Still bottoms from solvent recovery (dry cleaning operation)• Batch dumps of unused processing (finishing mixes) [5] 21
  • 22. Diagram 1: Cotton Fabric Production & Associated Water Pollutants: [18] 22
  • 23. Table 3.Effluent Characteristics From Textile Industry:[18]Heavy metals and its hazards: Metal pollution is a global concern. Input of chemical substances constitutes a greatpollution burden on natural ecosystems. When considering their impact, it is necessary todistinguish between poisonous substances, that have a toxic effect on organisms even at lowconcentrations, and burdening substances that bring about undesirable changes in eco systemsat higher concentration or load. The level of metals in all environments including air, waterand soil are increasing in some cases to toxic levels with contribution from a wide variety ofindustrial sources. Heavy metals are roughly defined as elements having a density over 6g/cm3. Amongthese elements, Co, Cu, Mn, Ni, Se and Zn are important in small amounts. some metals suchas Cu,Fe and Zn are essential at low concentrations and are toxic at high levels. The maincause of water pollution are household waste (As, Cr, Cu, Mn and Ni), coal fed powerstations (As, Hg and Se), iron and steel production (Cr, Mo, Zn and Sb), and metal smelters (Cd, Ni, Sb and Se). Of these metals, 25% are thought to enter rivers and lakes, and theirsurrounding soils are also heavily pollulated by these metals. 23
  • 24. Table 4. Substances present in industrial effluents: [23]Metals in the environment can be divided into two classes, bioavailable (soluble, nonsorbedand mobile) and non-bioavailable (precipitated, complexed, sorbed and non-mobile). It is thebioavailable metal concentration that is taken up and is thus toxic to biological systems.Some metals like Hg and Pb are highly toxic but many other metals are also of concern,including As, Br, B, Cd, Cr, Cu, Ni, Mn, Se, Ti and Zn. Elevated metal concentration in theenvironment has a wide ranging on animals, plants and microbial species. Toxicity to anorganism can be defined as the inherent potential or capacity of a material to cause adverseon living organisms and depends on the bioavailability of the toxicant. 24
  • 25. In textile industry wet processing, a large number of chemicals are used, such as causticsoda, hydrogen peroxide, formic acid, sodium nitrate, azo dyes, direct dyes, reactive dyes,and mordants. Correia et al. (1994), reported that problems associated with textile processingeffluents are heavy metal ions, which might rise from metal used in the dyeing process, or, inconsiderably higher amounts from metal containing dyes. For cotton, azoic, direct azo dyeand mordant are used, which contains metals in complexed form. Azo and direct azo dyecontains sodium salts, fixing agent and metallic compounds of Cu and Cr, whereas, themordants are the metallic salts of Al, Cr and Fe, which are used on cotton silk fibre.Commonly used metal containing pigments are chrome yellow (Cr), zinc yellow (Zn),chrome oxide green (Cr) and iron blue pigment (Fe) in textile industries.[14] It can be seen that of all of the heavy metals chromium is the most widely used anddischarged to the environment from different sources. However, it is not the metal that ismost dangerous to living organisms. Much more toxic are cadmium, lead and mercury. Thesehave a tremendous affinity for sulphur and disrupt enzyme function by forming bonds withsulphur groups in enzymes. Protein carboxylic acid (-CO2H) and amino (-NH2) groups arealso chemically bound by heavy metals. Cadmium, copper, lead and mercury ions bind to cellmembranes, hindering transport processes through the cell wall. Heavy metals may alsoprecipitate phosphate bio-compounds or catalyse their decomposition.Mercury: Because of its toxicity, its mobilisation as methylated forms by anaerobic bacteria, andother pollution factors, mercury generates a great deal of concern as a heavy-metal pollutant.Mercury is found as a trace component of many minerals, with continental rocks containingan average of around 80 ppb, or slightly less, of this element. Cinnabar, red mercuricsulphide, is the chief commercial mercury ore. Metallic mercury is used as an electrode in theelectrolytic generation of chlorine gas, in laboratory vacuum apparatuses and in otherapplications. Organic mercury compounds used to be widely applied as pesticides,particularly fungicides. Mercury enters the environment from a large number ofmiscellaneous sources related to human use of the element. These include discardedlaboratory chemicals, batteries, broken thermometers, lawn fungicides, amalgam toothfillings and pharmaceutical products. Sewage effluent sometimes contains up to 10 times thelevel of mercury found in typical natural waters. The toxicity of mercury was tragicallyillustrated in the Minamata Bay area of Japan during the period of 1953-1960. A total of 111 25
  • 26. cases of mercury poisoning and 43 deaths were reported among people who had consumedseafood from the contaminated bay. Among the toxicological effects of mercury wereneurological damage, including irritability, paralysis, blindness, insanity, chromosomebreakage and birth defects. The milder symptoms of mercury poisoning, such as depressionand irritability, have a psychopathological character and may escape detection. Theunexpectedly high concentrations of mercury found in water and in fish tissues result fromthe formation of soluble monomethylmercury ion, CH3Hg+, and volatile dimethylmercury,(CH3)2Hg, by anaerobic bacteria in sediments. Mercury from these compounds becomesconcentrated in fish lipid (fat) tissue and the concentration factor from water to fish mayexceed 103.[23]Zinc:Zinc is a trace element that is essential for human health. When people absorb too little zincthey can experience a loss of appetite, decreased sense of taste and smell, slow woundhealing and skinsores. Zinc-shortages can even cause birth defects. Although humans canhandle proportionally large concentrations of zinc, too much zinc can still cause eminenthealth problems, such as stomach cramps, skin irritations, vomiting, nausea and anaemia.Very high levels of zinc can damage the pancreas and disturb the protein metabolism, andcause arteriosclerosis. Extensive exposure to zinc chloride can cause respiratory disorders.In the work place environment zinc contagion can lead to a flu-like condition known as metalfever. This condition will pass after two days and is caused by over sensitivity.Zinc can be a danger to unborn and newborn children. When their mothers have absorbedlarge concentrations of zinc the children may be exposed to it through blood or milk of theirmothers.[33]Manganese:Manganese effects occur mainly in the respiratory tract and in the brains. Symptoms ofmanganese poisoning are hallucinations, forgetfulness and nerve damage. Manganese canalso cause Parkinson, lung embolism and bronchitis. When men are exposed to manganesefor a longer period of time they may become impotent. A syndrome that is caused bymanganese has symptoms such as schizophrenia, dullness, weak muscles, headaches andinsomnia. 26
  • 27. Manganese compounds exist naturally in the environment as solids in the soils and smallparticles in the water. Manganese particles in air are present in dust particles. These usuallysettle to earth within a few days. Humans enhance manganese concentrations in the air byindustrial activities and through burning fossil fuels. Manganese that derives from humansources can also enter surface water, groundwater and sewage water. Through the applicationof manganese pesticides, manganese will enter soils.For animals manganese is an essential component of over thirty-six enzymes that are used forthe carbohydrate, protein and fat metabolism. With animals that eat too little manganeseinterference of normal growth, bone formation and reproduction will occur. For some animalsthe lethal dose is quite low, which means they have little chance to survive even smallerdoses of manganese when these exceed the essential dose. Manganese substances can causelung, liver and vascular disturbances, declines in blood pressure, failure in development ofanimal foetuses and brain damage.When manganese uptake takes place through the skin itcan cause tremors and coordination failures. Finally, laboratory tests with test animals haveshown that severe manganese poisoning should even be able to cause tumor developmentwith animals.[34]Chromium:Chromium(VI) is known to cause various health effects. When it is a compound in leatherproducts, it can cause allergic reactions, such as skin rash. After breathing it in chromium(VI)can cause nose irritations and nosebleeds.Other health problems that are caused by chromium(VI) are:- Skin rashes- Upset stomachs and ulcers- Respiratory problems- Weakened immune systems- Kidney and liver damage- Alteration of genetic material- Lung cancer- DeathThe health hazards associated with exposure to chromium are dependent on its oxidationstate. The metal form (chromium as it exists in this product) is of low toxicity. The 27
  • 28. hexavalent form is toxic. Adverse effects of the hexavalent form on the skin may includeulcerations, dermatitis, and allergic skin reactions. Inhalation of hexavalent chromiumcompounds can result in ulceration and perforation of the mucous membranes of the nasalseptum, irritation of the pharynx and larynx, asthmatic bronchitis, bronchospasms and edema.Respiratory symptoms may include coughing and wheezing, shortness of breath, and nasalitch.[35]Aluminium :The water-soluble form of aluminum causes the harmful effects, these particles are calledions. They are usually found in a solution of aluminum in combination with other ions, forinstance as aluminum chlorine. The uptake of aluminum can take place through food, throughbreathing and by skin contact. Long lasting uptakes of significant concentrations ofaluminum can lead to serious health effects, such as:- Damage to the central nervous system- Dementia- Loss of memory- Listlessness- Severe tremblingThe concentrations of aluminum appear to be highest in acidified lakes. In these lakes thenumber of fish and amphibians is declining due to reactions of aluminum ions with proteinsin the gills of fish and the embryos of frogs. High aluminum concentrations do not onlycause effects upon fish, but also upon birds and other animals that consume contaminated fishand insects and upon animals that breathe in aluminum through air. The consequences forbirds that consume contaminated fish are eggshell thinning and chicks with low birth-weights. The consequences for animals that breathe in aluminum through air may be lungproblems, weight loss and a decline in activity. Another negative environmental effect ofaluminum is that its ions can react with phosphates, which causes phosphates to be lessavailable to water organisms. High concentrations of aluminum may not only be found inacidified lakes and air, but also in the groundwater of acidified soils. There are strongindications that aluminum can damage the roots of trees when its located in groundwater.[36] 28
