Bacterial growth and metabolism

In short

Bacterial growth means an increase in the number of cells, not in the size of one cell: a bacterium grows by binary fission, so a population doubles at a fixed interval called the generation time. Metabolism is the set of reactions that supply that growth with energy (ATP), reducing power (NADH, NADPH) and carbon skeletons — catabolism breaking substrates down, anabolism building cell material up. The topic is examined in B.Sc General Microbiology, M.Sc entrance tests (CUET-PG, JAM, GAT-B, ICMR, CSIR-NET life sciences) and in every fermentation or quality-control interview, because yield, batch time and sterility all follow from growth kinetics. It is used daily in Indian industry — Serum Institute, Biocon, Dr Reddy's, Hindustan Unilever, Amul and Mother Dairy, Nestlé India, water-testing labs under BIS and FSSAI — wherever cells are counted, grown in a fermenter or killed to a specification.

Explained

How bacteria grow, and how growth is measured

• Binary fission: DNA replication from *oriC*, chromosome segregation, septum formation by the FtsZ ring, then division into two nearly identical daughter cells. Growth is therefore exponential (geometric): 1 → 2 → 4 → 8.
• Generation (doubling) time g: time for the population to double. *E. coli* about 20 min in rich broth, *Mycobacterium tuberculosis* 15-20 h, *M. leprae* roughly 12-14 days — which is why a TB culture on Lowenstein-Jensen medium takes 6-8 weeks.
• Direct cell counts: haemocytometer / Petroff-Hausser chamber (total count, live + dead), direct microscopic count with a stain, Coulter counter, flow cytometry.
• Viable counts: standard plate count by serial dilution and spread/pour plate, reported as CFU/mL; membrane filtration for low-count water samples; MPN (most probable number) by multiple-tube dilution, standard for coliforms in water.
• Turbidity: optical density at 600 nm (OD₆₀₀) in a colorimeter/spectrophotometer; fast and non-destructive but counts dead cells too, and is linear only to about OD 0.4-0.8 (dilute above that).
• Mass and chemical methods: dry weight (for filamentous organisms), total nitrogen or protein, ATP assay; indirect: acid or gas production, substrate consumed, product formed.
• Colony-forming unit caution: one CFU may arise from a clump or chain, so CFU/mL ≤ total count.

The growth curve in batch culture

A closed system with fixed nutrients gives four phases on a plot of log₁₀(cell number) against time.

1. Lag phase: no increase in number; cells synthesise enzymes, ribosomes, ATP and cofactors and repair damage. Length depends on inoculum age, size and whether the medium is the same as before — a large, young, log-phase inoculum into identical medium gives almost no lag.
2. Log (exponential) phase: balanced growth at a constant maximum specific growth rate μ; cells are most uniform, physiologically active and most sensitive to antibiotics and disinfectants — so this is the phase used for inocula, transformation (competent cells), enzyme production and Gram staining.
3. Stationary phase: growth rate = death rate, number constant (typically 10⁹-10¹⁰ cells/mL for *E. coli*) because a nutrient is exhausted, oxygen is limiting or toxic products (acids, alcohols) accumulate. Cells become smaller, tougher, more resistant; secondary metabolites (most antibiotics), endospores and many exotoxins appear here. The stringent response (ppGpp) and σˢ (RpoS)-controlled genes switch on.
4. Death (decline) phase: exponential loss of viability; some cells persist as viable but non-culturable (VBNC) forms.
5. Diauxic growth: two log phases separated by a second lag, as in glucose + lactose for *E. coli* — glucose is used first (catabolite repression, low cAMP), then the *lac* operon is induced.
6. Cryptic growth / synchronous growth (all cells dividing together, obtained by filtration or temperature shift) are examined as special cases.

Continuous culture and the chemostat

• Chemostat: fresh medium enters and culture leaves at the same rate, holding volume constant; a single nutrient (glucose, ammonium, phosphate) is growth-limiting.
• Dilution rate D = F/V (h⁻¹). At steady state μ = D, so the experimenter sets the growth rate — the single most asked idea here.
• Washout occurs when D > μ_max; critical dilution rate D_crit ≈ μ_max.
• Turbidostat: feed rate is adjusted automatically to keep turbidity (cell density) constant.
• Monod equation links μ to the limiting substrate concentration (see Formulas).
• Uses: steady physiology for research, continuous production of single-cell protein, ethanol, lactic acid and waste-water treatment (activated sludge is effectively a continuous culture).
• Fed-batch, widely used industrially (penicillin, recombinant insulin at Biocon), feeds substrate without removing broth, avoiding substrate inhibition and overflow metabolism.

