Sustainability & Environment (Agri-Food Systems) · Foundations · also Microbial Ecology, Environmental Biotechnology (introductory), Soil and Water Microbiology
Microorganisms as the engines of nutrient cycling, wastewater treatment, bioremediation and disease. GATE ES Section 3 examines it from cell structure to Monod kinetics and coliform counts.
In the exams: GATE ES Section 3; UGC NET Environmental Sciences Unit 3; ICAR AICE subject 71 Units 4–5 (bioremediation, composting)
Environmental microbiology studies microorganisms (bacteria, archaea, fungi, algae, protozoa and viruses) in soil, water, air and engineered systems. Microbes are the engines of the biosphere: they fix nitrogen, decompose organic matter, drive the carbon, nitrogen, sulphur and iron cycles, produce and consume methane and nitrous oxide, treat wastewater, degrade pollutants, form symbioses with crops, and cause waterborne and foodborne disease. This manual covers their classification and diversity, cell chemistry and structure, metabolism, fermentation and anaerobic respiration, enzymes and their kinetics, growth and the Monod model, batch and continuous culture, physical and chemical control, plant- and soil-microbe interactions, roles in biogeochemical cycles, wastewater treatment and bioremediation, and pathogens, indicator organisms and coliform enumeration.
It supports and does not repeat several later subjects: water and wastewater treatment (reactor design and process engineering), water quality and nonpoint pollution (microbial standards for water bodies), soil science for sustainability (soil biology and soil health), pesticides and the environment (biodegradation of pesticides), and soil contamination and remediation. The ecology subject covers biogeochemical cycles at the ecosystem scale; here the focus is the microorganisms and their biochemistry.
GATE Environmental Science and Engineering Section 3 examines the subject from cell structure to Monod kinetics, specific growth rate and doubling time, chemostat washout, enzyme kinetics, disinfection, MPN and membrane filtration counts. UGC NET Environmental Sciences Unit 3 and ICAR AICE Environmental Sciences (bioremediation, composting and biofertilisers) test principles and applications. Calculations in this manual state the form of each equation used; constants such as yields and half-saturation coefficients vary with organism and substrate and are given as illustrative values.
Carl Woese's analysis of 16S/18S ribosomal RNA sequences (1977, formalised in 1990) divided life into three domains: Bacteria, Archaea and Eukarya. Bacteria and Archaea are prokaryotes, lacking a membrane-bound nucleus and organelles, typically 0.5–5 µm in size; they differ in membrane lipids (ester-linked in Bacteria, ether-linked in Archaea), cell walls (peptidoglycan in most Bacteria, absent in Archaea, some of which have pseudopeptidoglycan), and in aspects of transcription and translation that resemble eukaryotes in Archaea. Eukaryotic microorganisms, with nuclei and organelles, include fungi (yeasts and filamentous moulds), algae (photosynthetic, from unicellular to multicellular), protozoa (heterotrophic, often motile) and slime moulds. Viruses are acellular: genetic material (DNA or RNA) in a protein coat, replicating only inside host cells; bacteriophages infect bacteria. Bacteria are described by shape: cocci (spherical), bacilli (rods), spirilla and spirochaetes (spirals) and vibrios (curved rods), and by arrangement, such as chains (strepto-) or clusters (staphylo-). The Gram stain, developed by Hans Christian Gram (1884), separates bacteria with thick peptidoglycan walls that retain crystal violet (Gram-positive, purple) from those with thin walls and an outer membrane that lose it and take up safranin (Gram-negative, pink). Archaea include methanogens, which produce methane in anoxic environments, extreme halophiles and thermoacidophiles, but also many mesophilic forms in soils and oceans, including ammonia-oxidising archaea.
Microbial diversity is enormous. A gram of fertile soil may contain on the order of a billion bacterial cells and thousands to tens of thousands of species. Yet traditional culture methods recover only a small fraction, often cited as less than 1 per cent, of the cells seen under the microscope, a gap known as the 'great plate count anomaly'. Molecular methods have transformed the field: sequencing of the 16S rRNA gene (amplicon sequencing) identifies community members; metagenomics sequences all DNA in a sample to reveal genes and functions; metatranscriptomics and metaproteomics show which genes are active; and quantitative PCR measures specific genes, such as those for nitrogen fixation (nifH), ammonia oxidation (amoA) or antibiotic resistance. Microorganisms are classified by their environmental tolerances: by temperature as psychrophiles (optimum below about 15 °C), mesophiles (about 20–45 °C) and thermophiles (above about 45 °C, with hyperthermophiles above 80 °C); by oxygen as obligate aerobes, obligate anaerobes, facultative anaerobes (which can switch), microaerophiles and aerotolerant anaerobes; by pH as acidophiles, neutrophiles and alkaliphiles; and by salt tolerance as halophiles. Extremophiles are of interest for industrial enzymes; the thermophile Thermus aquaticus supplied the heat-stable DNA polymerase used in PCR. Nutritional classification depends on the energy source (light: photo-; chemical: chemo-), the electron source (inorganic: litho-; organic: organo-) and the carbon source (CO2: autotroph; organic compounds: heterotroph). Plants and cyanobacteria are photolithoautotrophs; most bacteria, fungi and animals are chemoorganoheterotrophs; nitrifying bacteria, which oxidise ammonia or nitrite and fix CO2, are chemolithoautotrophs.