  • 29. Lead: Inorganic lead arising from a number of industrial and mining sources occurs in waterin the +2 oxidation state. Lead from leaded gasoline used to be a major source of atmosphericand terrestrial lead, much of which eventually enters natural water systems. Despite greatlyincreased total use of lead by industry, evidence from hair samples and other sourcesindicates that body burdens of this toxic metal have decreased during recent decades. Thismay be the result of less lead used in plumbing and other products that commonly come incontact with food or drink. Acute lead poisoning in humans causes severe dysfunction in thekidneys, reproductive system, liver, and the brain and nervous system. [23] 29
  • 30. Table 5. Heavy Metals Found in Major Industries[23]Table 6. Comparative strengths of wastewater from industry:[23] 30
  • 31. Bioremediation:Heavy metals contaminate the aquatic environment and sources of potable water because oftheir known accumulation in food chain and their persistence in nature, when they aredischarged in small quantities by numerous industrial activities. The need for economical,effective and safe methods for removing heavy metals from waste water has resulted in thesearch for unconventional materials that may be useful in reducing the levels or accumulationof heavy metals in the environment. The newly developed metal sequestering properties ofcertain types of microbial biomass of fungi, yeast, bacteria, algae and plants, offerconsiderable promise in terms of providing a cost effective process, for removing toxic heavymetals from the industrial effluents.Microbial activities in the natural environment are the main process that remove, mobilize ordetoxify heavy metals and radionuclides. The activities can be harnessed to clean up toxicmetal wastes, before they enter the wider environment and such biotechnological processesare used to control pollution from diverse sources. The study of microorganisms that arecapable of resisting and surviving in polluted environments provides the basic knowledge ofbioremediation. Natural and genetically engineered species are used for protection andremediation of organic contaminants. Due to their non-biodegradability heavy metals cannotbe treated biologically in situ and must instead be extracted from contaminating streams. The utilization of microbial biomass for removal of metals from industrial waste water andpolluted waters is a well recognized method of remediation. Metals adversely influencemicroorganisms, effecting their growth, morphology and biochemical activities, resulting indecreased biomass and diversity. Despite these toxic stresses, numerous microorganismsdevelop metal resistance and detoxification mechanisms, which includes volatilization,extracellular precipitation and exclusion, intracellular sequestration, membrane associatedmetal pumps, entrapment of cellular component, cation exchange or complextion, adsorptionand degradation of organometals. The term biosorption is used to encompass uptakes by the whole biomass living or dead,via physiochemical mechanisms, adsorption, coordination and covalent bonding and ionexchange. Where living biomass is used, metabolic uptake mechanisms may also contributeto the process. The main chemical groups in biomass, which are able to partake in biosorptionare, electro negative groups such as hydroxyl or sulphydrl groups, anionic groups such ascarboxyl or phosphate groups and nitrogen containing groups such as amino groups. Dead 31
  • 32. microbial biomass, including fungal mycelium, can also adsorb metal ions, often in largerquantities than living biomass. Biodegradation is the microbially mediated decomposition of not only organic but alsoxenobiotic pollutants. Biodegradation can be a minor change in the molecule, fragmentationor complete mineraization. A range of current approaches to biodegradation are available,starting from identification of microbial strains that have the ability to break down or alterenvironmental pollutants such as petroleum products, polychlorinated biphenyls, halogenatedaromatic compounds,toluene, and nitrogen containing heterocyclic aromatic analogues ofpolycyclic aromatic hydrocarbons to screening for these isolates for degradation abilities byproviding target substances as carbon source or electron acceptor. [14]FACTORS OF BIOREMEDIATION:The control and optimization of bioremediation processes is a complex system of manyfactors. These factors include: the existence of a microbial population capable of degradingthe pollutants; the availability of contaminants to the microbial population; the environmentfactors (type of soil, temperature, pH, the presence of oxygen or other electron acceptors, andnutrients).MICROBIAL POPULATIONS FOR BIOREMEDIATION PROCESSESMicroorganisms can be isolated from almost any environmental conditions. Microbes willadapt and grow at subzero temperatures, as well as extreme heat, desert conditions, in water,with an excess of oxygen, and in anaerobic conditions, with the presence of hazardouscompounds or on any waste stream. The main requirements are an energy source and acarbon source. Because of the adaptability of microbes and other biological systems, thesecan be used to degrade or remediate environmental hazards. We can subdivide thesemicroorganisms into the following groups:Aerobic.: In the presence of oxygen. Examples of aerobic bacteria recognized for theirdegradative abilities are Pseudomonas, Alcaligenes, Sphingomonas, Rhodococcus, andMycobacterium. These microbes have often been reported to degrade pesticides andhydrocarbons, both alkanes and polyaromatic compounds. Many of these bacteria use thecontaminant as the sole source of carbon and energy.Anaerobic.: In the absence of oxygen. Anaerobic bacteria are not as frequently used asaerobic 32
  • 33. bacteria. There is an increasing interest in anaerobic bacteria used for bioremediation ofpolychlorinated biphenyls (PCBs) in river sediments, dechlorination of the solventtrichloroethylene (TCE), and chloroform.Ligninolytic fungi.: Fungi such as the white rot fungus Phanaerochaete chrysosporium havetheability to degrade an extremely diverse range of persistent or toxic environmental pollutants.Common substrates used include straw, saw dust, or corn cobs.Methylotrophs.: Aerobic bacteria that grow utilizing methane for carbon and energy. Theinitialenzyme in the pathway for aerobic degradation, methane monooxygenase, has a broadsubstrate range and is active against a wide range of compounds, including the chlorinatedaliphatics trichloroethylene and 1,2-dichloroethane.For degradation it is necessary that bacteria and the contaminants be in contact. This is noteasilyachieved, as neither the microbes nor contaminants are uniformly spread in the soil. Somebacteria are mobile and exhibit a chemotactic response, sensing the contaminant and movingtoward it. Other microbes such as fungi grow in a filamentous form toward the contaminant.It is possible to enhance the mobilization of the contaminant utilizing some surfactants suchas sodium dodecyl sulphate (SDS).ENVIRONMENTAL FACTORS:Although the microorganisms are present in contaminated soil, they cannot necessarily bethere in the numbers required for bioremediation of the site. Their growth and activity mustbe stimulated. Biostimulation usually involves the addition of nutrients and oxygen to helpindigenous microorganisms. These nutrients are the basic building blocks of life and allowmicrobes to create the necessary enzymes to break down the contaminants. All of them willneed nitrogen, phosphorous, and carbon. Carbon is the most basic element of living formsand is needed in greater quantities than other elements. In addition to hydrogen, oxygen, andnitrogen it constitutes about 95% of the weight of cells. Phosphorous and sulfur contributewith 70% of the remainders. The nutritional requirement of carbon to nitrogen ratio is 10:1,and carbon to phosphorous is 30:1.[10] 33
  • 34. Table 7. Environmental conditions affecting degradation:[10]Types of Bioremediation:The main types of bioremediation are as follows: Biostimulation -- Nutrients and oxygen - in a liquid or gas form - are added to contaminated water or soil to encourage the growth and activity of bacteria already existing in the soil or water. The disappearance of contaminants is monitored to ensure that remediation occurs. Bioaugmentation -- Microorganisms that can clean up a particular contaminant are added to the contaminated soil or water. Bioaugmentation is more commonly and successfully used on contaminants removed from the original site, such as in municipal wastewater treatment facilities. To date, this method has not been very successful when done at the site of the contamination because it is difficult to control site conditions for the optimal growth of the microorganisms added. Scientists have yet to completely understand all the mechanisms involved in bioremediation, and organisms introduced into a foreign environment may have a hard time surviving. Intrinsic Bioremediation -- Also known as natural attenuation, this type of bioremediation occurs naturally in contaminated soil or water. This natural bioremediation is the work of microorganisms and is seen in petroleum contamination sites, such as old gas stations with leaky underground oil tanks. Researchers are studying whether intrinsic bioremediation happens in areas with other types of chemical contamination. Application of this technique requires close monitoring of contaminant degradation to ensure that environmental and human health are protected.All three types of bioremediation can be used at the site of contamination (in situ) or oncontamination removed from the original site (ex situ). In the case of contaminated soil, 34
