1. Water as Biological solvent:

• Water is excellent solvent for polar substances. These includes ionic substances like salts, which contain charged particles and some non-ionic substances like sugar that contain polar groups (slightly negatively charged hydroxyl group)
• When polar substance dissolve in water, ions and polar groups are surrounded by water molecules which separates the ions or molecules from each other.
• Once the substance is in solution its molecules or ions can move freely thus making it more chemically reactive than if it is solid. Thus majority of biochemical reactions in cells take place in aqueous solution.
• The non-polar molecules such as lipid are repelled by water and usually group together forming bulky molecules. Thus these non-polar molecules are known as hydrophobic.
• Water is medium for diffusion

2. Water as transport medium:

• The solvent property of water make it as excellent transport medium in blood, lymphatic system, alimentary canal, xylem and phloem in plants.

3. Composition of cell:

• 70-95% of a typical cell contains water

4. Various biological functions of water:

• Water helps in dispersal of seeds, gametes and larval stages of aquatic organism and also fertilization by swimming gametes
• It also helps in germination of seeds by swelling and breaking open of testa and further development
• Helps in osmosis and turgidity which results in cell growth
• Helps in transpiration in plants
• Helps is osmoregulation in animals such as cooling by evaporation such as sweating and panting
• Water also helps in support mechanism, for example in annelida, water acts as hydrostatic skeleton
• Water also helps in protection. For example lacrimal fluids, mucus

5. High heat capacity of water:

• The heat capacity of water is the amount of heat required to raise the temperature of 1 kg of water by 1°C.
• Water has a high heat capacity. This means that a large amount of heat is require to rise relatively small temperature. This is because much of the energy is used in breaking the hydrogen bond which restrict the movement of molecules.
• Temperature change within water are minimized as a result of its high heat capacity. Biochemical processes therefore operate over a smaller temperature range, proceeding at more constant rates and less likely to be inhibited by extremes of temperature.
• Water also provides a very constant external environment for many cells and organisms

6. Water has high heat of vaporization:

• Latent heat of vaporization is a measure of the heat energy required to vaporize a liquid that is to overcome the attractive forces between its molecules so that they can escape as gas.
• A relatively large amount of heat energy is needed to vaporize water. This is due to the hydrogen bonding. As a result water has an unusually high boiling point for such a small molecule.
• The energy transferred to water molecules to allow then to vaporize results in loss of energy from their surroundings, thus cooling takes places. Sweating and panting of mammals, opening of mouth of some reptiles such as crocodile and transpiration of leaves helps in cooling of body.
• The high heat of vaporization means that a large amount of heat can be lost with minimal loss of water from the body.

7. Water has high heat of fusion:

• Latent heat of fusion is a measure of the heat energy required to melt a solid.
• Due to high heat capacity of water, ice requires relatively large amount of heat to thaw.
• Conversely, liquid water must lose a relatively large amount of heat energy to freeze.
• Contents of cells and their environments are therefore less likely to freeze. Ice crystals are particularly damaging if they develop inside the cells.

8. Density and freezing property of water:

• The density of water decrease below 4°C, therefore ice floats in water.
• Since ice floats, it forms at the surface first and the bottom last. If ponds froze from the bottom upwards, freshwater organisms could not exist in arctic climates.
• Ice insulates the water below it, thus increasing the chances of survival of the organism in water.
• Also ice thaw more rapidly at surface.
• Water below 4°C tends to rise upwards thus it helps in circulation of water resulting in nutrition cycling and colonization of water to greater depth.

9. High surface tension and cohesion:

• Cohesion is the force whereby individual molecules stick together.
• At the surface of a liquid, a force called surface tension exists between the molecules as a result of cohesive force between the molecules. These cause the surface of liquid to occupy the least possible surface area.
• Water has a higher surface tension than any other liquid. The high cohesion of water molecule is important in cells and in translocation of water through xylem in plants.
• Many small organism rely on surface tension of water to settle on water or to skate over its surface.

10. Water as reagent:

• Water is biologically significant as an essential metabolite for biochemical reaction in cell.
• Water is the source of hydrogen in photosynthesis and used in hydrolysis reaction.

Biological significance of water

The post Biological significance of water appeared first on Online Biology Notes.

• The purpose of waste water treatment is to remove contaminant from water so that the treated water can meet the acceptable quality standard. The quality standard usually depends whether the water will be reused or discharged into river.
• Methods of waste water treatment depends on composition of waste water and required quality for treated water. Treatment process are broadly classified as physical, chemical and biological treatments.
• Physical treatment methods utilizes physical separation of pollutant such as by filtration etc.
• Chemical treatment methods utilizes chemical characteristics of pollutant for purification. For eg. Coagulation etc
• Biological treatment methods utilizes biological characteristics of pollutants such as bacteria, viruses by purification.
• Other purpose of waste water treatment includes;
  • To reduce strength of sewage
  • To make waste water less offensive
  • To prevent public health from toxic effect of pollutant
  • To conserve nature

Steps of sewage treatment process:

I. Preliminary treatment of wastewater:

• The main objective of preliminary treatment is to remove gross solids (such as plastics, cloths, cans, dead body of animals etc), grits and fats from waste water.
• Some of the treatment technique applied for preliminary treatment purpose are;

i. Screening:

• Screening is used to remove gross solid waste like plastics, cloths, dead animals from waste water.
• For this purpose waste water is passed through a metal screen which consists of vertical or inclined steel bars usually set 5 cm apart.
• The removes gross solids are disposed by burning or composting.

ii. Grit removal:

• Grits are small, non-biodegradable particles which are heavier than suspended organic matters.
• Grits are removed by carefully regulating the flow velocity of sewage in grit removal tank

iii. Skimming:

• Skimming is the process of removal of fatty and oily material from sewage.
• In this method, sewage is placed in skimming tank and it is aerated from bottom so that fats and oils are collected at top of the liquid which are then removed by skimming.