Microbial cells are about 70–80 per cent water. Of the dry weight, protein is the largest component (roughly half), followed by nucleic acids (RNA more than DNA, especially in fast-growing cells), lipids, polysaccharides and small molecules and ions. Proteins are polymers of 20 amino acids joined by peptide bonds; their sequence determines folding into secondary (alpha helices, beta sheets), tertiary and quaternary structures, and their function as enzymes, transporters and structural components. Nucleic acids are polymers of nucleotides: DNA (deoxyribose, bases A, T, G, C, double helix) stores genetic information; RNA (ribose, uracil instead of thymine) includes messenger, transfer and ribosomal RNA. Lipids include phospholipids, which form membranes, and storage lipids such as poly-β-hydroxybutyrate in bacteria. Polysaccharides form cell walls (peptidoglycan, cellulose, chitin), capsules and storage polymers (glycogen, starch). Environmental engineers often represent bacterial biomass by the empirical formula C5H7O2N (Hoover and Porges, 1952), or C60H87O23N12P when phosphorus is included. From C5H7O2N, with a molar mass of 113 g/mol, cells contain about 12.4 per cent nitrogen (14/113) and about 53 per cent carbon, which is why nitrogen and phosphorus must be available in roughly a BOD:N:P ratio of 100:5:1 for aerobic treatment of nutrient-poor industrial wastes. Oxidising one gram of cells completely, C5H7O2N + 5O2 → 5CO2 + 2H2O + NH3, requires 160/113 ≈ 1.42 g of oxygen, the factor used to convert biomass to COD equivalents.
The cytoplasmic membrane, a phospholipid bilayer with embedded proteins, controls transport and, in prokaryotes, hosts the electron transport chain and ATP synthase. Outside it, the bacterial cell wall provides shape and resistance to osmotic lysis. Gram-positive walls have a thick peptidoglycan layer with teichoic acids. Gram-negative walls have a thin peptidoglycan layer within a periplasmic space, surrounded by an outer membrane containing lipopolysaccharide (LPS, or endotoxin), which makes them more resistant to some antibiotics, detergents and dyes. The antibiotic penicillin and the enzyme lysozyme target peptidoglycan. Mycobacteria have waxy mycolic acid walls and are 'acid-fast'. External structures include capsules and slime layers (polysaccharide), which protect against desiccation and phagocytosis and enable attachment and biofilm formation; flagella for motility; pili or fimbriae for attachment; and sex pili for conjugation. Inside, prokaryotes have a nucleoid (a circular chromosome not enclosed by a membrane), plasmids (small, often circular DNA molecules carrying genes such as antibiotic resistance or degradation of pollutants, transferable between cells), 70S ribosomes, and storage granules such as polyphosphate (volutin), sulphur and poly-β-hydroxybutyrate. Endospores, formed by Bacillus and Clostridium species, are dormant, highly resistant structures that survive heat, drying, radiation and disinfectants for years; they explain why sterilisation needs high temperatures under pressure and why Clostridium perfringens spores serve as indicators of past faecal contamination. Eukaryotic cells have a membrane-bound nucleus, 80S ribosomes, mitochondria, endoplasmic reticulum, Golgi apparatus and, in algae, chloroplasts; fungal walls contain chitin and algal walls cellulose or silica (diatoms). Biofilms, communities embedded in extracellular polymeric substances on surfaces, are the dominant microbial lifestyle in nature and in treatment systems such as trickling filters, and they are more resistant to disinfectants than free cells.
Metabolism is the sum of catabolism, which breaks down substrates to release energy, and anabolism, which uses energy to build cell components. Energy is captured mainly as ATP and as reduced electron carriers, NADH and FADH2. ATP is generated by substrate-level phosphorylation, in which a phosphate group is transferred directly from a high-energy intermediate to ADP, and by oxidative phosphorylation, in which electron transport creates a proton gradient across a membrane that drives ATP synthase (Peter Mitchell's chemiosmotic theory); phototrophs use light to drive the same machinery (photophosphorylation). Glycolysis (the Embden–Meyerhof–Parnas pathway) converts one glucose molecule to two molecules of pyruvate in the cytoplasm, with a net yield of 2 ATP (by substrate-level phosphorylation) and 2 NADH. Other pathways, such as the Entner–Doudoroff pathway used by Pseudomonas and the pentose phosphate pathway, also oxidise sugars. Under aerobic conditions, pyruvate is converted to acetyl-CoA (releasing CO2 and producing NADH), which enters the tricarboxylic acid (TCA, Krebs or citric acid) cycle. Each turn of the cycle oxidises one acetyl group to two CO2, producing 3 NADH, 1 FADH2 and 1 ATP (or GTP). For one glucose (two turns), the TCA cycle and pyruvate oxidation together yield 2 ATP, 8 NADH and 2 FADH2.
NADH and FADH2 donate electrons to the electron transport chain in the cytoplasmic membrane of prokaryotes or the inner mitochondrial membrane of eukaryotes. Electrons pass through carriers such as flavoproteins, quinones and cytochromes to a terminal electron acceptor, oxygen in aerobic respiration, which is reduced to water. The energy released pumps protons, and the proton motive force drives ATP synthesis. Modern estimates give about 2.5 ATP per NADH and 1.5 per FADH2, so complete aerobic oxidation of glucose yields about 30–32 ATP per glucose; older textbooks give 36–38 ATP, using 3 and 2 ATP per NADH and FADH2. In bacteria the yield varies with the composition of the electron transport chain. The energy available from a redox reaction depends on the difference in redox potential between electron donor and acceptor. Oxygen is the most favourable acceptor, so aerobic respiration yields the most energy; nitrate, manganese(IV), iron(III), sulphate and CO2 yield progressively less, the same sequence as the redox ladder in the chemistry subject. Microbes with less energy per unit of substrate produce less biomass; this is why anaerobic treatment processes produce far less sludge than aerobic ones. Chemolithotrophs obtain energy by oxidising inorganic compounds: ammonia (Nitrosomonas), nitrite (Nitrobacter), hydrogen sulphide and sulphur (Thiobacillus, Beggiatoa), ferrous iron (Acidithiobacillus ferrooxidans, important in acid mine drainage and bioleaching) and hydrogen. They usually fix CO2 by the Calvin cycle, which is energetically expensive, so their growth yields are low and their growth rates slow, which is why nitrifying bacteria need long solids retention times in activated sludge.