  • 35. sediments, and sludges, it can involve land tilling in order to make the nutrients and oxygenmore available to the microorganisms. [26]Table 8: Bioremediation strategies:[10] 35
  • 36. Microorganisms and its role:Level of microbial resistance is important criteria for evaluation of an isolate from metalcontaminated site, for use in bioremediation.Lead:Konopka et al (1999) analyzed Pb contaminated soil for microbial community diversitypotential microbial activity and metal resistance in three soils, having Pb in the range of0.00039-48.00 mM/kg of soil. In all samples, 10-15% of the total culturable bacteria wherePb resistant and had MRL of Pb of 100-150 µM. The Pb resistant isolates were all grampositive that were members of the genus bacillus Coryneform or Actinomycetes. Pb resistantbacteria were isolated from P contaminated sites of silver valley, Iadho (United States),containing 17.20-108.00 µM Pb/g dry soil. Pb resistant genera isolated includedPseudomonas, Bacillus, Corynebacterium and Enterobacter species. Among these isolates,Pseudomonas marginalis had a higher resistance at 2.5 mM total Pb, as compared to 0.625mM for Bacillus megaferium. However, resistance to soluble Pb was much low, 0.3 and 0.1mM, respectively. The degree of Pb resistance, and the mechanism of resistance for these twoisolates correspond with their environmental exposure. Pb biosorption by Pseudomonas aeruginosa PU21 was investigated along with effects ofenvironmental factorsand growth conditions. The results showed that, at pH 5.5, resting cellswere able to uptake Pb, 110mg/g dry cell. The resting cells held optimal Pb absorptioncapacity at the early stationary phase. By adjusting pH to 2.0, Pseudomonas aeruginosa PU21biomass removed 98% Pb. Niu te al. (1993), studied removal of lead ions from aqueous solution by adsorption on nonliving Penicillum Chrysogenum. Biosorption of the Pb2- ion was strongly affected by pH.Within a pH range of 4.0-5.0 the saturated sorption was 116mg/g dry biomass. This strainwas applied to treat industrial lead contaminated wastewater, in which lead concentration wasreduced to less than 1mg/l, after treatment.Chromium:Cr is the widespread industrial and nuclear waste, and several waste dumps serve as areservoir for Cr tolerant and accumulator microorganisms. A consortium of bacteria withtolerance towards high concentrations of Cr 6- (up to 2,500mg/L), and other toxic metal wasobtained from metal refinishing waste water in Chengdu, People ’s Republic of china. The 36
  • 37. consortium comprised of a range of Gram positive and Gram negative rods, and these rodshad capacity to reduce Cr6- to Cr3- . Cr removal was 80-90% from concentrations rangingfrom 50-2000mg/L. Removal was not totally passive because killed and gamma irradiatedcells could immobilize much of the metal concentration. This consortium was namedsulphate reducing bacteria SRB III. Several chromate reducing bacteria have been reported,from the genera Pseudomonas, Achromobacter, Aeromonas, Basillus, Enterobacter,Escherichia and Micrococcus. Experiments with free cell biomass (cells plus exoploysaccharides) of rhizobium BJVr12(mungbean isolate) showed that amount of Cr 3- ion sorbed was influenced by amount ofbiomass to Cr3- concentration ratio and time of contact. A reduction rate of 49.7% for Cr3-was observed for free cells, 95.6% for cells immobilized in ceramic bead and 94.6% for cellsin aquacell ( a porous cellulose career with a charged surface), was achieved after 48 hoursunder shaking conditions.Manganese:A large number of bacteria are reported to be involved in the oxidation of Mn2 and includethe genera Leptothrix, Pedomicrobium, Hypomicrobium, Caulobacter, Arthrobacter,Micrococcus, Bacillus, Chromobacterium, Pseudomonas, Vibrio, Oceanospiralium.Biosorption of toxic metals( Mn, Cu, Ni, Pb) by species of Arthrobacter showed highestvalue of specific uptake (mg/g) in case of Mn(406) followed by Cu(148), Pb(30), and theleast value observed was that of Ni, which was 13 mg/g. Pyrobaculum islandicum is ahyperthermophilic microorganism, which has the capacity to reduce Mn(IV) oxide to whiteprecipitate of manganese carbonate, when washed cell suspension was used at 100 0C. Fourstrains of Pseudomonas putida, Pseudomonas putida 06909, Pseudomonas putida 0690 s22x,Pseudomonas putida 06909s21x, Pseudomonas putida 0690 s23 were determined for theirMRL for Mn. All four strains showed MRL of more than 2.5mM. Saccharomyces cerevisiae,exhibits greater metal accumulation capacity, as compared to bacteria nd accumulate Mn 2cation. Immobilized Rhizopus arrhizus has also been shown to accumulate Mn2. 37
  • 38. Zinc:Alcaligenes eutrophus and Pseudomonas aeruginosa are Zn tolerant Gram negativebacteria.Strains of Pseudomonas putida, Pseudomonas putida 06909, Pseudomonas putida06909s21x Pseudomonas putida 0690s22x, , Pseudomonas putida 0690 s23 were evaluated forMRLof Zn on mannitol glutamate agar. These strains of Pseudomonas putida exhibiteddifferent values values, which were, Pseudomonas putida 06909(11.5mM), Pseudomonasputida 06909s21x(11.5mM), Pseudomonas putida 0690s22x(7.0mM),Pseudomonas putida0690s23(10.5mM). At Ingurtosa (southwestern Sardinia, Italy), due the mining in the past, mine tailingswere deposited along the Rio Naracaini creek. Rio Naracainli stream water was highlypolluted by heavy metals like Zn, Pb, Cd, Fe, Mn, Cu,and Ni. Podda et al.reported naturalbioremediation of heavy metals by a consortium of a photosynthetic filamentous bacterium,classified as scytonema spp, strain ING-1, associated with microalga chlorella spp, strainSA1. This microbial community was responsible for the natural polishing of heavy metals inthe water of stream Rio Naracainli by coprecipitation with hydrozincite [Zn 5(CO3)2(OH)6].Scytonema spp was responsible for formation of hydrozinciteduring photosynthesisofchlorella spp, thus removing Zn2+ in addition to Pb, Cd, Ni and Cu.[14]Metal Tolerance MechanismsIn high concentrations, heavy metal ions react to form toxic compounds in cells (Nies, 1999).To have a toxic effect, however, heavy metal ions must first enter the cell. Because someheavy metals are necessary for enzymatic functions and bacterial growth, uptake mechanismsexist that allow for the entrance of metal ions into the cell. There are two general uptakesystems — one is quick and unspecific, driven by a chemiosmotic gradient across the cellmembrane and thus requiring no ATP, and the other is slower and more substrate-specific,driven by energy from ATP hydrolysis. While the first mechanism is more energy efficient, itresults in an influx of a wider variety of heavy metals, and when these metals are present inhigh concentrations, they are more likely to have toxic effects once inside the cell (Nies andSilver, 1995). To survive under metal-stressed conditions, bacteria have evolved several typesof mechanisms to tolerate the uptake of heavy metal ions. These mechanisms include theefflux of metal ions outside the cell, accumulation and complexation of the metal ions insidethe cell, and reduction of the heavy metal ions to a less toxic state (Nies, 1999).[1] 38
  • 40. MATERIALS AND METHODS:A] Collection of wastewater and soil sample:Location:Wastewater and soil sample was collected from koperkhairane nullah near MIDCindustrial area besides Furnace fabrica Ltd & Alok industry- Navi Mumbai.Procedure: Water sample was collected in a plastic bottle and soil sample was collected in sterile Petri plates. pH and temperature of the water sample was checked. Water sample and soil sample was transported to college laboratory. Water sample and soil sample were kept in refrigerator (40C) until further analysis was carried out.B] Waste water analysis:1] Determinaton of total hardness of water:AIM: To determine the hardness of water by EDTA titrimetric method.Principle:EDTA forms chelated soluble complex when added to a solution of certain metal cations. Ifsmall amount of dye such as Eriochrome black T or calmagite is added to an aqueous solutioncontaining calcium and magnesium ions at pH 10.0 the solution turns wine red .If EDTA isadded as a titrant, the calcium and magnesium will be complexed. When all of the calciumand magnesium has been complexed, the solution turns wine red to blue, marking the endpoint of titration.Requirements:Equipments: Conical flask, burette, beakers etc. 40
  • 41. Reagents:  Buffer solution: Dissolve 16.9 gm of ammonium chloride in 143 ml conc Ammonium hydroxide and dilute to 250ml with distilled water.  Eriochrome black T indicator  Ethylene diamino tetra acetic acid [EDTA][0.01M]:weigh 3.723gm of disodium EDTA and dissolved in 50ml of distilled water and make the volume to 1000ml and store in a brown bottle.  Standard calcium :weigh 1gm of calcium carbonate powder into a 500ml conical flask. Place a funnel in the neck of the flask and slowly add conc. HCL drop wise and shake the flask repeatedly until the entire CaCO3 dissolved . Add 200ml D/W and boil for few minutes to expel CO2.Procedure:  Take 10ml of sample in a conical flask.  Add 1ml of buffer solution and shake well.  Add 1 to 2 drops of Eriochrome black T indicator, shake well to get wine red colour.  Titrate it against EDTA by adding drop wise from the burette until the colour changes wine red to blue.Calculation:1ml of 1m of EDTA =40.08 of Ca+2Since atomic weight of Ca+2 is 40.08, 1ml of 0.01M=0.4008mg of Ca+2When standardization is done with Std .Calcium,Hardness(EDTA) as mg Ca+2/L= BR x Normality of EDTA x 40.08 x1000 10[volume of sample taken] = BR x 0.01 x 40.08 x1000 10 = BR x 40.08mg Ca+2/L 41
