Environmental Microbiology: Microbial Groups, Cell Biology, Metabolism, Growth Kinetics and Water-Quality Microbiology

Prokaryotes — bacteria and archaea — have no nucleus or membrane-bound organelles; their DNA lies in a nucleoid, often with extra plasmids, and their ribosomes are 70S. Eukaryotes — fungi, algae and protozoa, and the helminths (worms) that are multicellular animals — have a nucleus, mitochondria (and chloroplasts in algae) and 80S ribosomes. Viruses are not cells: they are nucleic acid in a protein coat that replicates only inside a host. The modern three-domain classification (Bacteria, Archaea, Eukarya) rests on 16S/18S ribosomal RNA sequences, and the same sequencing reveals the microbial diversity of a sludge floc or a soil — thousands of taxa, most never grown in culture.

• In wastewater treatment: heterotrophic bacteria oxidise BOD and build flocs held together by extracellular polymers; nitrifiers (Nitrosomonas: NH₄⁺ → NO₂⁻; Nitrobacter: NO₂⁻ → NO₃⁻) and denitrifiers (NO₃⁻ → N₂ in the absence of oxygen) remove nitrogen; phosphate-accumulating organisms take up excess phosphorus after an anaerobic stage; methanogenic archaea finish anaerobic digestion; protozoa and rotifers graze free bacteria and signal a healthy sludge; filamentous bacteria in excess cause bulking.
• In bioremediation: microbes degrade or immobilise pollutants in place (in situ) or after excavation (ex situ). Biostimulation adds nutrients or oxygen to the native population; bioaugmentation adds selected organisms; bioventing, land farming, composting and biopiles are its engineered forms, and some bacteria reduce toxic Cr(VI) to Cr(III) or U(VI) to insoluble U(IV).
• In biogeochemical cycles: nitrogen fixation (N₂ → NH₃, by Rhizobium, Azotobacter and cyanobacteria), ammonification, nitrification, denitrification and anammox (NH₄⁺ + NO₂⁻ → N₂, anaerobically); sulphate reduction to H₂S (Desulfovibrio) and sulphide oxidation back to sulphuric acid (Thiobacillus — the cause of sewer crown corrosion); carbon fixation by photosynthesis and its return by respiration and methanogenesis; and phosphorus solubilisation from mineral phosphates.

Four classes of macromolecule build a cell. Proteins are chains of twenty amino acids joined by peptide bonds; their primary structure (sequence) folds into secondary structures (α-helix, β-sheet), a tertiary shape and, for multi-chain proteins, a quaternary arrangement — and the shape is what makes an enzyme work, which is why heat and extreme pH, by unfolding it, kill. Nucleic acids are chains of nucleotides: DNA is a double helix of deoxyribose nucleotides with the bases A, T, G and C paired A–T and G–C; RNA is single-stranded, uses ribose and U in place of T, and comes as messenger, transfer and ribosomal RNA. Lipids include the phospholipids whose bilayer is the cell membrane (archaea use distinctive ether-linked lipids). Polysaccharides store energy (glycogen, starch), build walls (peptidoglycan in bacteria, cellulose in algae and plants, chitin in fungi) and make up most of the extracellular slime.

Antimicrobial resistance arises when bacteria survive a drug that once killed them. The mechanisms are enzymatic inactivation (β-lactamases that open penicillin’s ring), efflux pumps that expel the drug, alteration of the drug’s target, and reduced permeability. Resistance genes spread vertically to daughter cells and horizontally between cells by conjugation (plasmid transfer through a pilus), transformation (uptake of free DNA) and transduction (carried by a bacteriophage). Wastewater treatment plants, where dense mixed populations meet sub-lethal antibiotic residues from hospitals and homes, are recognised hotspots for this exchange. Quorum sensing is cell-to-cell signalling by diffusible autoinducers — acyl-homoserine lactones in Gram-negative bacteria, short peptides in Gram-positive ones: when the population is dense enough for the signal to pass a threshold, genes for biofilm formation, bioluminescence or virulence switch on together. Blocking it (quorum quenching) is studied as a way to limit membrane biofouling.

Metabolism is catabolism, which breaks substrates down and releases energy, coupled to anabolism, which uses that energy to build cell material. The energy currency is ATP, made in three ways: substrate-level phosphorylation (a phosphate transferred directly from a high-energy intermediate, as in glycolysis), oxidative phosphorylation (driven by the proton gradient an electron transport chain creates) and photophosphorylation (the same, driven by light). Glycolysis turns one glucose into two pyruvate with a net gain of 2 ATP and 2 NADH. With oxygen, pyruvate becomes acetyl-CoA and enters the TCA cycle, each turn of which releases 2 CO₂ and yields 3 NADH, 1 FADH₂ and 1 ATP (or GTP). The NADH and FADH₂ pass their electrons down the electron transport chain to a terminal acceptor; the protons pumped out return through ATP synthase. Aerobic respiration of glucose therefore yields far more ATP than glycolysis alone — up to 38 in the classical prokaryotic accounting.