Fermentation is energy metabolism in which an organic compound serves as both electron donor and electron acceptor, without an external terminal acceptor or electron transport chain. ATP comes only from substrate-level phosphorylation, typically 2 ATP per glucose, so fermentation yields much less energy than respiration. NADH produced in glycolysis is reoxidised by reducing pyruvate or its derivatives to fermentation products. Lactic acid fermentation (by Lactobacillus, Streptococcus and other lactic acid bacteria) produces lactate and is used in yoghurt, silage and fermented foods. Alcoholic fermentation by yeasts such as Saccharomyces cerevisiae produces ethanol and CO2, used in brewing, baking and bioethanol production. Mixed-acid fermentation by enteric bacteria such as E. coli produces acetate, lactate, succinate, ethanol, formate, CO2 and H2, and butyric fermentation by Clostridium produces butyrate. Fermentation is important in silage preservation, in the rumen, in food processing and as the first stages of anaerobic digestion.
Anaerobic respiration uses an electron transport chain with an inorganic terminal acceptor other than oxygen. Denitrifying bacteria such as Pseudomonas and Paracoccus reduce nitrate to N2 and N2O; manganese- and iron-reducing bacteria such as Geobacter and Shewanella reduce Mn(IV) and Fe(III); sulphate-reducing bacteria such as Desulfovibrio reduce sulphate to hydrogen sulphide, causing odour, toxicity and corrosion of concrete sewers (via sulphuric acid formed when H2S is oxidised); and methanogenic archaea reduce CO2 with hydrogen to methane or split acetate into methane and CO2. Anaerobic digestion converts organic matter to biogas (methane and CO2) in four stages: hydrolysis of complex polymers (cellulose, proteins, lipids) to monomers by extracellular enzymes, often the rate-limiting step for solid wastes; acidogenesis, fermenting monomers to volatile fatty acids, alcohols, H2 and CO2; acetogenesis, converting these to acetate, H2 and CO2 (which requires low hydrogen partial pressure, maintained by hydrogen-consuming methanogens in syntrophy); and methanogenesis, in which acetoclastic methanogens (such as Methanosaeta, now Methanothrix, and Methanosarcina) produce roughly two-thirds to 70 per cent of the methane from acetate and hydrogenotrophic methanogens the rest from H2 and CO2. Methanogens are slow-growing and sensitive to pH (optimum about 6.8–7.4), temperature, ammonia, sulphide and oxygen; an accumulation of volatile fatty acids that lowers pH indicates process imbalance. The theoretical methane yield is 0.35 m³ of CH4 at standard temperature and pressure (0 °C, 1 atm) per kg of COD removed, because the COD of 1 mole of methane (16 g, 22.4 L at STP) is 64 g of oxygen; 22.4/64 = 0.35 L/g COD. Biogas typically contains 55–70 per cent methane. Anaerobic digestion is used for sewage sludge, animal manure (farm biogas plants), food waste and high-strength industrial wastewaters (UASB reactors), and it is covered further in the bioenergy and wastewater subjects.
Enzymes are biological catalysts, mostly proteins, that speed up reactions by lowering their activation energy without altering the equilibrium. They are highly specific for substrates, binding them at an active site, as described by the lock-and-key model (Emil Fischer) and the more flexible induced-fit model (Daniel Koshland). Many require cofactors: metal ions such as Mg²⁺, Fe²⁺, Mo (in nitrogenase) or Ni (in urease and methanogen enzymes), or organic coenzymes derived from vitamins, such as NAD⁺ (niacin) and FAD (riboflavin). Enzymes are classified by the reaction they catalyse into six main classes: oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases (a seventh, translocases, was added in 2018). Extracellular hydrolases such as cellulases, amylases, proteases and lipases are central to decomposition and to the hydrolysis stage of anaerobic digestion; urease hydrolyses urea fertiliser to ammonia; nitrogenase reduces N2 to ammonia and is irreversibly inactivated by oxygen, which is why nitrogen-fixing organisms protect it, for example with leghaemoglobin in legume nodules or heterocysts in cyanobacteria. Soil enzyme activities, such as dehydrogenase, phosphatase and β-glucosidase, are used as indicators of soil biological health. Enzyme activity depends on temperature, rising to an optimum and then falling as the enzyme denatures, and on pH, with an optimum characteristic of each enzyme. Activity is reduced by inhibitors, including heavy metals that bind sulphydryl groups, which is one reason metals are toxic to microbes in treatment plants.