Physical and chemical requirements

• Temperature: psychrophile (optimum below ~15 °C), mesophile (20-45 °C; all human pathogens, optimum ~37 °C), thermophile (45-80 °C), hyperthermophile (above 80 °C). Each has minimum, optimum and maximum — the optimum lies nearer the maximum because enzymes denature abruptly.
• pH: acidophile (below 5.5), neutrophile (5.5-8; most bacteria, optimum ~7), alkaliphile (above 8.5); fungi prefer acid pH, which is why Sabouraud medium is set at pH 5.6.
• Oxygen: obligate aerobe; obligate anaerobe (killed by O₂ — *Clostridium*, *Bacteroides*); facultative anaerobe (*E. coli*, *Staphylococcus*); aerotolerant anaerobe (*Lactobacillus*); microaerophile (*Campylobacter*, *Helicobacter*, ~2-10% O₂). Protection against reactive oxygen species needs superoxide dismutase, catalase, peroxidase; anaerobes are grown in McIntosh-Fildes jar, GasPak, Robertson's cooked meat medium or an anaerobic glove box.
• CO₂: capnophiles (*Brucella*, pneumococcus) need 5-10% CO₂, supplied by a candle jar.
• Water activity and solutes: halophiles need NaCl; halotolerant *Staphylococcus aureus* grows on mannitol salt agar at 7.5% NaCl; osmophiles and xerophiles tolerate low a_w.
• Nutrients: macronutrients C, H, O, N, S, P, K, Mg, Ca, Fe; micronutrients Zn, Mn, Mo, Co, Cu, Ni; growth factors (vitamins, amino acids, purines/pyrimidines) needed by auxotrophs and fastidious organisms.
• Radiation and pressure: UV and ionising radiation damage DNA; barophiles grow at deep-sea pressures.

Nutritional and energy classification

• By carbon source: autotroph (CO₂) vs heterotroph (organic carbon).
• By energy source: phototroph (light) vs chemotroph (chemical oxidation).
• By electron donor: lithotroph (inorganic — H₂, H₂S, NH₃, Fe²⁺, S⁰) vs organotroph (organic).
• The four combinations: photoautotroph (cyanobacteria, purple and green sulphur bacteria), photoheterotroph (*Rhodospirillum* in the light), chemoautotroph/chemolithoautotroph (*Nitrosomonas*, *Nitrobacter*, *Thiobacillus*, *Beggiatoa*), chemoheterotroph (most bacteria, all pathogens, all fungi).
• Saprophyte (dead organic matter) and parasite (living host) describe lifestyle, not energy metabolism.