  • 42. 2] Determination of total alkalinity of water:Principle:Generally pH of water remains neutral. Alkalinity of water represents the presence ofhydroxyl ions[-OH] in water; hence, it is capacity of water to neutralize a strong acid. Innatural or waste water alkalinity is due to the presence of free hydroxyl ions which causethrough hydrolysis of salts by weak acids and strong bases (e.g. carbonate and bicarbonates).Requirements:  Equipments: conical flasks, titration assembly etc.  HCL solution(0.1N)  Methylene orange indicatorProcedure:  Take 50ml of water sample in flask and add 2-3 drops of methyl orange into it. Clour turns to orange .  Transfer 0.1N HCL solution ito burette in titration assembly and titrate with the water sample[methylene orange added] untl yellow colour changes to pink. Note the end point.Calculation:Methylene orange alkalinity (mg/litre) =volume of 0.1 N HCL solution used as titrant x 1000 Volume of water sample3] Determination of chlorine in water:Principle:The potable water is chlorinated to make the water free from microorganisms. However,some times the concentration of chloride ions in water is increased than what is normallyrequired. Apart from this water also receives chloride ions from multifarious sources. Thechloride ions (Cl-) can be estimated by titrating with silver nitrate solution. 42
  • 43. Requirements:  Equipments : conical flask,pipette, titration assembly etcReagents:  Silver nitrate (AgNO3) solution (0.025N) –dissolve 3.4g dried AgNO3 in distilled water and dilute to make 1 litre.store in coloured bottle  Potassium dichromate solution (5%)- dissolve 5g of K2Cr2O7 in 100ml of distilled water.Procedure:  Take 50ml of sample in a conical flask and 2ml of K2Cr2O7 solution.  Pour 0.025N AgNO3 solution into burette set with titration assembly.  Titrate the water sample with AgNO3 solutio until reddish tinge appears. Note the end point (AgNO3 reacts with Cl- ions and forms very slightly solble white precipitate of AgCl2(silver chloride). Free silver ions (Ag++) react with chromate ions (Cr2O7) to form silver chromate of reddish rown colour).Calculation:Chloride (mg/litre) =volume of AgNO3 solution x 1000 x35.5 Volume of the water sample used 43
  • 44. 4] Determination of chemical oxygen demand(COD) of water:Principle:Due to gradually increasing population the number of industries is also increasing. Thisresults in increased pollution. The chemically oxidisable organic substances discharged inwater depletes the amount of oxygen. Estimation of BOD alone could not give the exact ideaof pollutants present in water, therefore , COD is estimated. COD refers to the oxygenconsumed by the oxidisable organic substances. The values of COD cannot be compareddirectly with that of BOD. The chemical oxidants such as potassium dichromate (K2Cr2O7) or potassiumpermanganate (KMnO4) are used to measure the oxidisability of the organic matter of waterwhere the oxidants oxidize the constituents (or the hydrogen but not nitrogen). Thenpotassium iodide (KI) is added. The excess amount of oxygen reacts with KI and liberatesequal amount of iodine. By using starch indicator, iodine is titrated with sodium thiosulfateand amount is estimated.Requirements: Potassium dichromate solution (0.1N): 3.676gm in 1 litre of distilled water. Sodium thiosulphate (0.1N): 15.811gm of sodium thiosulfate in 2 litre of distilled water. Sulphuric acid (2M): 10.8 ml of concentrated H2SO4 in 100ml of distilled water. Potassium iodide solution (10%) Starch solution (1%),water bath,conical flask,titration assembly etc.Procedure: Take three 100m conical flask and pour 50ml of water sample in each. Simultaneously run distilled water blanks standards (also in triplicates) Add 5ml of K2Cr2O7 solution in each of the six flask. Keep the flask in waterbath at 1000C (boiling temperature) for one hour. 44
  • 45.  Allow the sample to cool for 10 minutes. Add 5ml of potassium iodide in each flask. Add 10ml of H2SO4 in each flask. Titrate the contents of each flask with 0.1M sodium thiosulphate until the appearance of pale yellow colour. Add 1 ml of starch solution to each flask (solution turns blue). Titrate it again with 0.1 M sodium thiosulfate until the blue colour disappears.Result:COD of sample mg/litre= 8 x C x(A-B) SC = concentration of titrant (mmol/litre)A = volume of titrant used for blank (ml)B = volume of the totrant used for sample (ml)S = volume of water sample taken (ml)5] Determination of dissolved oxygen (DO) of water:Some amount of oxygen is dissolved in water which is used by the aquatic plants andanimals. The sources of dissolved oxygen in water are the autotrophic aquatic plants which asa result of photosynthesis evolve oxygen,and air where from oxygen is dissolved in waterdepending o salinity, temperature and water movement. Moreover, in an oligotrophic lake theamount of dissolved nutrients salts remains low, therefore ,it supports sparse plant and animallives. Tis results in high dissolved oxygen gradually increasing with depth. In addition, ineutrophic water reservoirs e.g. lakes, ponds, pools, etc. the organic nutrients accumulateabundantly which in turn are subjected to microbial decomposition. More growth ofmicroorganisms, plants and animals depletes oxygen. 45
  • 46. Dissolved oxygen is measured by titrimetric method. The theory behind this method is thatthe dissolved oxygen combines with magnous hydroxide which in turn liberates iodine(equivalent to oxygen fixed) after acidification with H 2SO4. The iodine can be titrated withsodium thiosulfate solution by using starch indicator.Requirements: BOD bottles (250 ml capacity),pippetes,titration set etc. Alkaline KI solution: Dissolve 100g of KOH and 50g of KI in 200ml of distilled water. Sodium thiosulphate (0.025N) Manganese sulfate(MnSO44H2O) solution: Dissolve 100g of manganese sulfate in 200ml of distilled water, filter and keep in stoppered bottle. Starch indicator: Dissolve 5g of starch in 100ml hot distilled water(boiled) and a few drops of formaldehyde(HCHO). Concentrated H2SO4Procedure: Collect water sample in a BOD glass bottle (250ml) in such a way that water bubble should not come out. Pipette separately 2ml of manganese sulfate and 2ml of alkaline iodine-azide solutions. Add these solutions in succession at the bottom of bottle and place the stopper of bottle. Shake the bottle upside down for about 6-8 times .There develops brown precipitate. Leave the bottle for few minutes , the precipitate settles down. Add 2ml of concentrated H2SO4 in the bottle. Shake properly so that brown precipitate may dissolve. Take a clean flask and pour 50 ml of this water sample. Titrate it against 0.025 N sodium thiosulphate solution taking in a burette until pale starw colour develops. Add 2 drops of starch solution to the flask. Colour of contents chages from ple to blue. 46
  • 47.  Again titrate against thiosulfate solution until the blue colour disappears. Note the volume of sodium thiosulfate solution used in titration.Calculation:Calculate the amount of dissolve oxygen (DO) (mg/litre) by sing the formula:DO (mg/l) = 8 x 1000 x N x v VWhere, V = volume of water sample for titration v = volume of sodium thiosulfate (titrant) N = normality of the titrant 8 = it is a constant since 1ml of 0.025N sodium thiosulfate solution is equivalent to0.2 mg oxygen.6] Determination of biological oxygen demand (BOD) of water :Principle:Biochemical Oxygen Demand (BOD) is the amount of oxygen, expressed in mg/L or partsper million (ppm), that bacteria take from water when they oxidize organic matter. Thecarbohydrates (cellulose, starch, sugars), proteins, petroleum hydrocarbons and othermaterials that comprise organic matter get into water from natural sources and from pollution.They may be dissolved, like sugar, or suspended as particulate matter, like solids in sewage.Organic matter can be oxidized (combined with oxygen) by burning, by being digested in thebodies of animals and human beings, or by biochemical action of bacteria. Because organicmatter always contains carbon and hydrogen, oxidation produces carbon dioxide (the oxygencombining with the carbon) and water (the oxygen combining with the hydrogen). Bacteria inwater live and multiply when organic matter is available for food and oxygen is available foroxidation. About one-third of the food bacteria consumed becomes the solid organic cellmaterial of the organisms. The other two-thirds is oxidized to carbon dioxide and water bythe biochemical action of the bacteria on the oxygen dissolved in the water. To determineBOD, the amount of oxygen the bacteria use is calculated by comparing the amount left at theend of five days with the amount known to be present at the beginning, whereas the same can 47
  • 48. be incubated at 270C for 3 days in tropical and subtropical regions where metabolic activitiesare higher. At room temperature, the amount of oxygen dissolved in water is 8 mg/L. Atfreezing, it increases to 14.6 mg/L; it also increases at high barometric pressures (lowaltitudes). At the boiling point, the solubility of oxygen is zero. [13][12]Requirements: BOD-free water BOD bottles ,Erlenmeyer flask, pipette, BOD incubator, pH meter Phosphate buffer solution (pH 7.4) Allythiourea (0.5%) Alkaline KI solution: Dissolve 100g of KOH and 50g of KI in 200ml of distilled water. Sodium thiosulphate (0.025N) Manganese sulfate(MnSO44H2O) solution: Dissolve 100g of manganese sulfate in 200ml of distilled water, filter and keep in stoppered bottle. Starch indicator: Dissolve 5g of starch in 100ml hot distilled water(boiled) and a few drops of formaldehyde(HCHO). Concentrated H2SO4Procedure: Add 1nacid/1N alkali in the water sample to adjust the pH to 7.0 Gently transfer this water inti BOD bottles so as bubbles should not come out. Add 1 ml of allylthiourea to each bottle to avoid nitrification. Measure dissolved oxygen following the steps as described for dissolved oxygen Incubate the other BOD bottle at 270C for 3 days in a BOD incubator. Measure the amount of oxygen as done earlier.Calculation:Calculate the BOD of water by using the following formula:BOD (mg/l) = D1-D2Where, D1 = initial dissolved oxygen (mg/l) in the first sample D2 = dissolved oxygen (mg/l) in the second sample after 3 days of incubation. [15] 48