Energy balances link this chemistry to sludge production. Writing the electron donor and acceptor as half-reactions per electron equivalent, the free energy released decides how much can be spent on synthesis: aerobic heterotrophs, with the most energy per electron, convert a large fraction of substrate into new cells (a high yield Y, of the order of 0.4–0.6 g VSS/g BOD), whereas methanogens, with little energy per electron, grow slowly and produce little sludge — the practical reason anaerobic treatment makes a fraction of the sludge of aerobic treatment. Enzymes are protein catalysts that lower activation energy without being consumed; many need cofactors (metal ions) or coenzymes (NAD⁺, FAD). Their rate follows Michaelis–Menten kinetics, v = V_max S/(K_m + S), where K_m is the substrate concentration giving half the maximum rate. The double-reciprocal Lineweaver–Burk plot, 1/v = (K_m/V_max)(1/S) + 1/V_max, turns it into a straight line. A competitive inhibitor raises the apparent K_m and leaves V_max unchanged; a non-competitive inhibitor lowers V_max and leaves K_m unchanged.

Microbes need carbon, an energy source, the macronutrients N and P (a BOD : N : P ratio of about 100 : 5 : 1 is the usual rule for aerobic treatment), sulphur, trace metals and sometimes growth factors such as vitamins. In a closed batch culture a population passes through the lag, exponential (log), stationary and death phases. In the exponential phase dX/dt = μX, where μ is the specific growth rate, so X = X₀e^(μt) and the doubling time is t_d = ln 2/μ. Counting by generations, the number of generations is n = (log N − log N₀)/log 2 and the generation time is g = t/n. Monod’s model relates μ to the limiting substrate: μ = μ_max S/(K_s + S), where K_s, the half-saturation constant, is the substrate concentration at which μ = μ_max/2. At S ≫ K_s growth is zero order in substrate; at S ≪ K_s it is first order.

In continuous culture a chemostat is fed at flow F into volume V, giving a dilution rate D = F/V. At steady state the cells grow exactly as fast as they are washed out, so μ = D, and Monod’s equation then fixes the residual substrate: S = K_s D/(μ_max − D). If D is raised past the maximum growth rate the feed allows, D_crit = μ_max S₀/(K_s + S₀), the culture washes out. This is the same logic by which an activated-sludge plant must hold its solids retention time above the minimum the slowest organisms need — nitrifiers wash out first. Culture media are defined (synthetic) or complex, liquid or solidified with agar, and are made selective (inhibiting unwanted organisms), differential (showing a reaction by colour) or enriched: MacConkey and EMB agars are both selective and differential for lactose-fermenting Gram-negatives, which is why they appear in coliform work.

Environmental factors shift growth: rate roughly doubles for each 10 °C rise within the organism’s range and collapses above its optimum; most bacteria prefer near-neutral pH (fungi tolerate acid); low water activity and high salt dehydrate cells; oxygen is required, tolerated or lethal according to the organism’s class. Control is physical or chemical. Physical: moist heat (autoclaving at 121 °C and about 15 psi gauge for 15 minutes kills spores), dry heat, pasteurisation, filtration through membranes of about 0.22–0.45 μm, and ultraviolet radiation near 254–260 nm, which damages DNA. Chemical: chlorine and its compounds, ozone, alcohols, phenolics, aldehydes and quaternary ammonium compounds. Killing is approximately first order, N = N₀e^(−kt), so each equal interval removes the same fraction; the Chick–Watson law adds the disinfectant concentration, ln(N/N₀) = −k′Cⁿt, which is the origin of the "C × t" design of disinfection.

Modes of transmission are classified by how water is involved: waterborne (the pathogen is drunk — cholera, typhoid, hepatitis), water-washed (spread by too little water for hygiene — trachoma, scabies, and faecal–oral diseases again), water-based (the pathogen spends part of its life cycle in an aquatic host — schistosomiasis in snails, guinea worm), and water-related insect vector (mosquitoes breeding in water — malaria, dengue, filariasis). The faecal–oral route through contaminated water, food and hands is the dominant one for enteric pathogens.

Pathogens are too many, too sparse and too hard to culture to test for routinely, so water is tested for indicator organisms instead. A good indicator is present whenever faecal contamination (and so possibly pathogens) is present and absent otherwise; is more numerous than the pathogens; survives at least as long in water and resists treatment at least as well; is not itself harmful; and is quick and cheap to count. Total coliforms (Gram-negative, non-spore-forming rods that ferment lactose with gas at 35 °C) include soil organisms; faecal (thermotolerant) coliforms, which do so at 44.5 °C, and above all E. coli are specific to faecal origin. Faecal streptococci (enterococci), spores of Clostridium perfringens (persistent, marking old or intermittent pollution) and coliphages (surrogates for viruses) complete the set. For drinking water the accepted criterion is that E. coli or thermotolerant coliforms are not detectable in any 100 mL sample.