Catabolism: central pathways and ATP

• Glycolysis (EMP, Embden-Meyerhof-Parnas): glucose → 2 pyruvate; net 2 ATP by substrate-level phosphorylation + 2 NADH. Key enzymes hexokinase, phosphofructokinase (rate-limiting), pyruvate kinase. Operates with or without oxygen.
• Pentose phosphate (HMP) pathway: oxidative branch gives NADPH for biosynthesis plus ribose-5-phosphate (nucleotides) and erythrose-4-phosphate (aromatic amino acids); CO₂ released. Used by many bacteria in parallel with EMP.
• Entner-Doudoroff pathway: glucose → KDPG → pyruvate + glyceraldehyde-3-P; net 1 ATP, 1 NADPH, 1 NADH per glucose. Characteristic of *Pseudomonas*, *Zymomonas*, *Azotobacter*, *Rhizobium* — a classic "which organism lacks phosphofructokinase" question.
• Phosphoketolase pathway in heterofermentative lactic acid bacteria (*Leuconostoc*): lactate + ethanol + CO₂.
• Pyruvate dehydrogenase complex: pyruvate + CoA + NAD⁺ → acetyl-CoA + NADH + CO₂ (needs thiamine pyrophosphate, lipoic acid, FAD).
• TCA (Krebs) cycle: per acetyl-CoA — 3 NADH, 1 FADH₂, 1 GTP/ATP, 2 CO₂. Also amphibolic: α-ketoglutarate and oxaloacetate are drawn off for amino acids, succinyl-CoA for porphyrins. Glyoxylate shunt (isocitrate lyase, malate synthase) lets bacteria grow on acetate or fatty acids by bypassing the two decarboxylations. Anaplerotic reactions (PEP carboxylase, pyruvate carboxylase) refill oxaloacetate.
• Electron transport chain: NADH dehydrogenase → flavoproteins → quinones (ubiquinone, menaquinone) → cytochromes (b, c, a, o, d) → terminal oxidase. Bacterial chains are branched and variable; the chain is in the cytoplasmic membrane (no mitochondria), protons are pumped out, and the proton motive force drives F₁F₀ ATP synthase (chemiosmotic hypothesis, Mitchell). Theoretical P/O about 3 for NADH and 2 for FADH₂, but real bacterial yields are lower and site-dependent.
• Aerobic respiration: terminal acceptor O₂, complete oxidation of glucose, maximum ATP. Anaerobic respiration: acceptor is NO₃⁻ (denitrification, *Pseudomonas*), SO₄²⁻ (*Desulfovibrio*), CO₂ (methanogens), Fe³⁺ or fumarate — less energy than O₂ but far more than fermentation.
• Fermentation: no external acceptor; an organic intermediate (pyruvate or its derivative) is reduced to reoxidise NADH; ATP only by substrate-level phosphorylation. Types: homolactic (*Lactobacillus*, *Streptococcus* — 2 lactate), heterolactic, alcoholic (*Zymomonas*, yeast — ethanol + CO₂), mixed acid (*E. coli* — methyl red positive), butanediol (*Klebsiella*, *Enterobacter* — Voges-Proskauer positive), butyric/acetone-butanol (*Clostridium*), propionic (*Propionibacterium* — Swiss cheese eyes). These reactions underlie IMViC and the whole of biochemical identification.

Anabolism and regulation

• Autotrophic CO₂ fixation: Calvin cycle (RuBisCO, phosphoribulokinase) in cyanobacteria and *Thiobacillus*; also reductive TCA and acetyl-CoA (Wood-Ljungdahl) pathways.
• Nitrogen assimilation: nitrate reduction, ammonia via GDH (high NH₄⁺) or the GS-GOGAT system (low NH₄⁺, costs ATP); nitrogen fixation by nitrogenase (Mo-Fe protein, O₂-sensitive, ~16 ATP per N₂) in *Rhizobium*, *Azotobacter*, *Anabaena*.
• Biosynthesis: amino acid families from glycolysis/TCA intermediates; peptidoglycan from UDP-NAG/UDP-NAM carried by bactoprenol; fatty acids by acyl carrier protein; nucleotides de novo and by salvage.
• Regulation: allosteric feedback inhibition, covalent modification, induction and repression (*lac* operon), end-product repression (*trp* operon attenuation), catabolite repression via cAMP-CAP, two-component signalling, quorum sensing and the stringent response.
• Growth yield Y_X/S links substrate to biomass and is the industrial measure of how well a pathway is being exploited.

Key points

• Binary fission - transverse division into two cells; produces exponential, not arithmetic, growth.
• Generation time g - time to double; constant only in the log phase of batch culture.
• Specific growth rate μ - h⁻¹, equals 0.693/g; the slope of ln(cells) against time.
• Log phase - balanced growth, uniform cells, maximum sensitivity to antibiotics and heat; best for inocula and staining.
• Stationary phase - nutrient exhaustion or toxic products; endospores, antibiotics and many secondary metabolites form here.
• CFU/mL - viable count from plates; always ≤ total microscopic count because clumps give one colony.
• OD₆₀₀ - quick turbidity estimate; includes dead cells, linear only at low density.
• Chemostat - at steady state μ = D = F/V; the operator fixes growth rate, washout above μ_max.
• Monod constant K_s - substrate concentration at which μ = μ_max/2; small K_s means high affinity.
• Diauxic growth - two log phases on glucose + lactose, caused by catabolite repression.
• EMP vs ED - EMP nets 2 ATP; Entner-Doudoroff nets 1 ATP and is the *Pseudomonas* route.
• HMP pathway - supplies NADPH and pentoses rather than bulk ATP.
• TCA cycle is amphibolic - oxidises acetyl-CoA and supplies carbon skeletons; glyoxylate shunt allows growth on acetate.
• Chemiosmosis - the proton motive force across the cytoplasmic membrane drives F₁F₀ ATP synthase; bacterial ETC is membrane-bound and branched.
• Fermentation vs anaerobic respiration - fermentation uses an internal organic acceptor and gives ATP only by substrate-level phosphorylation; anaerobic respiration uses an external inorganic acceptor and an ETC.
• Oxygen classes - obligate aerobe, facultative anaerobe, obligate anaerobe, aerotolerant, microaerophile; catalase and SOD decide tolerance.
• Four nutritional types - photoautotroph, photoheterotroph, chemoautotroph, chemoheterotroph, from energy source + electron donor + carbon source.
• Growth yield Y_X/S - g biomass per g substrate; typically 0.4-0.5 g/g on glucose aerobically, far lower in fermentation.