The Michaelis–Menten equation describes the rate of an enzyme-catalysed reaction as a function of substrate concentration: v = Vmax[S] ÷ (Km + [S]), where Vmax is the maximum rate when the enzyme is saturated and Km, the Michaelis constant, is the substrate concentration at which v = Vmax/2. A low Km indicates high affinity for the substrate. At substrate concentrations much below Km the rate is approximately first-order in [S]; at concentrations much above Km it is approximately zero-order. For an enzyme with Vmax = 100 µmol/min and Km = 5 mM, the rate at [S] = 5 mM is 50 µmol/min and at [S] = 15 mM is 75 µmol/min. The Lineweaver–Burk (double-reciprocal) plot, 1/v = (Km/Vmax)(1/[S]) + 1/Vmax, linearises the equation: the y-intercept is 1/Vmax and the x-intercept is −1/Km. It is convenient for illustration but distorts error; non-linear fitting is preferred for analysis. Inhibitors affect the parameters differently. A competitive inhibitor resembles the substrate and competes for the active site: it raises the apparent Km but does not change Vmax, and can be overcome by high substrate concentration. A non-competitive inhibitor binds elsewhere and reduces Vmax without changing Km. An uncompetitive inhibitor binds only the enzyme–substrate complex and lowers both Vmax and Km. In wastewater and bioremediation, substrate inhibition at high concentrations, described by the Haldane (Andrews) equation, occurs with toxic substrates such as phenol, which inhibit the organisms degrading them. The same mathematical form as Michaelis–Menten underlies the Monod model of microbial growth in the next chapter.
Bacteria reproduce mainly by binary fission. In a closed (batch) culture, growth follows four phases: the lag phase, when cells adapt to the new medium and synthesise enzymes; the exponential (log) phase, when cells divide at a constant maximum rate for the conditions; the stationary phase, when growth stops because a nutrient is exhausted or toxic products accumulate, and cell division balances death; and the death (decline) phase, when cells die, often exponentially. During exponential growth, the rate of increase of biomass X is proportional to biomass: dX/dt = μX, where μ is the specific growth rate (per unit time). Integrated, X = X0e^(μt). The doubling (generation) time td = ln 2 ÷ μ = 0.693/μ. In terms of numbers, N = N0 × 2ⁿ, where n is the number of generations (elapsed time ÷ td). For example, a culture of 1000 cells with a doubling time of 30 minutes undergoes 6 generations in 3 hours, giving 1000 × 2⁶ = 64,000 cells, and its specific growth rate is 0.693/0.5 h ≈ 1.39 per hour. Doubling times range from about 20 minutes for E. coli under ideal conditions to many hours or days for nitrifiers and methanogens. Growth is measured by direct microscopic counts, viable plate counts (colony-forming units, CFU), most probable number methods, turbidity (optical density), dry weight, volatile suspended solids in wastewater treatment, and molecular methods such as quantitative PCR.
Jacques Monod (1949) showed that the specific growth rate depends on the concentration of the growth-limiting substrate S in a form identical to the Michaelis–Menten equation: μ = μmax S ÷ (Ks + S), where μmax is the maximum specific growth rate and Ks the half-saturation constant, the substrate concentration at which μ = μmax/2. For μmax = 0.5 per hour and Ks = 20 mg/L, μ at S = 20 mg/L is 0.25 per hour; at S = 180 mg/L it is 0.45 per hour. The yield coefficient Y relates biomass produced to substrate consumed: Y = ΔX/ΔS (for example g of cells per g of BOD or COD removed). Typical values for aerobic heterotrophs on organic matter are about 0.4–0.6 g VSS per g BOD, while anaerobic organisms and nitrifiers have much lower yields (about 0.05–0.15). The substrate utilisation rate is therefore rsu = μX/Y, giving the maximum specific substrate utilisation rate k = μmax/Y. Cells also use energy for maintenance and die or are consumed by predators, described by an endogenous decay coefficient kd: the net specific growth rate is μ − kd, and the observed yield Yobs = Y/(1 + kd θc), where θc is the solids retention time (mean cell residence time). These relationships form the basis of the design equations for activated sludge and other biological treatment processes, developed in the wastewater treatment subject. The Monod model assumes a single limiting substrate; in reality multiple nutrients, inhibitors and mixed communities complicate kinetics, and parameters vary widely between studies.
In batch culture, all nutrients are supplied at the start and nothing is added or removed, so conditions change continuously as substrate is consumed and products accumulate; growth passes through the phases described above. Fed-batch culture adds substrate over time without removing culture, extending growth and avoiding substrate inhibition; sequencing batch reactors (SBRs) in wastewater treatment fill, react, settle and decant in a single tank on a timed cycle. Continuous culture supplies fresh medium and removes culture at the same rate, allowing steady state. In a chemostat, a completely mixed reactor of volume V receives medium at flow rate F, so the dilution rate D = F/V (per unit time) is the reciprocal of the hydraulic retention time. At steady state, ignoring decay, the specific growth rate equals the dilution rate: μ = D. Combining with the Monod equation, the steady-state substrate concentration is S = Ks D ÷ (μmax − D), which depends only on kinetic constants and D, not on the feed concentration; the biomass concentration is X = Y(S0 − S), where S0 is the feed substrate concentration. For example, with V = 50 L, F = 10 L/h, μmax = 0.5 per hour, Ks = 20 mg/L, Y = 0.5 and S0 = 500 mg/L: D = 0.2 per hour; S = 20 × 0.2 ÷ (0.5 − 0.2) ≈ 13.3 mg/L; and X = 0.5 × (500 − 13.3) ≈ 243 mg/L. If D is increased beyond the maximum growth rate the cells can attain at the feed concentration, Dc = μmax S0 ÷ (Ks + S0) (here 0.5 × 500/520 ≈ 0.48 per hour), cells are removed faster than they grow and the culture washes out.
