Microbial Metabolism and Growth

Exponential microbial growth

Exponential growth is the phase in which each cell divides by binary fission at a constant rate, so the population doubles over equal time intervals. After n generations the population is N = N_0 \times 2^{n}, so the number of generations is n = \dfrac{\log(N/N_0)}{\log 2}. The method is to form the ratio of the final to the initial count and express it as a power of two.

In the continuous form, the population follows N = N_0 e^{\mu t}, where \mu is the specific growth rate in \text{h}^{-1}. Setting N = 2N_0 gives e^{\mu t_d} = 2, so the doubling time is t_d = \dfrac{\ln 2}{\mu}. A rate and a time must never be equated directly, since t_d is inversely proportional to \mu.

The lag phase is a period with no net multiplication, so its duration must be subtracted before counting doubling periods. For example, if a culture has a 2 h lag and a 1 h doubling time, then in 6 h total there are (6-2)/1 = 4 doubling periods, giving a 16-fold increase.

Electron acceptors in microbial respiration

In respiration, microbes transfer electrons from a donor to a terminal electron acceptor, and the acceptor determines the energy yield. Oxygen gives the highest yield, so the choice of acceptor depends on the metabolic type of the organism.

• Dissolved oxygen (DO) — the preferred terminal acceptor of aerobic bacteria. Aerobes cannot use nitrite as an acceptor for respiration. e.g. An aerobic heterotroph oxidises glucose and reduces O_2 to water.
• Facultative bacteria — switch to nitrate or nitrite when DO is absent, which is the anoxic condition. e.g. A facultative denitrifier reduces nitrite to nitrogen gas in an anoxic tank.
• Anaerobes — strict anaerobes cannot use DO, which is toxic or inhibitory to them.
• Endogenous metabolism — when external substrate is exhausted, cells oxidise their own cell material, and the electron acceptor is located inside the cell.
• Exogenous metabolism — the electron acceptor is external, such as DO, and electron transfer to it occurs outside the cell.

In wastewater treatment, the donor and acceptor pairs differ by process. In acid fermentation, the same organic compounds act as both donor and acceptor. In methanogenesis, volatile fatty acids are donors and CO_2 is the acceptor reduced to methane. In denitrification, organic matter is the donor and nitrate or nitrite is the acceptor, while CO_2 is only a carbon source. In aerobic iron oxidation, Fe(II) is the donor and O_2 is the acceptor.

Photosynthesis and respiration basics

Photosynthesis converts light energy into chemical energy, and its location depends on cell type. In eukaryotes, the light reactions and the Calvin cycle take place in chloroplasts, whereas the breakdown of nutrients for energy takes place in the cytoplasm and mitochondria. Prokaryotes have no membrane-bound chloroplast, so the statement that prokaryotic photosynthesis occurs within chloroplasts is false.

Photophosphorylation is ATP synthesis from ADP and inorganic phosphate driven by light energy. Phosphorylation is the general term for ATP synthesis in mitochondria and chloroplasts. The Calvin cycle, consisting of carboxylation, reduction and regeneration, operates in all photosynthetic plants, including C3, C4 and other types.

C4 plants differ from C3 plants in leaf anatomy and performance. C4 plants have Kranz anatomy, which concentrates CO_2 around the Rubisco-containing cells. C3 plants lack this anatomy, have lower photosynthetic efficiency at high temperature, and show higher photorespiration. A statement that C3 plants have a special leaf anatomy and higher photosynthetic rate than C4 plants is therefore incorrect.

Heterotrophic and autotrophic bacteria

Bacteria are classified by their carbon source and energy source. Heterotrophs use organic matter as their carbon source, and they usually obtain energy by oxidising the same organic matter. Autotrophs use carbon dioxide as their carbon source, fixing it into cell material.

• Aerobic heterotrophs — use organic matter for both carbon and energy. Carbon dioxide is their product, not their carbon source.
• Chemolithoautotrophs — use CO_2 as carbon and oxidise reduced inorganic substances, such as ammonium, nitrite or sulfide, as energy sources. Organic matter is not their energy source.
• Nitrifiers — are autotrophs that carry out aerobic nitrification. Nitrosomonas oxidises ammonia to nitrite, and Nitrobacter oxidises nitrite to nitrate.

Biodegradation of organic matter, both aerobic and anaerobic, and denitrification are performed mainly by heterotrophic bacteria, since they require organic carbon and energy.

Microbial growth stoichiometry

Stoichiometry links substrate consumption, biomass formation and oxygen demand in a microbial reaction. The growth equation is written with a fixed cell formula, usually \text{C}_5\text{H}_7\text{O}_2\text{N}, and it balances carbon, nitrogen, electrons and charge.

The observed yield is the mass of biomass produced per mass of substrate consumed, expressed as Y_{obs} = \dfrac{\text{g VSS produced}}{\text{g COD consumed}}. The substrate mass is converted to COD using its oxygen equivalent, and the biomass moles from the balanced equation are converted to VSS using the molar mass of the cell formula.

Carbon balance gives the fate of the substrate carbon. Carbon not incorporated into the cell formula is oxidised to CO_2, so the percentage of carbon going to CO_2 equals (C_{substrate} - C_{cells})/C_{substrate} \times 100 when no endogenous metabolism occurs. For a yield coefficient given per gram of substrate, multiply by the molar mass of the substrate to get the cell mass per mole, then divide by the molar mass of the cell formula to get moles of cells.

Enzyme classification (EC classes)

Enzymes are classified by the type of reaction they catalyse, and each class carries a number in the Enzyme Commission system. The six main classes are the following.

• Oxidoreductases — catalyse oxidation-reduction reactions, such as dehydrogenases that transfer electrons to NAD^+. e.g. Lactate dehydrogenase converts lactate to pyruvate.
• Transferases — transfer a functional group from one molecule to another. e.g. Hexokinase transfers a phosphate from ATP to glucose.
• Hydrolases — cleave bonds by adding water. e.g. Lipase hydrolyses ester bonds in triglycerides.
• Lyases — cleave C–C, C–O, C–N or other bonds by elimination, leaving double bonds or rings, or add groups to double bonds.
• Isomerases — convert a molecule into its isomer, such as the racemisation of alanine.
• Ligases — join two molecules using energy from ATP.