Formulas and examples

Example 1 — generations, generation time and specific growth rate. A broth inoculated with 5 × 10³ cells/mL reaches 3.2 × 10⁸ cells/mL in 6 h of exponential growth.

• log₁₀(3.2 × 10⁸) = 8.5051; log₁₀(5 × 10³) = 3.6990.
• n = (8.5051 − 3.6990)/0.301 = 4.8061/0.301 ≈ 15.97 ≈ 16 generations.
• g = 360 min / 15.97 ≈ 22.5 min.
• μ = 0.693/0.375 h ≈ 1.85 h⁻¹. Check: 5 × 10³ × 2¹⁶ = 5 × 10³ × 65,536 = 3.28 × 10⁸ ✔.

Example 2 — predicting a count (food-safety style). *Staphylococcus aureus* at 10² cells/g in a mithai sample, g = 30 min at 37 °C, left out 5 h.

• n = 300/30 = 10 generations; N = 10² × 2¹⁰ = 10² × 1,024 = 1.024 × 10⁵ cells/g.
• Enterotoxin becomes a risk around 10⁵-10⁶ cells/g, so a 5-hour unrefrigerated hold is already at the limit — the basis of FSSAI hot/cold holding rules.

Example 3 — viable count from a dilution series. 0.1 mL of a 10⁻⁵ dilution spread-plated gives 148 colonies.

• Volume factor: 0.1 mL plated → multiply by 10.
• CFU/mL = 148/(0.1 × 10⁻⁵) = 148 × 10⁶ = 1.48 × 10⁸ CFU/mL.
• If the same dilution plated 1.0 mL by pour plate gave 148 colonies, the answer would be 1.48 × 10⁷ CFU/mL — the commonest exam slip.

Example 4 — chemostat. A 2 L chemostat is fed at 0.6 L/h; the organism's μ_max = 0.5 h⁻¹, K_s = 0.2 g/L.

• D = 0.6/2 = 0.3 h⁻¹, which is below μ_max, so a steady state exists and μ = 0.3 h⁻¹.
• Residual substrate: 0.3 = 0.5S/(0.2 + S) → 0.06 + 0.3S = 0.5S → S = 0.06/0.2 = 0.3 g/L.
• Steady-state doubling time = 0.693/0.3 ≈ 2.31 h.
• If feed were raised to 1.2 L/h, D = 0.6 h⁻¹ > μ_max → washout.

Example 5 — ATP and yield. Per glucose, aerobic respiration via EMP + TCA + ETC yields (theoretical maxima): 10 NADH × 3 + 2 FADH₂ × 2 + 4 substrate-level ATP = 30 + 4 + 4 = 38 ATP; homolactic fermentation yields 2 ATP.

• Ratio 38:2 = 19:1, so a facultative anaerobe must consume roughly 19 times the glucose anaerobically for the same ATP — the Pasteur effect, and why biomass yield collapses in fermentation.
• Modern estimates give about 30-32 ATP in mitochondria and less in bacteria (leaky membranes, lower P/O, transport costs) — quote 38 as the textbook maximum and say it is an upper limit.