  • 49. C] ISOLATION OF MICROORGANISMS:Suspensions of the soil sample were prepared using physiological saline. Spread platetechnique on Nutrient Agar plates was used for the isolation of microorganisms.Requirements:  Nutrient Agar medium. Composition/litre: SR NO. COMPOSITION g/l 1. AGAR 15.0 2. PEPTONE 5.0 3. NaCl 5.0 4. YEAST EXTRACT 2.0 5. BEEF EXTRACT 1.0 Table 9: Nutrient Agar Composition. The pH is adjusted to 7.4(+/-) 0.2 at 250C.Preparation: Add components to Distilled water and bring the volume to 1.0L. Mixthoroughly. Gently heat and bring to boiling. Distribute into tubes or flasks. Autoclave for 15mins at 15 psi pressure and -1210C. Pour into sterile Petri plates.  Physiological saline. Add 0.85g of NaCl in 100ml Distilled water. Autoclave for 15 mins at 15 psipressure and -1210CProcedure:0.1ml of soil suspensions were spread onto the Nutrient Agar plates under aseptic conditionsusing a sterile glass spreader. These Petri plates were incubated overnight at roomtemperature. 49
  • 50. D] CHARACTERIZATION OF THE MICROORGANISMS: GRAM STAINING: Gram staining (or Grams method) is an empirical method of differentiatingbacterial species into two large groups: Gram-positive and Gram-negative, based on thechemical and physical properties of their cell walls. The Gram stain named after theDanish scientist Hans Christian Gram, is almost always the first step in the identification of abacterial organism. Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50-90% of cell wall), which stains purple while Gram-negative bacteria have a thinner layer(10% of cell wall), which stains pink. Gram-negative bacteria also have an additional outermembrane which contains lipids, and is separated from the cell wall by the periplasmic space.There are four basic steps of the Gram stain, which include applying a primary stain (crystalviolet) to a heat-fixed smear of a bacterial culture, followed by the addition of a trappingagent (Gramsiodine), rapid decolorization with alcohol or acetone,and counterstaining with safranin. Basic fuchsin is sometimes substituted for safranin since itwill more intensely stain anaerobic bacteria but it is much less commonly employed as acounterstain. Crystal violet (CV) dissociates in aqueous solutions into CV+ and chloride (Cl – )ions. These ions penetrate through the cell wall and cell membrane of both Gram-positive andGram-negative cells. The CV+ ion interacts with negatively charged components of bacterialcells and stains the cells purple. Iodine (I – or I3 – ) interacts with CV+ and forms large complexes of crystal violet andiodine (CV–I) within the inner and outer layers of the cell. Iodine is often referred to asa mordant, but is a trapping agent that prevents the removal of the CV-I complex andtherefore color the cell. After decolorization, the gram-positive cell remains purple and thegram-negative cell loses its purple color. Counterstain, which is usually positively chargedsafranin or basic fuchsin, is applied last to give decolorized gram-negative bacteria a pink orred color.Requirements:  Nutrient Agar slants of the isolates. 50
  • 51.  Reagents: Crystal violet, Gram’s iodine, 95% ethyla alcohol and safranin.  Eqiupments: Inoculating loop, glass slides, Bunsen burner, staining tray,bibulous paper, lens paper, immersion oil and microscope.Method:  A bacterial suspension is prepared and loopful of suspension is used to make a smear on a clean glass slide under sterile conditions.  The smear is allowed to air dry, then it is heat fixed and the slide is transferred to a staining tray.  On the tray, smear is gently flood with crystal violet for 1 min.Wash briefly in water to remove excess crystal violet.  Flood with Gram’s iodine 1 min. Wash briefly in water, to remove excess of stain.  Decolourise with 95% ethanol until the moving dye front has passed the lower edge of the section. Wash immediately in tap water.  Counterstain with safranine for 45 sec. and wash with tap water.  The slide is blotted dry using bibulous paper and examined under oil immersion lens of microscope.[37]E] BIOCHEMICAL CHARACTERIZATION:1]Catalase test: Catalase is a common enzyme found in nearly all living organisms that are exposed tooxygen, where it functions to catalyze the decomposition of hydrogen peroxide towater and oxygen. H2O2 ------catalase--------> H2O + O2 Catalase test is used to detect the presence of catalse enzyme. Hydrogen peroxide isformed by some bacteria as an oxidative end product of the aerobic breakdown of sugars. Ifallowed to accumulate it is highly toxic to bacteria and can result in cell death. Catalase eitherdecomposes hydrogen peroxide or oxidizes secondary substrates, but it has no effect on otherperoxides. 51
  • 52. Catalase production is indicated bubbles of free oxygen gas upon addition of substrate to aculture. The absence of bubble formation is a negative test.Requirements:  Nutrient Aagr slants of the soil isolates.  3% Hydrogen peroxide solution.  Equipments: Inoculating loop, Cavity slide.Method:  A drop of 3% Hydrogen peroxide solution is placed onto a watch glass.  A loopful of culture is taken out under sterile conditions and placed on the drop and results are observed. [38]2] Citrate Utilization test: Simmons citrate agar tests the ability of organisms to utilize citrate as a carbon source.Simmons citrate agar contains sodium citrate as the sole source of carbon, ammoniumdihydrogen phosphate as the sole source of nitrogen, other nutrients, and the pH indicatorbromthymol blue. Organisms which can utilize citrate as their sole carbon source use the enzyme citrase orcitrate-permease to transport the citrate into the cell. These organisms also convert theammonium dihydrogen phosphate to ammonia and ammonium hydroxide, which creates analkaline environment in the medium. At pH 7.5 or above, bromthymol blue turns royal blue.At a neutral pH, bromthymol blue is green, as evidenced by the uninoculated media. If the medium turns blue, the organism is citrate positive. If there is no color change, theorganism is citrate negative. Some citrate negative organisms may grow weakly on thesurface of the slant, but they will not produce a color change.Requirements:  Simmon’s Citrate Agar slants. Composition/l: 52
  • 53. SR NO. COMPOSITION g/l 1. AMMONIUM DIHYDROGEN 1.0 PHOSPHATE 2. DIPOTASSIUM PHOSPHATE 1.0 3. NaCl 5.0 4. SODIUM CITRATE 2.0 5. MAGNESIUM SULFATE 0.2 6. AGAR 15.0 7. BROMOTHYMOL BLUE 0.08 Table 10: Simmon’s Citrate Agar Composition.  Nutrient Agar slants of the soil isolates.  Equipments: Inoculating loop, Bunsen burner.Method:  A loopful of culture is taken and streaked on Simmon’s citrate agar slant.  After 24 hr. incubation period, results are observed. [39]3] Nitrate Utilization Test: Nitrate may be reduce to multiple compounds by two processes. Anaerobic respirationand denitrification. In anaerobic respiration the bacterium uses nitrate as its terminal electronacceptor, reducing nitrate to a variety of compounds, while denitrification reduces nitratesolely to molecular nitrogen. Sulfanilic acid and dimethyl 1-naphthylamine are added todetect nitrite, which will complex with these molecules forming a red color. If no red color isobserved there are two possibilities; the nitrate has not been reduced, or it has been reducedfurther than nitrite. To differentiate between these two possibilities, zinc powder is added,which will complex with nitrate forming a red color. Thus if the tube turns red after zinc, 53
  • 54. nitrate has not been reduced and the result is negative. If no red color is observed then thenitrate has been reduced further than nitrite and the result is positive.Requirements:  Nutrient Agar slants of the soil isolates.  Equipments: Inoculating loop, Bunsen burner.  Compounds: Nitrate broth, sulfanilic acid, dimethyl 1-naphthaline and zinc powder.Method:  Inoculate a trypticase-nitrate tube with the organism to be tested.  Incubate for 24 hours, test for results.  Add 1ml of sulfanilic acid to each tube, then add 1ml of dimethyl 1-naphthylamine solution, Mix  Development of a red color indicates a positive test.  If no red color develops add a small amount of zinc powder.  If a red color develops, nitrate is present and the test is negative. If no red color develops, nitrate has been reduced and the test is positive.4] MRVP Test: This test is used to determine two things. The MR portion (methyl red) is used todetermine if glucose can be converted to acidic products like lactate, acetate, and formate.The VP portion is used to determine if glucose can be converted to acetoin. Methyl red-positive organisms produce high levels of acid during fermentation ofdextrose, overcome the phosphate buffer system and produce a red color upon the addition ofthe methyl red pH indicator. In the Voges-Proskauer test, the red color produced by the addition of potassiumhydroxide to cultures of certain microbial species is due to the ability of the organisms toproduce a neutral end product, acetoin (acetylmethylcarbinol), from the fermentation ofdextrose.3 The acetoin is oxidized in the presence of oxygen and alkali to produce a redcolor.3 This is a positive Voges-Proskauer reaction. 54
  • 55. These tests are performed by inoculating a single tube of MRVP media with a transferloop and then allowing the culture to grow for 1 day. After the culture is grown, about half ofthe culture is transferred to a clean tube. One tube of culture will be used to conduct the MRtest, the second tube serves as the VP test.Requirements:  Nutrient Agar slants of the soil isolates.  Equipments: Inoculating loop, Bunsen burner.  Chemicals: MRVP medium, Methyl Red(MR), 5% alpha-naphthol, 40% potassium hydroxide.Method:  Inoculate the tubes with MRVP media with Organism to be tested.  Incubate the tubes at room temperature for 24hrs.  Methyl Red Test: Add 5 drops of methyl red indicator to the broth under sterile conditions. Interpret the colour result immediately.  Voges-Proskauer’s Test: First 5% alpha-naphthol and then 40% potassium hydroxide are added to the VP tube. The culture is allowed to sit for 15 mins for colour development to occur. Interpret the result. [40]5] Indole Test: The Indole test determines the ability of an organism to produce indole from thedegradation of amino acid trytophan. Tryptophan is hydrolysed by trytophanase to producethree possible end products- one of which is indole. Organisms possessing the enzyme trytophanase cleave tryptophan, producing 3 endproducts. Amyl alcohol in Kovac’s reagent acts as a solvent for indole which then reacts withp-dimethylaminobenzaldehyde to produce a red rosindole dye. Organisms which don’tproduce the enzyme, produce no colour change in the medium upon addition of Kovac’sreagent. 55