A simple chemostat has equal hydraulic retention time (HRT) and solids retention time (SRT), so slow-growing organisms wash out at short HRTs. Biological treatment processes decouple the two by retaining biomass: activated sludge settles biomass in a secondary clarifier and recycles it to the aeration tank; biofilm processes such as trickling filters, rotating biological contactors and moving-bed biofilm reactors attach biomass to surfaces; membrane bioreactors retain it behind membranes; and upflow anaerobic sludge blanket (UASB) reactors retain it as dense granules. Long SRTs allow slow growers such as nitrifiers and methanogens to persist and improve treatment stability, while short HRTs keep reactors small. Key operating parameters in activated sludge are the SRT (mean cell residence time, typically about 5–15 days for conventional plants and longer for nitrification), the food-to-microorganism ratio (F/M, kg BOD applied per kg MLVSS per day, roughly 0.2–0.5 for conventional activated sludge) and the mixed liquor suspended solids concentration. Reactors are also characterised by their hydraulics: completely mixed reactors dilute shock loads but give lower efficiency for a given volume than plug-flow reactors, whose concentration falls along the length. The wastewater treatment subject derives design equations; here the point is the microbial basis: the SRT must exceed the minimum needed for the slowest important organisms, with a safety factor, or they will be lost.
Sterilisation destroys or removes all microorganisms, including endospores; disinfection kills or inactivates pathogens on objects or in water, but not necessarily all spores; antisepsis applies disinfecting agents to living tissue; and sanitisation reduces microbial numbers to safe levels. The effectiveness of a treatment is expressed as log reduction: a reduction from 10⁶ to 10² organisms is a 4-log (99.99 per cent) reduction. Heat is the most widely used physical method. Moist heat is more effective than dry heat because it coagulates proteins. The autoclave sterilises with steam at 121 °C and about 15 psi (103 kPa) above atmospheric pressure, typically for 15 minutes or longer depending on load. The decimal reduction time (D-value) is the time at a given temperature to reduce a population by 90 per cent (one log); a 6-log reduction therefore takes 6 D-values. The z-value is the temperature change needed to change the D-value tenfold. Pasteurisation, which kills pathogens and reduces spoilage organisms in milk without sterilising it, uses 63 °C for 30 minutes (batch) or 72 °C for 15 seconds (high-temperature short-time). Dry heat in hot-air ovens (about 160–170 °C for two hours) sterilises glassware, and incineration destroys contaminated material. Filtration through membranes with pores of about 0.2–0.45 µm removes bacteria from heat-sensitive liquids and air (HEPA filters). Radiation is also used: ultraviolet light at about 254 nm (near the DNA absorption peak of about 260 nm) causes thymine dimers that block replication, and UV is widely used for water disinfection because it is effective against chlorine-resistant protozoa such as Cryptosporidium and Giardia and forms no chlorinated by-products, though it leaves no residual. Ionising radiation (gamma rays) sterilises medical supplies and some foods. Low temperatures (refrigeration and freezing) and drying slow or stop growth but do not reliably kill.
Chemical agents include halogens (chlorine, chloramines, iodine), ozone, chlorine dioxide, alcohols, phenolics, quaternary ammonium compounds, heavy metals, aldehydes and peroxides. In water treatment, chlorine forms hypochlorous acid (HOCl), which dissociates to hypochlorite (OCl⁻) with a pKa of about 7.5; HOCl is the far more effective disinfectant, so chlorination works better at lower pH. Chlorine reacts with ammonia to form chloramines (combined chlorine), weaker disinfectants that persist longer in distribution systems. Breakpoint chlorination adds enough chlorine to oxidise ammonia and leave a free chlorine residual. Chlorine reacts with natural organic matter to form disinfection by-products such as trihalomethanes, which are regulated. Ozone is a very strong oxidant and disinfectant but leaves no residual. Disinfection kinetics are described by Chick's law (1908), a first-order decay of organisms with time, and the Chick–Watson model, ln(N/N0) = −k Cⁿ t, where C is disinfectant concentration and n a coefficient often near 1. This underlies the Ct concept: the product of residual disinfectant concentration (mg/L) and contact time (minutes) needed for a given log inactivation of a given organism at given pH and temperature. For example, 0.5 mg/L of free chlorine for 30 minutes gives a Ct of 15 mg·min/L. Required Ct values are much higher for Giardia cysts than for bacteria and far higher still for Cryptosporidium oocysts, which are practically resistant to chlorine at normal doses. Resistance to control agents follows an approximate order from most resistant to least: prions, bacterial endospores, mycobacteria, protozoan cysts and oocysts, non-enveloped viruses, fungi, vegetative bacteria and enveloped viruses. Antibiotics are chemicals that inhibit or kill bacteria by targeting cell walls, protein synthesis, nucleic acids or membranes; their widespread use in medicine and livestock has selected for antibiotic-resistant bacteria and resistance genes, which spread in wastewater, manure and soils and are an emerging environmental concern.