II. Primary treatment of wastewater:

• After removal of gross solids, grits and fats, next step in treatment is removal of remaining suspended solids as much as possible.
• The main objective of primary treatment is to reduce strength of sewage by removing suspended materials.
• Some common technique applied for primary treatment of sewage are:

i. Sedimentation:

• Sedimentation tank is used for removal of suspended solids and some organic matters.
• There are different types of sedimentation tank.
• Common example is rectangular horizontal flow tank. In this tank sewage flow very slowly (1-2 feet/min) such that solids present is waste water settle at bottom.
• Settled solids are periodically removed by sludge scrapper.
• This technique removes about 90% of suspended solids and about 40% of organic matters from sewage.

ii. Mechanical flocculation:

• In this method sewage is paced in a flocculation tank, then sewage is rotated at an speed of 0.43m/sec with the help of rotating paddles
• While sewage rotates in circular motion, small size dissolved solids attached to each other to form large size solids and settles at the bottom which is then removed out.

iii. Chemical flocculation:

• In this method, sewage is placed in coagulation tank and then some precipitating agents such as alum is added.
• Alum forms precipitate of Al(OH)3, suspended solids attached to the precipitate such that size of precipitate increase gradually to settle down at bottom.

iv. Neutralization:

• If sewage is highly acidic or basic, it is neutralized by adding base or acid to facilitate growth of microorganisms during secondary treatment process.

III. Secondary treatment of waste water:

• In secondary treatment, dissolved or colloidal organic matters are present in sewage are removed by utilizing microorganisms. In this steps, microorganisms utilizes organic matter and converts them into inorganic minerals.
• Following changes occurs in sewage during secondary treatment;
  • Organic matter (carbon) is oxidized into CO2 and H2O
  • Organic nitrogen compounds are first converted into NH3 and then into NO3
  • Colloidal matters are coagulated or precipitated out.

• Thus main purpose of secondary treatment of sewage is to reduce BOD level.
• Various techniques are used in secondary treatment of sewage. Some of them are;

i. Trickling filter:

• Trickling filter consists of filtering bed, spraying arm and water collecting chamber.
• Filtering bed consists of well graded gravel, broken stone of size (40-150mm diameter).
• Effluent or sewage from primary treatment tank is sprayed uniformly over the filter bed. During filtration a gelatinous layer of bacteria, algae, protozoa and some fungi is produced on the surface of filter bed. This layer is called Zoogleal layer.
• As the water trickles through the filter bed, organic matter present in it are oxidized by microorganism of zoogleal layer.
• Although trickling filter is classified as aeration process of sewage treatment, it is facultative system. It is because aerobic bacteria lies on the top of the filter bed whereas anaerobic bacteria lies in middle or bottom of filter bed.
• Trickling filter can reduce BOD of sewage by about 65-85% depending on the rate of filtration.

ii. Oxidation ditch:

• Oxidation ditch consists of circular canal with inlet and outlet.
• In this method, sewage from primary treatment plant is placed in oxidation ditch and then it is agitated with the help of mechanical rotator and then left for a period of about 12-24 hours.
• During the period of oxidation, microorganism present in sewage oxidize the organic matter.
• Finally the sewage is removed from oxidation ditch through outlet for tertiary treatment.
• Oxidation pond or lagoon:
• Oxidation pond is also known as lagoon or reduced pond or stabilization pond.
• It is an aerobic method of sewage treatment technique.
• In this treatment method, sewage from primary treatment plant is placed in an oxidation pond and left there for 10-40 days.
• During this period in oxidation pond, microorganisms oxidize the organic matter present in sewage. Oxygen released by algae during photosynthesis is utilized by microorganism for oxidation of organic compounds. During oxidation CO2 and H2O are released which are utilized by algae for photosynthesis. Therefore there is mutually beneficial relationship between algae and bacteria.
• Some oxygen is also derived from atmosphere for oxidation because oxidation pond is open system.
• The oxidation pond remains aerobic during day time and first hours of night. During this period oxidation of organic compound (aerobic decomposition) takes place. During rest hours of night condition become anaerobic and anaerobic decomposition of organic compound takes place.
• Advantage of oxidation pond;
  • It is very simple and easy technique
  • Treated sewage can be utilized for irrigation
• Limitation of oxidation tank:
  • Holding time is very long (10-40 days)
  • It require large area
  • It creates bad odor. Furthermore it may become breading place for mosquitoes and other vectors
  • It is influenced by seasonal temperature. It is effective only in warm climate but not in cold and rainy season.

iii. Activated sludge system:

• Activated sludge system, consists of aeration tank, settling tank and sludge return system.
• At first sewage from primary treatment plant is mixed with sludge drawn from previous batch, which is known as activated sludge or return sludge.
• The activated sludge contains large number of microorganisms and serves as inoculum of microorganisms.
• After mixing of activated sludge, sewage is placed in aeration tank. In aeration tank. Sewage is continuously aerated for 6-8 hours. During this period microorganisms oxidizes the organic compounds to form CO2, H20 and NO3 etc.
• After oxidation, sewage is passed to settling tank and left undisturbed for 2-3 hours. Sludge settle to the bottom. This sludge is called activated sludge which is fully oxidized and is very offensive. This activated sludge can be used as inoculum for next batch of sewage.
• Most of the sludge is removed and some is returned to aeration tank for next round of treatment.
• By sludge digestion process, BOD of sewage is reduced by 5-15%.

iv. Septic tank:

• Septic tank is used for disposal of content of toilet where sewage system is not available for disposal.
• Septic tank is prepared under the ground.
• Sewage along with toilet content is placed into septic tank where heavier solid wastes settle down to from sludge whereas lighter solids including fats form layer on top of sewage called scum.
• In septic tank organic compounds in sewage is anaerobically digested by anaerobic microorganisms such as Methanogenic bacteria.
• After anaerobic decomposition, the sludge become stable and inoffensive whereas liquids in sewage percolates into soil from septic tank.