  • 56. Requirements:  Nutrient Agar slants of the soil isolates.  Equipment: Bunsen burner, Inoculating loop.  Chemicals: Tryptic Soy Broth, Kovac’s reagent.Method:  Inoculate the Tryptic soy broth with organism to be tested and incubate at room temperature for 24hrs.  Add 5 drops of Kovac’s reagent and gently agitate.  Examine the upper layer of liquid.  Positive result: red/pink colour.  Negative result: No colour change.[41][42]6] Gelatin Test: Gelatin causes liquids to solidify at temperatures below 28 degrees Celsius. Attemperatures above 28 degrees C. gelatin is a liquid. Some bacteria produce gelatinase, aproteolytic enzyme that hydrolyzes gelatin. The hydrolyzed gelatin no longer has the abilityto gel and thus remains a liquid, even if placed at temperatures below 28 degrees. Thepresence or absence of gelatinase can be used to aid in identification of certain bacteria.Requirements: Nutrient agar slants of the soil isolates. Equipments: Bunsen burner, Inoculating loop. Medium for gelatin: Nutrient gelatin deep tubes containing 12% gelatin Method:  An inoculum of test organism is stabbed into the nutrient gelatin deep tubes.  The media is incubated at room temperature for 24 hrs. and hen refrigerated for approximately 30 mins. 56
  • 57.  If the gelatin is still intact ( organism did not produce gelatinase), the media will solidify in the refrigerator and a negative result is recorded.  If organism has produced sufficient gelatinase then the tube will remain liquid (atleast partially) and a positive result is recorded. 7] Utilization of carbon sources: Carbohydrates are the chief energy providers of the organism. They are made up ofcarbon, hydrogen and oxygen. The bonds between carbon and hydrogen are broken downwith the help of oxygen to give energy (oxidation). Bacteria produce acidic products whenthey ferment certain carbohydrates. The carbohydrate utilization tests are designed to detectthe change in pH which would occur if fermentation of the given carbohydrate occurred.Acids lower the pH of the medium which will cause the pH indicator (Andrade’s solution) toturn dark pink. If the bacteria do not ferment the carbohydrate then the media remains lightpink.[43]Requirements:  Nutrient agar slants of the soil isolates.  Equipments: Bunsen burner, Inoculating loop.  Chemicals: Different sugars- Glucose, Lactose, Xylose, Mannitol, Fructose, Sucrose, Inositol, Galactose (All 5g): Peptone (10g), NaCl (5g).Preparation of media: 1% peptone, 0.5% NaCl, 1% sugar. After autoclaving the media, add 2-3 drops of Andrade’s indicator.Method:  The media is prepared as mentioned above.  The media is then inoculated with the test organism and incubated at room temperature for 24 hrs.  Results are observed. [44] 57
  • 58. 8] Starch hydrolysis test:Principle:Starch agar is a differential medium that tests the ability of an organism to produce certainexoenzymes, including a-amylase and oligo-1,6-glucosidase, that hydrolyze starch. Starchmolecules are too large to enter the bacterial cell, so some bacteria secrete exoenzymes todegrade starch into subunits that can then be utilized by the organism.Starch agar is a simple nutritive medium with starch added. Since no color change occurs inthe medium when organisms hydrolyze starch, we add iodine to the plate after incubation.Iodine turns blue, purple, or black (depending on the concentration of iodine) in the presenceof starch. A clearing around the bacterial growth indicates that the organism has hydrolyzedstarch.In this test, starch agar is inoculated with the species in question. After incubation at anappropriate temperature, iodine is added to the surface of the agar. Iodine turns blue-black inthe presence of starch. Absence of the blue-black color indicates that starch is no longerpresent in the medium. Bacteria which show a clear zone around the growth produce theexoenzyme amylase which cleaves the starch into di- and monosaccharides. These simplersugars can then be transported into the cell to be catabolized. Bacillus species are known toproduce the exoenzyme, amylase.[27][28]Requirements: Sterile starch agar plates. Nutrient Agar slants of the soil isolates. Equipments: Inoculating loop, Bunsen burner. Iodine solution. Starch agar medium 58
  • 59. Composition per litre Sr No. Chemicals Qty(gms) 1. Beef Extract 3.0 2. Soluble Starch 10.0 3. Agar 12.0 [29] Table 11: Starch agar mediumMethod: Starch media are prepared according to the above mentioned composition. It is then autoclaved at 15lbs at 1210C for 20 minutes. Sterile starch agar plates are prepared The plates are inoculated with the culture on surface by spot inoculation. They are incubated for 24 hrs at room temperature. After incubation iodine solution is added to surface of the plates and colour is observed9] Casein Hydrolysis Test:Principle:Casein hydrolysis test is used to determine if an organism can produce the exoenzymecasesase.Casease is an exoenzyme that is produced by some bacteria in order to degrade casein. Caseinis a large protein that is responsible for the white color of milk. This test is conducted on milkagar which is a complex media containing casien, peptone and beef extract. If an organismcan produce casein, then there will be a zone of clearing around the bacterial growth.Requirements: Nutrient Agar slants of the soil isolates. Equipments: Inoculating loop, Bunsen burner Sterile petri plates Microbial cultures. Milk agar medium : 59
  • 60. Composition per litre: Sr No Chemicals Qty (gms) 1. Agar 15.0 2. Peptone 5.0 3. Yeast extract 3.0 4. Milk solids 1.0 The pH is adjusted to 7.2 +/- 0.2 at 250c Table 12: Milk agar mediumMethod: Milk agar media are prepared according to the above mentioned composition. It is then autoclaved at 15lbs at 1210C for 20 minutes. Sterile milk agar plates are prepared The plates are inoculated with the culture on surface by spot inoculation. They are incubated for 24 hrs at room temperature, observe results. Clearing of the agar around the bacterial growth indicates casein hydrolysis.[30]10] Macconkey agar test:MacConkey Agar (MAC) is a selective and differential medium designed to isolate anddifferentiate enterics based on their ability to ferment lactose. Bile salts and crystal violetinhibit the growth of Gram positive organisms. Lactose provides a source of fermentablecarbohydrate, allowing for differentiation. Neutral red is a pH indicator that turns red at a pHbelow 6.8 and is colorless at any pH greater than 6.8.Organisms that ferment lactose and thereby produce an acidic environment will appear pinkbecause of the neutral red turning red. Bile salts may also precipitate out of the mediasurrounding the growth of fermenters because of the change in pH. Non-fermenters willproduce normally-colored or colorless colonies.[31] 60
  • 61. Requirements : Macconkey agar medium Composition per litre: Sr No. Chemicals Qty(gms) 1. Peptic digest of animal tissue 17.0 2. Proteose peptone 3.0 3. Lactose 10.0 4. Bile salts 1.5 5. Sodium chloride 5.0 6 Neutral red 0.03 7 Agar 15.0 [19] The pH is adjusted to 7.1 Table 13: Macconkey agar mediumMethod: Macconkey media are prepared according to the above mentioned composition. It is then autoclaved at 15lbs at 1210C for 20 minutes. Sterile macconkey agar plates are prepared The plates are inoculated with the culture on surface by spot inoculation or streaking They are incubated for 24 hrs at room temperature, observe results. 61
  • 62. 11] Protease test: This test is performed to determine the presence of enzyme protease in the bacterialcell. Requirements:  Gelatin Agar medium. Sr No. Chemicals Qty(grams) 1. Gelatin 30 2. Agar Agar 15 3. Pancreatic digest of casein 10 4. Sodium Chloride 10 Table 14: Gelatin Agar medium Method:  Gelatin Agar media was prepared according to the above mentioned composition.  It was then autoclaved at 15lbs at 1210C for 20 minutes.  Sterile Gelatin Agar plates were prepared.  The plates were inoculated with the culture on surface by spot inoculation or streaking.  They were incubated for 24 hrs at room temperature.  10% HgCl2 was then added to the plates and the clear zone formation around growing colony was considered as positive. 12] Lipase test: Many microbes hydrolyse fats, resulting in rancidity.This is accomplished by an enzyme, Lipase. Fats are usually broken down to glycerol and fatty acids. Lipase is one of the most important class of industrial enzymes and has been explored in oleochemical, paper, organic chemical processing, agrochemical industry,etc. This test qualitatively determines the presence or absence of lipase in the bacterial cell. 62
  • 63. Requirements: Peptone Agar medium. Sr No. Composition Qnty(grams) 1. Peptone 5 2. Agar Agar 15 Table 15: Peptone Agar medium.Method: Peptone Agar media was prepared according to the above mentioned composition. It was then autoclaved at 15lbs at 1210C for 20 minutes. Sterile Peptone Agar plates were prepared. The plates were inoculated with the culture on surface by spot inoculation or streaking. They were incubated for 24 hrs at room temperature.Formation of opaque whitish zone around colony was taken as a positive result.F] STUDY OF EFFECTS OF VARIOUS PARAMETERS ON THE SOIL ISOLATES.1] Effect of different temperatures: Overview: An effort was taken to study the effects of varying temperatures on the growth of the soil isolates. The temperatures selected for this purpose were 4 0C, 100C, 280C(R.T), 370C and 550C. Method:  Nutrient broth was prepared with its pH adjusted to 7.2 and was dispensed into test-tubes.  The test-tubes containing the media were then autoclaved at 121 0C at 15lbs for 15mins. 63