The rhizosphere, the narrow zone of soil influenced by plant roots, a term coined by Lorenz Hiltner in 1904, is enriched with microbes feeding on root exudates (sugars, amino acids, organic acids) and sloughed cells. Microbial numbers are often many times higher there than in bulk soil, a ratio called the rhizosphere effect (R:S ratio). Plants shape their rhizosphere microbiome, and the microbiome in turn affects plant nutrition, health and stress tolerance. Plant growth-promoting rhizobacteria (PGPR) benefit plants by fixing nitrogen, solubilising phosphate, producing phytohormones such as indole-3-acetic acid, producing siderophores that sequester iron (depriving pathogens of it), producing the enzyme ACC deaminase that lowers stress ethylene, and inducing systemic resistance against pathogens. Biofertilisers are preparations of such living microorganisms: Rhizobium for legumes; free-living Azotobacter and associative Azospirillum for cereals; Gluconacetobacter (Acetobacter) diazotrophicus for sugarcane; cyanobacteria (blue-green algae) and Azolla–Anabaena for rice; phosphate-solubilising bacteria such as Bacillus and Pseudomonas species; potassium- and zinc-solubilising bacteria; and arbuscular mycorrhizal fungi (such as Glomus and Rhizophagus species) that improve phosphorus and water uptake. India promotes biofertilisers under its fertiliser control rules, which set quality standards such as minimum viable cell counts. Field responses vary with soil, crop, inoculant quality and existing native populations. Symbiotic nitrogen fixation in legumes involves signalling: plant flavonoids induce rhizobial nod genes, whose Nod factors trigger root hair curling, infection threads and nodule formation; inside nodules, bacteroids fix nitrogen, protected from oxygen by leghaemoglobin, which gives effective nodules their pink colour.
Soil and plant-associated microbes also include plant pathogens: fungi such as Fusarium, Rhizoctonia, Pythium (an oomycete) and rusts; bacteria such as Xanthomonas, Pseudomonas syringae and Ralstonia; and viruses often transmitted by insects. Suppressive soils, in which disease fails to develop despite the presence of a pathogen, owe their suppression to microbial communities. Biological control uses antagonistic microorganisms: Trichoderma species (fungi that parasitise other fungi, compete and produce antibiotics) against soil-borne pathogens; Pseudomonas fluorescens, which produces antibiotics and siderophores; Bacillus subtilis; and Bacillus thuringiensis (Bt), whose crystal (Cry) proteins are toxic to specific insect larvae and are used as biopesticide sprays and, through their genes, in insect-resistant crops. Entomopathogenic fungi such as Beauveria bassiana and Metarhizium and viruses such as nucleopolyhedroviruses also control insect pests. The pesticides subject compares biological and chemical control. Soil microbes drive decomposition and humus formation, aggregate stability (through fungal hyphae and microbial polysaccharides), nutrient mineralisation and immobilisation, and pesticide degradation. When residues with a high carbon-to-nitrogen ratio (above about 25–30:1) are added, microbes immobilise mineral nitrogen to build biomass, temporarily reducing nitrogen available to crops; low C:N residues release nitrogen (net mineralisation). Soil microbial biomass carbon, basal respiration and enzyme activities are used as indicators of soil health, as the soil science subject explains.
Microorganisms carry out most transformations in biogeochemical cycles, which the ecology subject describes at ecosystem scale. In the carbon cycle, photosynthetic microbes (cyanobacteria and algae) fix roughly half of global CO2 through ocean phytoplankton; heterotrophic bacteria and fungi decompose organic matter, returning CO2 by respiration; fungi such as white-rot basidiomycetes are the main degraders of lignin, using extracellular peroxidases and laccases; and in anoxic environments, fermenters and methanogens convert organic carbon to methane, while methanotrophic bacteria (such as Methylococcus and Methylosinus) oxidise methane to CO2, consuming much of it before it escapes from soils and sediments. Anaerobic oxidation of methane by archaea in partnership with sulphate reducers removes most methane in marine sediments. In the nitrogen cycle, nitrogen fixation by bacteria and archaea with nitrogenase converts N2 to ammonia; ammonification by many decomposers releases ammonium from organic nitrogen; nitrification oxidises ammonium to nitrite (ammonia-oxidising bacteria such as Nitrosomonas and ammonia-oxidising archaea, which are often more abundant in soils) and nitrite to nitrate (Nitrobacter and Nitrospira, some of which perform complete ammonia oxidation, 'comammox'); denitrification reduces nitrate to N2O and N2 under low oxygen; dissimilatory nitrate reduction to ammonium (DNRA) conserves nitrogen; and anammox bacteria convert ammonium and nitrite directly to N2. Nitrification inhibitors such as nitrapyrin and dicyandiamide slow the first step to reduce nitrate leaching and nitrous oxide emissions, as the nutrient stewardship subject explains.
In the sulphur cycle, sulphate-reducing bacteria (Desulfovibrio, Desulfobacter) produce hydrogen sulphide in anoxic sediments and waterlogged soils; sulphur-oxidising bacteria such as Thiobacillus and Beggiatoa oxidise sulphide and elemental sulphur to sulphate, a process used to lower the pH of alkaline soils with elemental sulphur and responsible for acid formation in acid sulphate soils when sulphidic coastal soils are drained. Green and purple sulphur bacteria use sulphide as an electron donor in anoxygenic photosynthesis. Iron-oxidising bacteria, such as Acidithiobacillus ferrooxidans in acidic conditions and Gallionella and Leptothrix at neutral pH, oxidise Fe²⁺ to Fe³⁺, producing orange iron deposits in wells and drains and, with sulphur oxidisers, generating acid mine drainage; the same organisms are used in bioleaching to extract copper and gold from low-grade ores. Iron-reducing bacteria such as Geobacter reduce Fe(III) in anoxic sediments and aquifers, releasing iron and associated arsenic, as the chemistry subject explains, and can transfer electrons to electrodes in microbial fuel cells. Microbes solubilise phosphate from minerals by producing organic acids and phosphatases, and some accumulate polyphosphate, the basis of enhanced biological phosphorus removal. Microbes also transform metals and metalloids: they methylate mercury (sulphate- and iron-reducing bacteria in anoxic sediments) and arsenic, reduce soluble chromium(VI) to less toxic chromium(III) and uranium(VI) to insoluble uranium(IV), and precipitate metals as sulphides, processes used in bioremediation. Microbial processes thus control both nutrient availability and the mobility and toxicity of contaminants.