IV. Tertiary or final treatment of waste water:

• Tertiary treatment of waste water is final treatment process in which all the chemical and biological agents are completely removed from sewage before disposal into river.
• The main objectives of tertiary treatment process;

i. Removal of suspended solids:

• Suspended solids are removed by two methods:
  • Microstraining:
    • In this method, sewage is placed in rotating drum filter of pore size 25-35 µm and then drum is rotated,
    • During rotation, clear water comes out of drum and suspended solids remains inside drum.
  • Chemical coagulation and filtration:
    • Chemical coagulation and filtration:
      • In this method, precipitating agents such as alum is added in sewage. Fine suspended solids adsorbs to the surface of Al(OC)3 precipitate, finally precipitate with adsorbed solids are separated by filtration.

ii. Removal of dissolved solids like salts:

• Various technique are used for this purpose such as adsorption by activated carbon, reverse osmosis
  • Adsorption by activated carbon:
    • Dissolved solids can be removed by filtering the water through filter containing activated carbon particle.
  • Reverse osmosis:
    • Reverse osmosis removes dissolved solids like NaCl and microbial cells

iii. Removal of nitrate and phosphate

• If sewage after treatment is to be discharge into river, nitrate and phosphate should be removed from sewage before disposal. It is because nitrate and phosphate causes eutrophication.
• These plant nutrients are removed by biological process. At first sewage is placed in a tank containing nitrifying bacteria. These bacteria converts ammonium salt and nitrite into nitrate
• Then the sewage is placed into second tank containing denitrifying bacteria. These bacteria converts nitrate into Nitrogen gas that leaves the sewage.
• Phosphate is also removed by bacteria by microbial assimilation process.

iv. Killing of microorganisms

• Finally microorganisms in sewage are killed by disinfection like chlorination.

Sewage treatment; Process of wastewater treatment

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• There are two broad categories of waste water on the basis of their origin. They includes sewage and industrial effluent.
• Sewage is waste water generated from residential areas like community whereas industrial effluent is waste water generated from various industries.
• Domestic sewage and industrial effluent differ in their composition and nature of pollutant. For example, microorganisms and organic matter are main pollutant in sewage whereas various toxic chemicals are main pollutants in industrial effluent.

I. Industrial effluent:

• Waste water generated from various industries is called industrial effluent.
• In general various toxic chemicals alike acid, alkali, coloring agents etc are main pollutants. Microorganisms and organic matters are usually lower in industrial effluent.
• Actual composition and characteristics of industrial effluent depends on type of industry and nature of raw materials of industry.
• Pollutants in industries are generated during processing of raw materials and manufacturing of products.

Some of the common industries and characteristics of their effluents:

1. Leather industry effluent:

• In leather industry raw skin is processed to make leather tough. Steps in making leather from raw skin includes-removal of hairs, liming of skin with Na2S, deliming by NH4Cl or (NH4)2SO4, tanning with chromium and coloring.
• Each steps in skin processing adds pollutants in effluent.
• Some important characteristics of these leather industries effluent are:
  • Organic matter; mainly keratin protein, hairs, some fats and coloring dyes
  • BOD: 700mg/liter
  • Color: strong dark
  • Dissolved and suspended solids: high
  • pH: 9.5

2. Paper and pulp industry effluent:

• In paper and pulp industries, paper is manufactured from woods.
• Various operations are carried out during paper making such as- preparation of raw materials, pulping, washing, coloring and paper making.
• All of these steps adds some pollutants in effluent.
• Characteristics of these paper industries effluent are:
  • Organic matters; mainly cellulose, lignin and coloring agents
  • Inorganic matters: acids, alkali, bleaching agents etc
  • Color: dark brown
  • Suspended solids: 1350mg/ltr
  • Dissolved oxygen (DO): 1650mg/ltr
  • Effluent is highly resistant to biological oxidation.

3. Dairy industry effluent:

• In dairy industry, various products like cheese, yoghurt, butter etc are produced from raw milk.
• Some of the characteristics of these dairy industry effluent are:
  • Organic matter: very high, mainly casein and lactose
  • Odor: highly putrescible and bad odorous due to anaerobic decomposition of casein
  • BOD: high
  • Effluent is highly susceptible for microbial decomposition
  • Toxic inorganic matter: very low or absent

4. Distillery and wine industry effluent:

• In distillery and wine industries, carbohydrate is fermented to produce alcoholic products.
• Operations carried out in distillery industries includes- preparation of raw materials, fermentation and down streaming process like distillation of alcohol.
• Pollutants are generated in each steps.
• Some characteristics of these distillery industry effluent are;
  • Organic matters: mainly carbohydrates, organic acids, and some spill alcohol
  • BOD: high (29000mg/ltr)
  • COD: high (80000 mg/ltr)
  • Suspended solids:high (4000mg/ltr)
  • Color: strong dark
  • pH: Acidic

5. Edible oil manufacturing industry effluent:

• In edible oil industry, pollutant are generated during neutralization of excess fatty acids, filtration and distillation of oil.
• Characteristics of these edible oil industry effluent are;
  • Organic matter: high content of free and saponified (hydrolyzed) fat
  • Suspended solids: high content of suspended and dissolved solids
  • BOD: high
  • COD: high

6. Detergent industry effluent:

• Detergent industry effluent mainly contains emulsifying agents like phosphorus, borax, alkyl benzene, sulphonate etc
• Effluent is usually alkaline and are non-biodegradable and toxic to aquatic biota.