  • 64.  Keeping one control tube for every temperature, rest of the tubes were inoculated with the 14 soil isolates under sterile conditions and incubated for 24hrs. at respective temperatures.2] Effect of different pH: Overview: An effort was made to study the effects of varying pH on growth of the soil isolates. Thedfifferent pH selected for this purpose were 3 and 5 Method:  Nutrient broth was prepared in 2 conical flasks and pH of the media in each conical flask was adjusted to 3 and 5 respectively.  The media from all the flasks was then dispensed into test-tubes.  The test-tubes containing the media were then autoclaved at 1210C at 15lbs for 15mins.  Keeping one control tube for every pH, rest of the tubes were inoculated with the soil isolates under sterile conditions and incubated for 24hrs.3] Effect of varying concentrations of different heavy metals: An effort was made to study the effects of varying concentrations of different heavy metals on the growth of the soil isolates. The heavy metals used for the purpose were aluminium(AlCl3), chromium(KCrO4), manganese(MnSO4), zinc(ZnSO4) and mercury(HgCl2).Method:  15ml of Nutrient agar media was prepared per butt tube.  The butt tubes were then autoclaved at 1210C at 15lbs for 20 mins.  The media was then slightly cooled and following concentrations of heavy metals were added per tube as follows:  A stock of 10,000ppm of heavy metal was prepared. (10mg/ml) 64
  • 65.  It was diluted to 1000ppm by adding 9ml of distilled water. (making it to 10mg/10ml). Thn using this as the stock solution, required quantities of heavy metal concentrations wee prepared using the formula RT/G.  For eg: To make 10ppm conc. of a heavy metal, R= Required concentration, T= Total quantity and G= given conc. of heavy metal. Therefore, 10*15/1000 = 0.15.  After adding the heavy metal, the media was poured into petri plates and allowed to solidify.  The soil isolates were then streaked onto the plates under sterile conditions and incubated at room temperature for 24 hrs.  The results were observed.4] To study the growth curve of the organisms at different concentrations of phenol:The growth curve is studied of the organisms at various concentrations of 50 ppm and100ppm of phenolMethod : Three flask of 100ml capacity were taken and nutrient broth is prepared (100ml) in each flask. The media was then autoclaved at 1210C at 15lbs for 20 mins. Phenol solution of 50 ppm and 100 ppm concentration is prepared by formula RT/G and added to respective flask, and 7ml isolate suspension of 0.05 O.D at 540nm is inoculated in each flask. The flask were kept in shaker at room temperature for 24 hours. The growth curve is studied for period of 24 hours at regular time intervals and absorbance is measured at 540nm. 65
  • 66. RESULTS 66
  • 67. RESULTS:A] Waste water analysis:Sr No. Test Parameters Results Units1 Total Dissolve Solids 1423 mg/l2 COD 1963 mg/l3 BOD 3 days, 270C 1076 mg/l4 Cyanide Absent mg/l5 Zinc 0.59 mg/l6 Copper BDL mg/l7 Aluminium 58 mg/l8 Manganese BDL mg/l9 Mercury 0.017 mg/l10 Iron 0.19 mg/l11 Chromium 0.23 mg/l12 Oil & Grease 12 mg/l13 Methylene orange alkalinity 66 mg/l14 Chloride 23252.5 mg/l15 Hardness (EDTA) 148.2 mg Ca+2/L Table 16: Waste water analysis 67
  • 68. Some of the parameters of wastewater analysis like heavy metal ion detection were given inlaboratory and reports were obtained. pH of water was 9 and temperature around 300C. Thecolour of wastewater was found to be black and reddish brown.B] Isolation of microorganisms:1] Soil microorganisms:Sr No. Dilutions cfu/ml Average cfu/ml1 10-3 3570 x 1042 10-4 2840 x 104 ≈ 10 x 1073 10-5 2740 x 104 Table 17: Soil microorganisms2] Waste water microorganisms:Sr No. Dilutions cfu/ml Average cfu/ml1 10-3 880 x 1042 10-4 790 x 104 ≈ 20 x 1063 10-5 500 x 104 Table 18: Wastewater microorganisms 68
  • 69. Figure 1: Citrate Utilization testFigure 2: Glucose Utlization Test 69
  • 70. C] Gram staining:Organism No. Gram Nature MorphologyKN1. Negative RodsKN2. Positive RodsKN3. Positive RodsKN4. Positive CocciKN5. Positive RodsKN6. Positive CocciKN7. Positive RodsKN8. Positive RodsKN9. Negative Rods Table 19: Gram stainingD] Biochemical Tests:Sr Test KN1 KN2 KN3 KN4 KN5 KN6 KN7 KN8 KN9No.1. Citrate Utilization + - + + - + + + -2. Nitrate Utilization + + + - - - + + -3. Indole test - - - - - - - - -4. Gelatin - - - - - - + + +5. Methyl red test + - - - - - - - -6. Voges-Proskauer - - - - - - - - - test Table 20:Biochemical Tests 70
  • 71. Figure 3: Sucrose Utlization TestFigure 4: Nitrate Utlization Test 71
  • 72. Figure 5: Casein Hydrolysis Test:Figure 6: Casein Hydrolysis Test: 72
  • 73. Figure 7: Starch Hydrolysis Test: 73
  • 74. E] Acid production:Sr Sugars KN1 KN2 KN3 KN4 KN5 KN6 KN7 KN8 KN9No.1. Glucose - + + - - - + + +2. Lactose - - - - + - - - -3 Xylose + - - - - - - - -4 Manitol + + + - - + - - -5 Inositol - - - + - - - - -6 Fructose + + + + - + + + +7 Sucrose + + + + - + - - -8 Galactose + + + + - - - - - Table 21: Acid productionF] Enzyme assay:Sr Test KN1 KN2 KN3 KN4 KN5 KN6 KN7 KN8 KN9No.1. Starch + + - + - - - - - hydrolysis2. Casein + + - - + + + - - Hydrolysis3. Catalase + + - + + + + + - test4. Protease - - - - - - - - -5. Lipase - - - - - - - - - Table 22: Enzyme assay 74
  • 75. Figure 8: ZnSO4Tolerance:Figure 9: KCrO4Tolerance: 75
  • 76. G] Probable organisms:The biochemical test, acid production test and enzyme assay were then compared to differentspecies in the bergy’s manual and following organisms characteristics were matched withstudied isolates. Pseudomonas spp Bacillus spp Micrococcus spp Arthrobacter sppH] Heavy Metal Tolerance:1] ZnSO4Tolerance:The soil isolates were tested against varying concentrations of different heavy metals tocheck their tolerance against these heavy metals. The results were confirmed by comparingturbidity of the inoculated tubes with the control tubes.Sr Concentration KN1 KN2 KN3 KN4 KN5 KN6 KN7 KN8 KN9No. (ppm)1 10 + + + + + + + + +2 50 + + + + + + + + +3 100 + + + + + + + + +4 300 + + + + + + + - -5 500 + + + + - - - - -6 700 - + - + - - - - -7 1000 - - - - - - - - - Table 23: ZnSO4 Tolerance: 76
  • 77. 2] KCrO4Tolerance:Sr Concentration KN1 KN2 KN3 KN4 KN5 KN6 KN7 KN8 KN9No. (ppm)1 10 + + + + + + + + +2 50 + + + + + + + + +3 100 + + + + + + + + +4 300 + + + + + + + + +5 500 + + + + + + + + +6 700 - + + + + + + + +7 1000 - + + + + + + + + Table 24: KCrO4 Tolerance 77
  • 78. 3] MnSO4Tolerance:Sr Concentration KN1 KN2 KN3 KN4 KN5 KN6 KN7 KN8 KN9No. (ppm)1 10 + + + + + + + + +2 50 + + + + + + + + +3 100 + + + + + + + + +4 300 + + + + + + + + +5 500 + + + + - + + - +6 700 - + + + - + + - +7 1000 - + + + - + - - + Table 25: MnSO4Tolerance 78
  • 79. Figure 10: HgCl2Tolerance:Figure 11: HgCl2Tolerance: 79
  • 80. 4] HgCl2Tolerance:Sr Concentration KN1 KN2 KN3 KN4 KN5 KN6 KN7 KN8 KN9No. (ppm)1 10 + + + + + + - + +2 50 - + + - + - - - +3 100 - + - - + - - - +4 300 - + - - + - - - -5 500 - + - - - - - - -6 700 - - - - - - - - -7 1000 - - - - - - - - - Table 26: HgCl2 Tolerance 80
  • 81. 5] AlCl3 Tolerance:Sr Concentration KN1 KN2 KN3 KN4 KN5 KN6 KN7 KN8 KN9No. (ppm)1 10 + + + + + + + + +2 50 + + + + + + + + +3 100 + + + + + + + + +4 300 - - + + + - + - -5 500 - - - - - - - - -6 700 - - - - - - - - -7 1000 - - - - - - - - - Table 27: AlCl3 Tolerance 81
  • 82. I] Growth at different pH:Sr pH KN1 KN2 KN3 KN4 KN5 KN6 KN7 KN8 KN9No.1 5 + + + + + + + + +2 3 - - - - - - + + + Table 28: Growth at different pHJ] Growth at different Temperatures:Sr Temperature KN1 KN2 KN3 KN4 KN5 KN6 KN7 KN8 KN9No. [0C]1 4 - - - - - - - - -2 10 - - - - - - - - -3 28 + + + + + + + + +4 37 + + + + + + + + +5 55 - - - - - - - - - Table 29: Growth at different Temperatures 82
  • 83. K] Effect of various concentration of phenol on growth curve of the organism:Time (Hrs) Optical Optical Optical density(540nm) density(540nm) density(540nm) Without Phenol Phenol(50ppm) Phenol(100ppm)0 0.0 0.0 0.02 0.0 0.0 0.04 0.20 0.14 0.106 0.35 0.23 0.138 0.56 0.41 0.2910 0.77 0.65 0.5612 0.85 0.73 0.6514 0.95 0.79 0.7316 1.00 0.86 0.8318 1.05 0.94 0.8920 1.06 0.95 0.8922 1.09 1.03 0.9824 1.09 1.06 1.01 Table 30: Effect of various concentration of phenol on growth curve of the organism 83
  • 84. Graph 1: Effect of phenol on growth curve 84
  • 85. DISCUSSION 85
  • 86. DISCUSSION:Due to rapid industrialization, different types of pollution have emerged and recognized,which include air, soil and ground water pollution, caused by gaseous, liquid and solid formsof chemicals. Soil degradation from various inorganic and organic contaminants, is not onlyan ecological risk, but simultaneously it is also a social, health and economic issue. Such soilsbecome poor in physiochemical properties, susceptible to erosion; result in loss of productionand sustainability, diminished food chain quality, and tainting of water resources. Restorationof such degraded land depends on one hand upon action of nature itself, which is timeconsuming, and on the other hand, it also depends upon human intervention. Amongavailable remediation technologies, bioremediation is a useful approach to clean upcontaminated soil and wastewaters, which is based upon utilization of natural or engineeredbiota( Rani Faryal 2003)[14] In the present study, an attempt has been made to analyze physiochemical properties ofwastewater collected from koperkhairane nullah near MIDC industrial area besides Furnacefabrica Ltd and Alok industry, Maharashtra. Analysis of effluent showed that effluentsamples were highly coloured like dark black and reddish brown, foul smelling, alkaline andhaving temperature approximately 300C. Colour is the first contaminant to be recognized inthe textile industry waste water, due to presence of unused dye in water, which affectsaesthetic as well as biological activity of the ecosystem (sarnaik and kanekar, 1995; bant et al1996). Total dissolved solids (TDS) of the effluent were high (1423 mg/l) as compared to theIndian standard of specification requirement but in the permissible limit. BOD of the effluentwas exceeding Indian standard specifications(appendix 1). These results were in line with thework of Gowrisankar et al.(1997) on textile mill effluent in India, with BOD ranging between400-1000mg/L. In general high BOD reflects high concentrations of substances that can bebiologically degraded, thereby consuming oxygen and potentially resulting in dissolvedoxygen. (Rani Faryal 2003)[14] COD of the effluents was also high (1963mg/L) as compared to Indian standardspecifications. In textile effluent, pollution is aggravated by the presence of free chorine andtoxic heavy metals, which causes rapid depletion of dissolved oxygen leading to an “oxygensag” in receiving waters. In other words, a high degree of pollution is associated with highCOD.[14] Alkalinity and Hardness of water were under normal range, but chlorides were 86