Where it is used

• Vaccines and biologics: Serum Institute of India (Pune), Bharat Biotech, Biological E — growth curves and fed-batch kinetics fix harvest time; roles as upstream process associate and QC microbiologist.
• Fermentation industry: antibiotics and enzymes (Aurobindo, Cipla, Advanced Enzymes), ethanol distilleries, lactic acid and citric acid plants; stationary-phase control is what maximises secondary-metabolite titre.
• Dairy and food: Amul, Mother Dairy, Nestlé India — starter cultures (*Lactobacillus delbrueckii* subsp. *bulgaricus*, *Streptococcus thermophilus*) for curd and dahi; shelf life predicted from lag-phase extension by chilling; FSSAI microbiological limits in CFU/g.
• Water and environment: coliform MPN and membrane-filtration counts under IS 1622 (sampling and microbiological examination of water) and the drinking-water specification IS 10500; activated-sludge plants run as continuous cultures; the exact clause numbers are revised periodically, so verify the current edition.
• Clinical and pharma QC: blood-culture turnaround, MIC and MBC determination, antibiotic susceptibility (which assumes log-phase cells), sterility testing and growth-promotion tests per the Indian Pharmacopoeia; D-value calculations for autoclave validation.
• Research and teaching: CSIR labs, NCCS Pune, ICAR institutes — chemostats for steady-state physiology, isotope labelling of central pathways, biofertiliser and biogas (methanogen) development.

Interview questions

• Q: What is generation time and what does it depend on? A: The time a population takes to double. It depends on the species, medium composition, temperature, pH and oxygen — *E. coli* needs about 20 min in rich broth, *M. tuberculosis* 15-20 h.
• Q: Why does the growth curve use a logarithmic y-axis? A: Because growth is exponential; plotting log₁₀ cell number gives a straight line in the log phase whose slope yields μ, and it fits several orders of magnitude on one graph.
• Q: Why are log-phase cells used for antibiotic susceptibility tests? A: They are actively synthesising cell wall, protein and DNA, so they are uniformly susceptible; stationary cells are physiologically heterogeneous and far more resistant.
• Q: In a chemostat, who controls the growth rate? A: The operator — at steady state μ equals the dilution rate D = F/V, provided D is below μ_max.
• Q: Difference between fermentation and anaerobic respiration? A: Fermentation reduces an internal organic intermediate and makes ATP only by substrate-level phosphorylation; anaerobic respiration passes electrons down an ETC to an external inorganic acceptor such as nitrate or sulphate.
• Q: Which pathway does *Pseudomonas* use for glucose and why does it matter? A: The Entner-Doudoroff pathway, because it lacks phosphofructokinase; it nets only 1 ATP per glucose instead of 2.
• Q: Why is the TCA cycle called amphibolic? A: It both oxidises acetyl-CoA for energy and supplies intermediates (α-ketoglutarate, oxaloacetate, succinyl-CoA) for amino acid, pyrimidine and porphyrin synthesis.
• Q: What is diauxic growth? A: Two successive log phases with a lag between them when two sugars are present — *E. coli* uses glucose first because catabolite repression keeps cAMP low and the *lac* operon off, then adapts to lactose.
• Q: Why can CFU/mL be lower than the microscopic count? A: Clumps or chains form a single colony, some cells are dead, and fastidious or VBNC cells do not grow on the medium used.
• Q: Why do most antibiotics appear in stationary phase? A: They are secondary metabolites, not needed for growth; their synthesis is switched on when a nutrient becomes limiting and growth slows.
• Q: How do obligate anaerobes die in air? A: They lack superoxide dismutase and catalase, so superoxide and hydrogen peroxide accumulate and damage enzymes, membranes and DNA.
• Q: What is the glyoxylate shunt for? A: It bypasses the two decarboxylation steps of the TCA cycle so that bacteria growing on acetate or fatty acids can make oxaloacetate for biosynthesis without losing carbon as CO₂.

Common mistakes

• Treating growth as an increase in cell size instead of cell number, or plotting cell number on a linear axis and reporting a curve where a straight line is expected.
• Forgetting the 0.1 mL plating factor in CFU/mL, or counting plates outside the 30-300 colony range.
• Writing μ = 1/g instead of μ = 0.693/g, and mixing minutes with hours in the same calculation.
• Saying growth stops in stationary phase — division and death are balanced, and the culture is metabolically very active.
• Confusing the net ATP of glycolysis (2) with the gross (4), and quoting 38 ATP per glucose as a measured bacterial value rather than a textbook maximum.
• Calling nitrate respiration "fermentation", or claiming fermentation needs the absence of an ETC rather than the absence of an external acceptor.
• Assuming bacterial respiration happens in mitochondria; the chain lies in the cytoplasmic membrane.

Related topics

• Bacterial structure and classification · Culture media and staining · Sterilisation and disinfection · Microbial genetics · Industrial microbiology and fermentation technology · Enzymology and bioenergetics