II. Sewage:

• Wastewater generated from residential area is called sewage.
• Sewage is extremely unpleasant in odor which contains large number of microorganisms, organic matters and other pollutants.
• Pure domestic sewage consists of discharge from bathroom, toilet, kitchen and laboratories. However sometimes industrial effluent are also mixed with sewage.
• Actual composition of sewage depends on type, condition and age of sewage.
• In general sewage contains:
  • Sullage: it is waste water generated from kitchen, bathroom, laboratories etc
  • Human and animal excreta from toilet and animal farm
  • Run-off waste water from street during raining
  • Some industrial effluent

Wastewater; Industrial effluent and domestic sewage

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Microbiological or bacteriological analysis of water is performed in 2 steps:

Step I: Water sample collection:

• Collection of water sample is important step in bacteriological examination of water.
• If sample collection process is not appropriate, it gives false result and raises question about validity and liability of the test.
• Water sample for analysis should be collected in sterile bottle made up of neutral glass of capacity 200-500 ml.
• Methods of water sample collection vary depending on the source of water.

i. Bottled or mineral water sample:

• In case of bottled water, at first outside of the bottle is disinfected or sterilized with chemicals like chlorine or phenol before sample collection.
• Water sample is taken out by sterile syringe through sterile portion of bottle.

ii. Jar water sample:

• In case of jar water sample, at first the covering of jar is sterilized by chemicals and then covering is removed using sterile knife.
• Finally sample water is taken out from jar using sterile pipette.
• Tap water sample:
• In case of tap water sample, at first tap is open fully for about 2 minutes to remove the microorganisms present in internal nozzle.
• Then water sample is collected in sterile collecting bottle.

iii. River, pond or lake water:

• In case of river, pond or lake water sample, sterile collecting bottle with string at neck is used to collect sample.

Step II: Water sample examination:

• There are different methods utilized for microbiological examination of water. Some of them are:

1. Standard plate count method
2. Most probable number method
3. Membrane filtration technique

1.  Standard plate count (SPC) method:

• Pour plate technique is commonly used method for standard plate count to count the microorganism (bacteria) in water.
• SPC is not considered satisfactory for bacteriological analysis of drinking water because it gives total count of all bacteria present in water but not specifically of coliform and pathogenic one. However this method is commonly used to check efficiency of water treatment plant.
• In water treatment plant, standard plate count method is carried out after each step of treatment.
• To count coliform in water, media such as crystal violet neutral red bile lactose agar (VBRL agar) is used.
• In this method, at first sample (water) is serially diluted in different test tubes, then fix volume (1ml) of diluted sample from each dilution is placed in sterile petriplate.
• Then molted agar media is poured in the petriplate, mixed with inoculum, allowed to solidify and then incubated for appropriate time and temperature.
• Colony count is made and number of bacteria in water sample is calculated with the formula:
  • Colony forming unit (cfu/ml) = number of colonies * dilution factor /Volume of sample

2. Most probable number (MPN) method: multiple tube test

• MPN method is a statistical technique for counting bacteria present in water sample. This test is mainly used to count coliforms but it can also be used to count pathogens like Salmonella, Vibrio etc.
• This MPN technique gives most probable number of pathogens in water but not actual number.
• In this technique, microbial number is not directly counted but recorded from previous MPN chart.

3. Membrane filtration (MF) method:

• In membrane filtration method, water sample is first filtered through membrane filter of pore size 0.45µ
• During filtration bacterial cells are trapped on filter paper. Then the filter paper is removed and placed over the solidified agar media and incubated for 24 hours at suitable temperature.
• Bacteria form colony and these colonies are counted. Finally number of bacteria in original sample is calculated by correlating the number of colonies with volume of water filtered.

Bacteriological water analysis

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I. Physical characteristics of sewage:

1. Temperature:

• Temperature of sewage depends upon season. However temperature is slightly higher than that of ground water.
• High temperature of sewage is due to evolution of heat during decomposition of organic matter in sewage.

2. Color:

• Color of sewage indicates its strength and age.
• Fresh domestic sewage is grey in color but septic sewage is dark in color
• When industrial effluent is mixed it give characteristic color to sewage

3. Odor:

• Fresh domestic sewage is almost odorless.
• Septic or stale sewage is putrid in odor which is due to generation of H2S during anaerobic decomposition of organic matters.
• When industrial effluent is mixed, it give characteristics odor to sewage

4. Turbidity:

• Sewage is highly turbid.
• Turbidity of sewage is due to dissolved substances, colloidal matters, suspended solids and microbial cells.

 II. Chemical characteristics of sewage:

1. Organic matter:

• In general sewage contains large amount of organic matters. However amount of organic matter depends on types and condition of sewage.
• Organic matter in sewage may be found in the form of dissolved substances, colloidal matter, suspended or sedimented form.

2. Chloride:

• Human beings discharge large amount (8-15gm/day) of chloride in the form of NaCl, especially through urine and sweat. So domestic sewage from toilet and bathroom contains higher level of chloride.
• Sulfite:
• In sewage sulfite in the form of H2S (hydrogen sulfite) is generated during anaerobic decomposition of organic matters by anaerobic bacteria.
• H2S gives putrid odor to sewage.