  • 87. present beyond the permissible limit. Except for Aluminum, all other heavy metals like Zinc,Copper, Manganese, Mercury, Iron, and Chromium are under desirable limits of Indianstandard specifications.An attempt has been made to isolate and enrich microbes from soil of koperkhairane nullahnear MIDC industrial area besides Furnace fabrica Ltd and Alok industry, Maharashtra.Identification and characterization of the isolates was based on gram staining, biochemicaltests like citrate utilization, nitrate utilization, indole test, gelatin test, MR-VP test and acidproduction. All the isolates appeared to be Gram positive with most of them being purplerods, a few purple cocci and only two isolates KN 1 & 2 were Gram negative rods (Table 19).Most of the isolates were found to be citrate positive indicating that they could use sodiumcitrate as a carbon source.All the isolates showed negative results for Indole and MRVP test.This could infer that these organisms did not possess the enzyme tryptophanase and do notproduce high levels of acid during fermentation of glucose. However, few organisms didshow the presence of gelatinase and hence tested positive for gelatin test. Out of 9 isolates 5isolates showed positive nitrate reduction test and rest were negative. Out of the varioussugars used as carbon sources like, glucose, lactose, xylose, manitol, inositol, fructose,sucrose, galactose, the isolated colonies successfully fermented most of the sugars exceptlactose, inositol and xylose. Only few isolates were able to ferment lactose, inositol andxylose.( Table 20, 21).Most of the result for Qualitative enzyme assay i.e. strach hydrolysis, casein hydrolysis andcatalase test were positive which indicates that isolates can produce enzyme required fordegradation of respective substrates. But results were negative for protease and lipasetest.(Table22). All the biochemical characteristics were compared with Bergey’s Manualchart, from which few organisms were matching in their characteristics with the studiedisolates in the said work Hence, it could be said that the organisms isolated from soil nearbywaste water could belong to any one of Pseudomonas spp, Bacillus spp, Micrococcus spp, orArthobacter spp. Isolates of the said project can be further confirmed upto species level bysequencing and morphological study.In the next half of the study, an attempt was made to study heavy metal tolerance of the soilisolates and optimize various parameters like pH, temperature and growth curve studies of thesoil microorganism. 87
  • 88. To survive under metal-stressed conditions, bacteria have evolved several types ofmechanisms to tolerate the uptake of heavy metal ions. These mechanisms include the effluxof metal ions outside the cell, accumulation and complexation of the metal ions inside thecell, and reduction of the heavy metal ions to a less toxic state (Nies, 1999). [1]All the isolated strains (KN 1-9) were tolerant towards five different heavy metals. Most ofthe isolates are tolerant to ZnSO4 up to 300ppm-500ppm, only two KN2 & KN3 tolerated upto 700ppm(Table 15). For K2CrO4 all isolates tolerates up to 1000ppm except KN1(Table 16).5 isolates KN 2, 3, 4, 6& 9 are tolerant towards MnSO4 up to 1000ppm(Table 17). Theisolates are least tolerant to HgCl2, only KN 5 tolerates up to 500ppm(Table 18). Most of theisolates are tolerant towards AlCl3 up to 100ppm. Only KN 3, 4, 5 AND 7 tolerates up to300ppm(Table 19).All the isolates showed full growth at 280C and 370C suggesting these temperatures to betheir optimum temperatures. None could tolerate freezing temperatures of 4 0C and 100C andthermophilic range of 550C (Table21). The pH study was carried for pH 5 & 3. All theisolates showed growth at pH 5, only KN 7, 8 and 9 showed growth at pH 3(Table 21).Effects of various concentration of phenol on growth curve of the microorganism wereobserved. The growth curve was optimum for organism without phenol concentration. Thegrowth curve slightly decreased with increasing concentrations of phenol (Graph 1). Thus the said project paves the ways to study the wastewater of this region further.Analysis of various dyes and attempt to study the degradation of dyes with soilmicroorganisms would be helpful to decrease the pollution of water. 88
  • 89. CONCLUSION 89
  • 90. Conclusions:1] Textile effluents are highly polluted. Such polluted effluents must be treated properly before their discharge into the drainage channel to minimize the effect of various pollutants on the environment.2] Although some heavy metals are important and essential trace elements, at high concentrations, such as those found in many environments today, most can be toxic to microbes. Microbes have adapted to tolerate the presence of metals or can even use them to grow. Thus, a number of interactions between microbes and metals have important environmental and health implications. Some implications are useful, such as the use of bacteria to clean up metal-contaminated sites.3] Wastewater collected from Koperkhairane Nullah is a source of heavy metal Contamination, containing Aluminium, Iron, Mercury.4] High BOD and COD of effluent samples is an indicator of high load of organic pollutants.5] The isolated microbes showed remarkable tolerance against various heavy metals. 90
  • 92. Future Prospects: Further research can be carried out to enhance detoxification and biodegradation capabilities of these strains. Genetic engineered strains can be developed from these isolates, to enhance bioremediative abilities. Analysis of various dyes and attempt to study the degradation of dyes with soil microorganisms would be helpful to decrease the pollution of water. 92
  • 94. BIBLIOGRAPHY:1] Anne Spain, Communicated by: Dr. Elizabeth Alm (2003). Implications of Microbial Heavy Metal Tolerance in the Environment. Reviews in Undergraduate Research, Vol. 2, 1-6,2003.2] ADEL AL-KDASI, AZNI IDRIS, KATAYON SAED, CHUAH TEONG GUAN(2004). TREATMENT OF TEXTILE WASTEWATER BY ADVANCED OXIDATION PROCESSES – A REVIEW. Global Nest: the Int. J. Vol 6, No 3, pp 222- 230, 20043] Asamudo, N. U., A.S. Daba and O.U. Ezeronye (2005). Bioremediation of textile effluent using Phanerochaete chrysosporium. African Journal of Biotechnology Vol. 4 (13), pp. 1548-1553, December 2005.4] Clifford C. Hach, Robert L. Klein, Jr., Charles R. Gibbs. Introduction to BIOCHEMICAL OXYGEN DEMAND Technical Information Series—Booklet No. 7.5] Dr. Subrata Das. Textile Effluent Treatment – A Solution to the Environmental Pollution.6] Irina-Isabella Savin, Romen Butnaru(2008). WASTEWATER CHARACTERISTICS IN TEXTILE FINISHING MILL. November/December 2008, Vol.7, No.6, 859-864.7] John G. Holt, Noel R. Krieg, Peter H.A. Sneath, James T. Staley, Stanley T. Williams. Bergey’s Manual of Determinative Bacteriology. 93-94,530-583.8] Lars Järup (2003). Hazards of heavy metal contamination. British Medical Bulletin 2003; 68: 167–182. 94
  • 95. 9] Muhammad Masud Aslam, M A Baig2, Ishtiaq Hassan, Ishtiaq A Qazi, Murtaza Malik, Haroon Saeed (2004). TEXTILE WASTEWATER CHARACTERIZATION AND REDUCTION OF ITS COD & BOD BY OXIDATION. Electron. J. Environ. Agric. Food Chem. ISSN 1579-4377. 804-811.10] M. Vidali (2001). Bioremediation. An overview. Pure Appl. Chem., Vol. 73, No. 7, pp. 1163-1172.11] Naeem Ali (2006). Biotechnological Approaches for the Treatment of Textile Dyes by Indigenous Microorganisms.12] Nupur Mathur, Anuradha Singh. Industrial Microbiology A Laboratory Manual. 217- 22013] Olukanni O. D., Osuntoki, A. A. and Gbenle, G. O.( 2006). Textile effluent biodegradation potentials of textile effluent-adapted and non-adapted bacteria.14] Rani Faryal (2003). Role of microorganisms in bioremediation of heavy metal ions and organic pollutants present in textile industry.15] R.C.Dubey, D.K.Maheshwari. Practical Microbiology.162-182,286-301.16] RONALD M. ATLAS, Edited by Lawrence C.Parks. Handbook of Microbiological Media.17] R. Baskar, K.M. Meera Sheriffa Begum, S.Sundaram (2006). CHARACTERIZATION AND REUSE OF TEXTILE EFFLUENT TREATMENT PLANT WASTE SLUDGE IN CLAY BRICKS. Journal of the University of Chemical Technology and Metallurgy, 41, 4, 2006, 473-478.18] R.O. YUSUFF, J.A. SONIBARE (2004). CHARACTERIZATION OF TEXTILE INDUSTRIES’ EFFLUENTS IN KADUNA, NIGERIA AND POLLUTION IMPLICATIONS. Global Nest: the Int. J. Vol 6, No 3, pp 212-221, 2004. 95
  • 96. 19] Seema Jilani (2004). Biodegradation of hazardous waste during during biological treatment Process.20] SOFIA NOSHEEN, HAQ NAWAZ AND KHALIL-UR-REHMAN(2000). Physico- Chemical Characterization of Effluents of Local Textile Industries of Faisalabad– Pakistan. INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY 1560–8530/2000/02–3–232–233.Web Links:21] http://www.water-pollution.org.uk/health.html22] http://www.tutorvista.com/content/biology/biology-iv/environmental-pollution/water- pollution-effects.php23] http://www.balticuniv.uu.se/teacher/index.php/resources/downloads/doc_download/683- 18- industrial-wastewater-characteristics24] http://www.fao.org/docrep/t0551e/t0551e03.htm25] http://en.wikipedia.org/wiki/Industrial_wastewater_treatment26] http://www.biobasics.gc.ca/english/View.asp?x=741#top27] http://www.austincc.edu/microbugz/starch_hydrolysis.php28] http://www.microbelibrary.org/asmonly/details.asp?id=2846&Lang=29] http://www.bd.com/ds/technicalCenter/inserts/Starch_Agar.pdf30]http://homepages.wmich.edu/~rossbach/bios312/LabProcedures/Casein%20hydrolysis%2 0te t%20results.html31] http://www.austincc.edu/microbugz/macconkey_agar.php32] http://www.himedialabs.com/TD/M008.pdf33] http://www.lenntech.com/periodic/elements/zn.htm34] http://www.lenntech.com/periodic/elements/mn.htm35] http://www.lenntech.com/periodic/elements/cr.htm36] http://www.lenntech.com/periodic/elements/al.htm37] http://www.ihcworld.com/_protocols/special_stains/gram_ellis.htm38] http://www.hpa-standardmethods.org.uk/documents/bsopTP/pdf/bsoptp8.pdf39] http://www.austincc.edu/microbugz/citrate_test.php40] http://www.bd.com/ds/technicalCenter/inserts/MR_VP_Medium.pdf 96
  • 97. 41] http://www.hpa-standardmethods.org.uk/documents/bsopTP/pdf/bsoptp19.pdf42] http://www.pmlmicro.com/assets/TDS/845.pdf43] http://web.fccj.org/~lnorman/unknowns.htm?index=244] http://www.hpa-standardmethods.org.uk/documents/bsopTP/pdf/bsoptp27.pdf 97
  • 98. APPENDIX 98
  • 99. Appendix: 99
  • 100. 100
  • 101. 101
  • 102. 102
  • 103. 103
  • 104. 104
  • 105. 105