Secondary treatment of wastewater uses microorganisms to oxidise dissolved and colloidal organic matter. In the activated sludge process, developed by Edward Ardern and William Lockett in Manchester (1914), wastewater is aerated with a suspended mixed culture that forms flocs held together by extracellular polymers; the flocs settle in a clarifier and part of the sludge is recycled. Floc-forming bacteria such as Zoogloea, protozoa that graze on free bacteria and clarify the effluent, and rotifers are characteristic; a healthy community includes stalked ciliates. Sludge bulking, poor settling caused by excessive filamentous organisms such as Sphaerotilus natans, Microthrix parvicella and type 021N, is a common operating problem, monitored with the sludge volume index; foaming is often caused by Nocardia-like actinomycetes. Biological nutrient removal adds anoxic zones in which denitrifiers reduce nitrate (produced by nitrification in aerobic zones) to N2, using organic carbon, and anaerobic–aerobic sequences that favour polyphosphate-accumulating organisms (PAOs, such as Candidatus Accumulibacter), which take up volatile fatty acids anaerobically and then take up excess phosphate aerobically, allowing phosphorus to be removed with the waste sludge (enhanced biological phosphorus removal). Biofilm processes include trickling filters, rotating biological contactors and moving-bed biofilm reactors. Waste stabilisation ponds rely on a symbiosis of algae, which produce oxygen by photosynthesis, and bacteria, which oxidise organic matter and release CO2 and nutrients, and they remove pathogens through long retention, sunlight and predation. Anaerobic processes, such as UASB reactors developed by Gatze Lettinga and colleagues in the Netherlands and widely used in India, treat high-strength wastes and produce biogas. Constructed wetlands use plants, biofilms and sediments. The wastewater treatment subject covers design.
Bioremediation uses microorganisms or plants to degrade or immobilise pollutants. Strategies include natural attenuation (relying on intrinsic degradation with monitoring), biostimulation (adding nutrients, oxygen or electron donors to stimulate native degraders), bioaugmentation (adding specialised microbial cultures), and engineered approaches: landfarming (spreading contaminated soil and tilling it), biopiles and composting of contaminated soil, bioventing (supplying air to unsaturated soil), biosparging (injecting air below the water table) and bioreactors. Petroleum hydrocarbons are readily biodegraded aerobically by bacteria such as Pseudomonas, Rhodococcus and Alcanivorax; chlorinated solvents such as trichloroethylene are degraded by reductive dechlorination under anaerobic conditions, notably by Dehalococcoides. Ananda Chakrabarty's genetically engineered Pseudomonas, carrying plasmids for degrading several hydrocarbons, was the subject of the 1980 US Supreme Court decision Diamond v. Chakrabarty allowing patents on living organisms. Phytoremediation uses plants to extract (phytoextraction, using hyperaccumulators), stabilise (phytostabilisation), degrade (phytodegradation, rhizodegradation with root microbes) or volatilise contaminants. Effectiveness depends on contaminant bioavailability, environmental conditions and time; the soil contamination subject covers site remediation. Composting is the controlled aerobic decomposition of organic wastes to a stable, humus-like product. It proceeds through a mesophilic phase, a thermophilic phase in which temperatures rise to about 55–65 °C as heat from microbial respiration accumulates (killing pathogens and weed seeds), and a cooling and maturation phase dominated by fungi and actinomycetes. Good composting requires a C:N ratio of about 25–30:1, moisture of about 50–60 per cent, adequate aeration (by turning or forced air) and suitable particle size. Pathogen reduction standards, such as maintaining 55 °C or above for several days with turning in windrows, are set in national guidelines. Vermicomposting uses earthworms such as Eisenia fetida at lower temperatures and produces a fine, nutrient-rich product, though it lacks the thermophilic phase's pathogen kill.
Waterborne and water-related diseases are caused by bacteria (Vibrio cholerae, causing cholera; Salmonella Typhi, typhoid; Shigella, dysentery; pathogenic E. coli such as O157:H7; Campylobacter; Leptospira; Legionella, spread by aerosols from cooling towers and plumbing), viruses (hepatitis A and E, rotavirus, norovirus, enteroviruses including poliovirus, adenoviruses), protozoa (Giardia lamblia, Cryptosporidium parvum, Entamoeba histolytica) and helminths (Ascaris, hookworm, Schistosoma, whose larvae penetrate skin in infested water). Most are transmitted by the faecal–oral route, summarised in the 'F-diagram' of faeces reaching new hosts through fluids, fields, flies, fingers and food. John Snow's investigation of the Broad Street pump during the 1854 London cholera outbreak is a founding study of waterborne disease epidemiology. Diarrhoeal disease from unsafe water, sanitation and hygiene remains a major cause of child death in low-income countries. Because pathogens are diverse, intermittent and hard to detect, water quality is monitored with indicator organisms. An ideal indicator is present whenever faecal pathogens are present and in greater numbers, survives at least as long as pathogens, does not multiply in the environment, is non-pathogenic and is easy and cheap to measure. Total coliforms (Gram-negative, non-spore-forming rods that ferment lactose with gas or acid at 35–37 °C within 48 hours) indicate treatment efficiency and distribution system integrity but include environmental species. Faecal (thermotolerant) coliforms grow at 44.5 °C and are more specific to faecal contamination, and Escherichia coli, identified by the enzyme β-glucuronidase (detected with substrates such as MUG), is the preferred indicator of recent faecal contamination. WHO guidelines and India's IS 10500 require that E. coli (or thermotolerant coliforms) not be detectable in any 100 mL sample of drinking water. Intestinal enterococci are used for recreational and marine waters; bacteriophages (coliphages) as indicators of viruses; and Clostridium perfringens spores as indicators of remote or intermittent contamination and of protozoan removal. Indicators have limits: chlorine-resistant protozoa and viruses can be present when coliforms are absent.