3. Biological oxygen demand (BOD):

• Sewage usually have high BOD due to presence of large amount of organic matters.
• Value of BOD ranges from 100mg/ltr for very dilute sewage to 600mg/ltr or more for concentrated sewage containing industrial effluent mix.

4. Dissolved oxygen (DO):

• Due to high level of microbial cells and biodegradable organic matters, sewage have very low level of dissolved oxygen.
• In some sewage, DO is completely absent.
• Level of Do depends on age and condition of sewage. Low level DO is also due to lower solubility of oxygen in sewage. Oxygen is only 95% soluble in sewage than in pure water).

5. pH:

• sewage is slightly alkaline in pH

6. Nitrogen:

• In sewage nitrogen is found in variety of form like organic nitrogen, ammonia, nitrite, nitrate etc
• Fresh sewage mainly contains organic nitrogen and very little inorganic form of nitrogen. On the other hand organic septic sewage contains high inorganic nitrogen and low organic nitrogen.
• In sewage nitrite never accumulate in concentration greater than 1mg/ltr because it is intermediate product during conversion of ammonia into nitrate (NO3).
• In sewage treatment plant, NH3 and NO2 are finally converted to NO3.

7. Oxidation-Reduction (O-R) potential:

• Oxidation-Reduction potential indicates energy state of sewage in terms of its oxidizing or reducing potential.
• O-R potential is very valuable index to monitor sewage treatment plant.
• In aerobic treatment process like tripling filters, positive OR potential of about +2—to +600 is needed. In anaerobic treatment process like sludge digestion, negative OR potential of about -100 to -200 is needed.

III. Biological characteristics of sewage:

1. Bacteria:

• Two types of bacteria are found in sewage.
  • Intestinal bacteria:
    • Non-pathogenic intestinal bacteria are normal flora of gastointestinal tract of human and animals and enter into sewage together with stool. Examples; faecal coliform, faecal streptococci, Clostridium perfingens, et
    • Pathogenic intestinal bacteria such as Salmonella, Shigella, Vibrio cholera, Yersenia enterocolitica etc enter into sewage through stool of patients.
  • Real sewage bacteria
    • The natural habitat of these bacteria is sewage.
    • Both aerobic as well as anaerobic are found in sewage.
    • Aerobic bacteria play important role in oxidation of organic matter during aerobic process.
    • Common anaerobic bacteria includes;
      • Clostridium sporogens
      • Bifidobacterium
      • Peptococcus
      • Methanogenic bacteria like Methanobacterium, methanosarcina
    • Common aerobic bacteria includes;
      • Zeoglea remigera
      • Noacrdia
      • Flavobacterium
      • Achromobacter
      • Nitrosomonas
    • ** Zeoglea remigera is the main organism found in trickling filter.

2. Algae:

• Some algae found in sewage includes Chlorella phormidum, Ulothrix etc
• Algae are used in trickling filter in sewage treatment plant

3. Fungi:

• Fungi like Fusarium and Sporotricum are found in sewage which play important role in trickling filter.

4. Virus:

• Some viruses causing human disease such as Poliovirus, Rotavirus, Hepatitis A and E etc are found in sewage which get access through stool of patients.

5. Protozoa:

• Some protozoa that cause disease of intestinal tract enter into sewage together with stool of patient.
• Examples: Entamoeba histolytica, Giardia, Balantidium coli etc are pathogenic protozoa
• Few protozoa such as Vorticella and Opercularia are found in trickling filter.

Physical, chemical and Biological characteristics of sewage

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• MPN method utilizes 3 set method or 4 set method for counting coliform in water. However three set method is commonly used.
• MPN method for counting coliform in water is completed in three steps:

step I: Presumptive test:

• At first three set of 5 test tubes are taken.
• 10 ml of double strengthen liquid media (MacConkey broth) is placed in each test tubes of 1^st set
• Similarly, 10 ml of single strengthen liquid media is placed in each test tubes of 2^nd set and 3^rd
• ** double strengthen broth refers to broth made up using twice the normal amount of broth powder.
• Lactose broth or lauryl sulphate broth or tryptose lauryl broth is used as liquid media for the test.
• Then, Durham’s tube is inserted in inverted position in each test tubes of all sets.
• All the test tubes are then cotton plugged and sterilized using autoclave for 15 minutes at 15 lb/inc pressure at 121 °
• After cooling water sample is added in each test tubes as follows;
  • Add 10 ml water sample in each test tubes of 1^st set
  • Add 1 ml water sample in each test tubes of 2^nd set
  • Add 0.1ml water sample in each test tubes of 3^rd
• Then incubate all test tubes at 35.5 °C for 24 hours. After incubation gas production in Durham’s tubes is observed.
• Tubes in which gas production is 10% or more is recorded as positive tube and tubes in which gas production is less than 10% is recorded as doubtful.
• Doubtful test tubes are further incubated for 24 hours and again gas production is noted. If gas production is still less than 10%, then tube is recorded as negative and are discarded
• ** doubtful result is given by other gas producing lactose fermenting bacteria other than coliforms such as Lactobacillus, Streptococci, Bacillus, Clostridium Clostridium produces more than 10% gas but only after 48 hours of incubation.
• All the positive test tubes are taken for confirmatory test.

step II: Confirmatory test for MPN method:

• Positive tubes obtained from presumptive test are now confirmed for coliform.
• For confirmation of coliform, brilliant green lactose bile (BGLB) broth is used as culture media, because BGLB broth inhibits growth of gram positive bacteria such as lactobacillus, Streptococci, Bacillus and Clostridium
• Coliforms can grow in this BGLB medium
• For confirmation, one loopful of sample from each positive tubes obtained from presumptive test is inoculated in respective tubes containing Brilliant green lactose bile broth and incubated for 24 hours at 35.5 °
• Gas production 10% or more are recorded as positive while less than 10% is recorded as doubtful. Doubtful tubes are again incubated and the result is recorded.
• All the positive test tubes are now confirmed for presence of coliforms.
• Finally the number of bacteria present in water sample is determined from previous MPN chart. Alternatively number of coliforms can also be calculated by the formula;
  • Coliforms/100ml = numbers of positive tubes
• _/ volume of samples in negative tubes * volume of samples in whole experiments

step III: Completed test for MPN method:

• It is a final test in which a loopful of sample from positive confirmatory tubes is streaked on Eosin methylene blue agar or M-endo agar and incubated for 24 hours.
• Three types of colonies are obtained in culture media;
  • Typical colony: they are pink colored with greenish metallic appearance or nucleated colony. Coliforms gives typical colony
  • Atypical colony: they are pink and non-nucleated colony. Coliforms as well as other lactose fermenting organisms gives atypical colony
  • Non-typical colony: they are non-pink colony given by non-coliforms.

MPN method for faecal coliforms; Eijkman test:

• Eijkman test or confirmed E.coli test or differential coliform test is MPN test used to detect faecal coliforms in water,
• This test is similar to MPN test for total coliforms but incubation temperature is slightly higher.
• Faecal coliforms are thermotolerant and can grow at temperature of 44.5° Therefore if incubation temperature is maintained at this point, only faecal coliforms can grow, but not other coliforms.

step I: Presumptive test:

• Media: lactose broth or Macconkey broth
• Incubation temperature: 35.5°C

step II: Confirmatory test:

• Media used: E. coli media (EC media)
• Incubation temperature: 44.5 °C

step III: Completed test:

• Media used: M-endo agar or Eosin methylene blue agar
• Incubation temperature: 44.5 °C

Most probable number (MPN) method for counting coliform

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• Detail step and methods of water treatment depends on nature of raw water and required standard of water quality.

General steps in purification of drinking water includes

1. Aeration:

• Raw water is first collected in large aeration tank and the water is aerated by bubbling compressed air through perforated pipes.
• Aeration removes bad odors and CO2. It also removes metal such as iron, manganese by precipitating then as their respective hydroxides.

2. Storage or settling:

• Aerated water is then placed in settling tank and stored for 10-14 days.
• During storage about 90% of suspended solids settle down within 24 hrs and the water becomes clear.
• Certain heavier toxic chemicals also settle down during storage.
• Similarly pathogenic bacteria gradually die and bacterial count decreases by 90% in first in first 5-7 days of storage.
• During storage organic matter present in water is oxidized by microorganisms. Similarly NH3 present is oxidized into nitrate by microorganisms during storage.

3. Coagulation:

• Water from storage tank is then placed in coagulation tank and then some precipitating agents such as alum, lime etc are added in water and mixed.
• These precipitating agents form precipitate of Al(OH)3 when dissolved in water.
• Suspended solids absorbs on the surface of precipitate, so gradually mass of precipitate becomes heavier and finally settle down.
• This technique is used to remove very light suspended solids that do not settle by themselves during storage. Furthermore, if negatively charged colloidal impurities are present, they are neutralized by Al+++ ions and settle down.

4. Filtration:

• Partially clarified water is then passed through sand gravity filter which removes 98-99% of microorganisms and other impurities.
• Sand gravity water filter:
  • Sand filter is a rectangular tank in which filter bed is made up to 3 layers.
  • Top layer: fine layer of 1 meter thick
  • Middle layer: 0.3-0.5 meter thick layer of coarse sand
  • Bottom layer: 0.3-0.5 meter thick layer of gravel

• There is a collection tank at the bottom of the filter bed to collect filtered water. During filtration filter bed soon gets covered with a slimy layer called vital layer.
• Vital layer consists of thread like algae, diatoms and bacteria.
• During filtration microorganisms presents in vital layer oxidize organic and other matter present in water. For example if NH3 is present, it is oxidized into nitrate.
• Vital layer also helps in filtration of microbial cells.
• If water contains unpleasant odor, activated carbon may be placed in filter bed that removes bad odors.

5. Disinfection:

• The filtered water is finally purified by using disinfectants. Eg. Chlorination
• Disinfectant kills pathogenic as well as other microorganism in water.
• After disinfection water is pumped into overhead tank for subsequent domestic distribution.

Steps of water purification process

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• Enrichment of water system with plant nutrients such as nitrate and phosphate is known as eutrophication.
• The word eutrophication refers to nutrient rich condition of water system.
• On the basis of nutrition status and productivity, water systems are classified as;
  • Oligotrophic: water with poor nutrient content and poor productivity
  • Mesotrophic: water with moderate nutrient content and moderate productivity
  • Eutrophic: water with rich nutrient content and very high productivity

• Nitrate and phosphate are key plant nutrient. Therefore, if nitrate and phosphate enter into natural water system, they facilitates heavy growth of aquatic plants like algae causing Algal bloom.
• Waste water such as Sewage contains high concentration of nitrate and phosphate. Similarly, death of aquatic plants and animals also increases nitrate and phosphate content of water.
• Nutrition status of water system such as lakes gradually increases with increase in age of lake as they gradually changes from oligotrophic to eutrophic water system.

