The four stages of methane fermentation: from hydrolysis to methane

Methane fermentation is a four-stage, anaerobic breakdown of organic matter: hydrolysis splits polymers into soluble molecules, acidogenesis converts them into organic acids and alcohols, acetogenesis turns these products into acetate, hydrogen and CO₂, and methanogenesis produces methane from them. Each stage is carried out by different groups of microorganisms that must work in balance — and it is this balance, not any single parameter, that determines the stability of a biogas plant.

Stage 1: hydrolysis

Hydrolytic bacteria secrete extracellular enzymes that break large molecules down into soluble ones: proteins into amino acids, complex carbohydrates into simple sugars, fats into glycerol and long-chain fatty acids. This is the limiting step for fibrous materials, because cellulose, hemicelluloses and especially lignin are hard to access. Under anaerobic conditions lignin is practically not degraded, and it encases the cellulose, making it harder for enzymes to reach. That is why size reduction, retention time and pretreatment (described in separate articles) have the greatest influence on exactly this stage.

Stage 2: acidogenesis

The products of hydrolysis are fermented into volatile fatty acids (acetic, propionic, butyric, valeric), alcohols, hydrogen and CO₂. Acidogenic bacteria grow quickly and tolerate wide pH ranges, so when an easily degradable substrate is overfed, this stage "gets ahead" of the following ones. Acids accumulate and the pH falls — this is the mechanism behind most acidification events.

Stage 3: acetogenesis

Acetogenic (syntrophic) bacteria oxidise propionic and butyric acids and alcohols to acetate, hydrogen and CO₂. These reactions are thermodynamically feasible only when the partial pressure of hydrogen is very low — which is maintained by methanogens consuming H₂. This dependence is called interspecies syntrophy; it is why propionate is often the first sign of disturbance.

Stage 4: methanogenesis

Methanogens are archaea, not bacteria. There are two main pathways:

• acetoclastic — the breakdown of acetate into CH₄ and CO₂ (genera including Methanosaeta and Methanosarcina); according to commonly cited estimates it accounts for the larger share of the methane formed,
• hydrogenotrophic — the reduction of CO₂ with hydrogen to CH₄; it dominates, among other conditions, at high ammonia concentrations, when acetoclastic methanogens are inhibited and acetate is oxidised syntrophically to H₂ and CO₂.

Methanogens grow slowly (doubling times are measured in days) and are sensitive to changes in temperature, pH, oxygen and inhibitors. Washing them out of the digester, when retention time is too short, ends the process.

What this means in practice

Biogas composition

In agricultural biogas plants the biogas usually contains about 50–65% CH₄ and 35–50% CO₂, plus trace amounts of H₂S (from several dozen to several thousand ppm, depending on the substrate), water vapour and small amounts of NH₃. The methane content depends on the substrate composition: fats and proteins give a gas richer in CH₄ than carbohydrates.

How to apply this knowledge

1. Feed gradually — introduce changes in feed rate and mix composition over days, not hours.
2. Measure several indicators at once (pH, FOS/TAC, acids, gas composition), because each points to a different stage.
3. Match the pretreatment to the substrate — a fibrous one needs size reduction and a longer time, an easily degradable one needs a lower load and more buffering capacity.
4. Look after trace elements — methanogen enzymes contain, among others, nickel and cobalt, and a deficiency limits the last stage.

The ADM1 model (Batstone et al., 2002) formalises this scheme in equations and is still the basis of most process simulations.

Energy and mass balance in brief

Anaerobic digestion retains most of the substrate's chemical energy in methane: only a small part goes into biomass growth, which is why sludge accumulation in the digester is small compared with aerobic processes. The gas composition depends on the substrate: carbohydrates give a biogas poorer in CH₄ than fats and proteins. Methods for estimating the theoretical potential (the Buswell equation) are described in a separate article on BMP.

Common misconceptions about the process

• "More substrate means more gas." Up to a point, yes, but overloading acidogenesis disrupts methanogenesis and the yield falls.
• "One indicator is enough." pH, FOS/TAC, acids and gas composition describe different stages.
• "Microbes will get used to anything." Adaptation is possible, but it takes time and gradual changes.
• "Lignin can be degraded with a longer retention time." Under anaerobic conditions practically not; only physical and chemical processes help.

A good understanding of the stages makes it possible to plan the substrate mix, choose pretreatment and read BMP test results, in which both the lag phase (adaptation) and the gentle gas release from the less degradable fractions are visible.

Checklist: is the process in balance?

• pH within the range typical for the plant and without a downward trend,
• FOS/TAC stable or falling after changes in feed rate,
• propionate below the alarm level set for the plant,
• CH₄ share in the biogas consistent with the history for the given mix,
• digester temperature held to within about ±1 °C,
• no foam and no scum layer hindering mixing.