The multiple-tube fermentation method estimates the most probable number (MPN) of coliforms statistically. In the standard procedure, sample volumes (for example five tubes each of 10 mL, 1 mL and 0.1 mL) are inoculated into lactose or lauryl tryptose broth and incubated at 35 °C for 24–48 hours (presumptive test: gas production). Positive tubes are transferred to brilliant green lactose bile broth (confirmed test), and the completed test streaks confirmed cultures onto eosin methylene blue (EMB) agar and checks Gram stain and gas production. The pattern of positive tubes is converted to an MPN per 100 mL using statistical tables; for five tubes each of 10, 1 and 0.1 mL with a 5-2-0 result, the table gives about 49 per 100 mL. Thomas's approximate formula, MPN/100 mL = 100 × P ÷ √(N × T), where P is the number of positive tubes, N the millilitres of sample in negative tubes and T the millilitres in all tubes, gives a similar estimate: with P = 7, N = 3.5 and T = 55.5, MPN ≈ 50 per 100 mL. MPN methods suit turbid samples and those with low counts but are slow and imprecise, with wide confidence intervals. The membrane filtration method passes a measured volume of water through a sterile membrane filter with 0.45 µm pores, which retains bacteria; the filter is placed on a selective medium and incubated, and colonies are counted directly. Total coliforms form red colonies with a green metallic sheen on m-Endo medium at 35 °C, and faecal coliforms blue colonies on m-FC medium at 44.5 °C. The count per 100 mL = colonies counted ÷ volume filtered × 100: 35 colonies from 50 mL is 70 CFU per 100 mL. Volumes are chosen so that the filter has a countable number of colonies (typically 20–80 for coliforms). Membrane filtration is faster and more precise than MPN but unsuitable for very turbid samples. Enzyme–substrate methods (such as defined-substrate tests detecting β-galactosidase for total coliforms and β-glucuronidase for E. coli) give results in 18–24 hours and are widely used. For wastewater reuse in agriculture, WHO's 2006 guidelines use a multiple-barrier approach and health-based targets, including limits on helminth eggs (no more than one egg per litre for unrestricted irrigation in many applications) and on E. coli depending on the crop and exposure. Pathogen removal in treatment, die-off in fields, withholding periods before harvest, and protective equipment together protect farm workers and consumers, as the wastewater subject explains.
A student uses this subject to master both concepts and the numerical core of GATE Section 3: doubling time and specific growth rate, generations and cell numbers, Monod and Michaelis–Menten rates, chemostat substrate, biomass and washout dilution rate, log reductions, D-values and Ct, theoretical methane yield from COD, biomass nitrogen content and oxygen demand from C5H7O2N, and MPN and membrane filtration counts. Common confusions include fermentation versus anaerobic respiration, competitive versus non-competitive inhibition, and total versus faecal coliforms.
A five-year practitioner, such as a wastewater treatment plant microbiologist, a water quality laboratory analyst, a biofertiliser production manager or a bioremediation consultant, uses the subject to run coliform and E. coli tests to accredited methods, diagnose sludge bulking from microscopy, control SRT to keep nitrifiers, troubleshoot anaerobic digesters from volatile fatty acids and alkalinity, check biofertiliser quality, and design biostimulation for hydrocarbon-contaminated soil.
A senior professional with 25 years' experience, such as a head of a state water testing network, a utility's process director or a research station director, uses microbiology to set monitoring and disinfection policies (including for chlorine-resistant protozoa), choose treatment technologies, manage wastewater reuse in agriculture safely, scale biofertiliser and biocontrol programmes, and address emerging concerns such as antimicrobial resistance in wastewater and manure.
A researcher with a PhD and 25 years' experience works on microbial community ecology with metagenomics, nitrogen and methane cycling organisms and greenhouse gas emissions, kinetics and process modelling of biological treatment, bioremediation of persistent contaminants, rhizosphere microbiomes for sustainable agriculture, and the environmental dimension of antimicrobial resistance. Such a researcher develops methods and trains laboratory and modelling teams.
Kinetic and yield coefficients in this manual are illustrative; real values vary widely with organism, substrate, temperature and system, and should be taken from design references or measured. It does not cover microbial genetics and molecular methods in depth, immunology and epidemiology of individual diseases, food microbiology, industrial fermentation engineering, or detailed design of treatment reactors, which belong to other subjects or tracks. Biofertiliser quality standards and coliform limits are national rules that change; current Fertiliser (Control) Order specifications and drinking water standards should be checked. Pathogen removal credits for treatment processes and composting time–temperature requirements differ between national guidelines and are summarised rather than specified.
Original practice questions written to the syllabus and pattern of the exams named in each question's examTags. They are not past papers and do not reproduce any board's questions.
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