Undesirable effects of eutrophication:

• It facilitates rapid growth of aquatic plants causing algal blood. Growth of algae on surface of water prevents penetration of light into deeper layer.
• When aquatic life dies, microorganisms consume dissolved oxygen to oxidize their dead bodies. Loss of dissolved oxygen inhibits growth of aquatic organisms.
• Loss of dissolved oxygen creates anaerobic conditions that facilitates anaerobic decomposition of remaining organic matters. Compounds such as H2S, CH4, NH3 etc generated during anaerobic decomposition creates odor and taste problem in water.
• Over growth of algae and other plants causes clogging of water filters during water treatment process.

Control measures of eutrophication:

• Waste water like sewage should be discharge into river or other water system only after proper treatment. For example nitrate and phosphate should be removed from waste water before disposal into river.
• Algal bloom should be removed from water. Algae should not be disposed into water after killing them because it further generate plant nutrient during decomposition.
• Use phosphate free detergents to reduce eutrophication
• Growth of algae in water can also be controlled by applying algicides such as CuSO4.
• Prevents flooding of water from agricultural soil containing fertilizers to water system.

Eutrophication: undesirable effects and control measures

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• Quality of water is determined by its physical, chemical and biological parameters.

I. Physical parameters of water quality

1. Color: The guideline value (maximum acceptable level) for color of drinking water is 15 TCU (True color unit).
2. Turbidity: Drinking water should have turbidity less than 5 NTU (Naphthalometric turbidity unit)
3. Taste and odor: Pure water is always tasteless and odorless. Therefore if any types of taste and odor is present, it indicates water pollution.
4. Temperature: There is no guideline value for temperature for drinking water.
5. Foam
6. Conductivity
7. Total dissolved solid (TDS): TDS should not exceeds 300mg/ltr

II. Chemical parameters of water quality

1. Chloride ion: Maximum permissible limit of chloride ion in drinking water is 200mg/ ltr.
2. Ammonia: Concentration of NH3 in ground water system is usually 3mg/ltr. If its concentration is greater than 50mg/ltr, it gives characteristic taste and odor.
3. Nitrite: Level of nitrite in drinking water should not exceed 3mg/ltr.
4. Nitrate: It is most stable oxidized form of nitrogen. Like nitrite Nitrate should not exceed 3mg/ltr in drinking water.
5. Phosphate
6. Hardness: Water is classified as 9on the basis of concentration of calcium carbonate)
   • Soft water:<5omg/ltr
   • Moderately hard water: 50-150 mg/ltr
   • Hard water: 150-300 mg/ltr
   • Very hard water:>300 mg/dl

7. Biological oxygen demand (BOD)
8. Chemical oxygen demand (COD)
9. Dissolved oxygen (DO)
10. pH: pH of drinking water; 6.5-8.5

III. Biological parameters of water quality

1. Bacteriological aspects of water pollution
2. Viral aspects of water pollution
3. Parasitological aspects of water pollution
4. Algae

Water quality criteria/ Parameter of water quality

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• These are some chemical aspects of water quality that helps to determine whether water is polluted or not.

1. Chloride ion:

• Normally all types of water contains chloride ion but its concentration is very low in natural water system.
• Chloride ion concentration increases in case of urine and sewage contaminated water.
• High concentration of chloride ion give salty taste and also corrodes pipelines of water.
• Normally 150mg/ltr of chloride ion is harmless.
• Maximum permissible limit of chloride ion in drinking water is 200mg/ ltr.

2. Ammonia:

• In water ammonia come from decomposition of organic matter like protein, amino acids etc. Its concentration also increases during water disinfection process using chloramine.
• In water Ammonia (NH3) is first oxidized into nitrite and then into nitrate. Therefore by measuring the concentration of NH3, nitrite and nitrate, we can predict the time of contamination of organic matter in water.
• In recently contamination, concentration of NH3 is very high than nitrite and nitrate.
• Concentration of NH3 in ground water system is usually 3mg/ltr. If its concentration is greater than 50mg/ltr, it gives characteristic taste and odor.

3. Nitrite:

• It is very unstable intermediate formed during conversionof NH2 into nitrate.
• In aerobic condition nitrite is oxidized into nitrate whereas in anaerobic condition, nitrite is reduced to ammonia.
• If concentration of nitrite is greater in drinking water, it brings serious health hazard to the consumers.
• Disease caused by high concentration of nitrite in infants is called Blue baby syndrome, which is characterized by blue coloration of skin
• Level of nitrite in drinking water should not exceed 3mg/ltr.
• **Note; blue baby syndrome: hemoglobin has greater affinity for nitrite than oxygen. Therefore, if level of nitrite is high in blood due to consumption of contaminated water then it inhibit formation of oxyhemoglobin, instead it form nitrosomyoglobin. This prevent transport of Oxygen to tissue giving characteristic blue color. In infants nitrite poisoning occurs even due to high concentration of nitrate in drinking water because stomach of infant is less acidic. In this situation nitrate is reduced by intestinal bacteria into nitrite causing blue baby syndrome.

4. Nitrate:

• It is most stable oxidized form of nitrogen. In water nitrate comes from organic matter decomposition and from atmospheric nitrogen fixation.
• Like nitrite Nitrate should not exceed 3mg/ltr in drinking water. It is because nitrate can be reduced into nitrite in gut of infants and causes nitrite poisoning.
• Nitrate is very important in natural water system like lake and pond because high concentration of nitrate facilitates heavy growth of aquatic plants causing eutrophication.

5. Phosphate:

• In water phosphate is present in the form of H2PO4-, polyphosphate and as organic phosphate.
• Phosphate in water sources comes from agricultural wastes, sewage and from industrial effluent.
• Phosphate is not toxic to human being but it is important chemical in natural water system like pond because its high concentration facilitates eutrophication.

6. Hardness:

• Hardness of water is merely due to salt of calcium and magnesium.
• Temporary hardness is due to carbonate and bicarbonate of calcium and magnesium while permanent hardness is due to chloride and sulfate of calcium and magnesium.
• Water is classified as 9on the basis of concentration of calcium carbonate)
  • Soft water:<5omg/ltr
  • Moderately hard water: 50-150 mg/ltr
  • Hard water: 150-300 mg/ltr
  • Very hard water:>300 mg/dl
• Hard water is not suitable for industrial use. But hard water is usually beneficial for drinking purposes. However hardness caused by MgSO4 give some serious health effects. So, concentration of Mg++ should not exceeds 50 mg/lt in drinking water.

7. Biological oxygen demand (BOD):

• Biological oxygen demand represents amount of oxygen required by living organism (microorganism) for oxidation of biodegradable organic matters present in water under aerobic condition.
• BOD is a direct measurement of quality of oxygen needed for biodegradation of organic matter and indirect measurement of quantity of biodegradable organic matter in water.
• When biodegradable organic matter is added in water. Microorganism utilizes dissolved oxygen to oxidize organic matter. If the rate of consumption of oxygen during biodegradation is greater than solubilization of atmospheric Oxygen into water, level of dissolved oxygen gradually decreases.
• If organic matter content is very high, complete loss of dissolved oxygen occurs. This creates anaerobic environment in water. In this case aerobic aquatic organism cannot survive.
• Furthermore, if dissolved oxygen is absent then organic matter starts to decompose anaerobically that creates taste and odor problem.
• High value of BOD is an indicator of water pollution.

Determination of BOD

• To determine BOD, water sample is placed in a 300ml BOD bottle, seal it and incubated at 20C for 5 days in dark room.
• Light must be excluded to prevent the growth of algae that may produce O2 inside bottle.
  • BOD = (DO1-DO5) * dilution factor * temperature factor

Limitation of BOD measurement

• For complete oxidation of organic matter, it will take about 20-30 days which is practically difficult. Therefore BOD is calculated by 5 days incubation method.
• One of the major limitation pf BOD calculation is that it gives quantity of only biodegradable organic matte but not non-biodegradable organic matters.
• BOD cannot determine if water contains toxic chemicals or antimicrobial substances. It is because microorganism are killed by these toxic chemicals.

8. Chemical oxygen demand (COD):

• COD is amount of oxygen needed for oxidation of organic matter present in water by strong chemical oxidizing agents such as K2Cr2O7.
• COD measurement is particularly valuable to determine amount of organic matter in water which contains toxic or antimicrobial chemicals.
• K2Cr2O7 is strong oxidizing agents and it oxidizes both biodegradable and non-biodegradable organic matters. Therefore the value of COD is always greater than BOD for particular water sample.
• COD gives amount of total organic (biodegradable + non-biodegradable) matter present in water.

Measurement of COD:

• To determine COD, water sample is mixed with excess of K2Cr2O7 in sulfuric acid solution. At the same time add AgSO4 (as catalyst) and HgSO4 to eliminate interference by chloride ion in water.
• K2Cr2O7 oxidizes organic matter into water, CO2 and NH3. Therefore, level of K2Cr2O7 decreases, then remaining K2Cr2O7 is measured by titration with ferrous ammonium sulfate.
  • COD = (V1-V2)*N*8*1000/X
  • Where, V1= Initial volume of ferrous ammonium sulfate
  • V2= final volume of ferrous ammonium sulfate remaining
  • N= normality of ferrous ammonium sulfate
  • X= volume of water sample used during titration

9. Dissolved Oxygen (DO):

• Oxygen is highly soluble and get dissolved in water in the form of dissolved oxygen (DO).
• DO is a source of oxygen for aquatic aerobic organism like fishes, aerobic bacteria etc. therefore, DO is an important characteristics of natural water system that determines its quality.
• If DO is absent in water, aquatic organism cannot survive. Total absent of Dissolved oxygen in water creates anaerobic condition which facilitates anaerobic decomposition of organic compounds.
• Dissolved oxygen in water is affected by many factors like temperature, organic matter content etc.
• Temperature affects DO by two ways- first, solubility of oxygen in water decreases with increase in temperature of water. Second, high temperature promotes growth and activity of aquatic microorganisms so that they consume oxygen faster.
• If organic content is high, microorganisms utilizes DO for its oxidation causing depletion of dissolved oxygen level.

Measurement of Dissolved oxygen (DO) by modified Winkler’s method:

• DO content of water is determined iodometrically.
• At first water sample is taken and alkaline Potassium iodide (KI) is added
• When MnSO4 is added, dissolved oxygen in water oxidize MnSO4 into basic manganese oxide {Mn(OH)2}.
• Adding H2SO4 in the solution liberates iodine as gas. Amount of iodine gas is estimated by titration with sodium thiosulfate containing starch indicator.
• The amount of iodine liberated is equivalent to amount of dissolved oxygen present in water.

10. pH:

• Water become alkaline due to presence of Nh3, OH-, Ca++, Mg++ etc. in alkaline water certain minerals are deficient. Alkaline pH also decreases efficiency of disinfection process (chlorination).
• Water become acidic due to presence of dissolved acids and dissolved carbon-dioxide. Acidic pH corrodes pipeline. Similarly certain minerals are more than excess in acidic water.
• pH of drinking water should be around 6.5-8.5

Chemical parameters of water quality/ Chemical characteristics of water

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