The four phases of anaerobic digestion: hydrolysis, acidogenesis, acetogenesis, methanogenesis

Anaerobic digestion is often described as turning organic matter into biogas, but inside it is an ordered sequence of four biochemical phases. Each one is carried out by a different group of microorganisms, and every phase depends on the previous one.

Knowing these phases is not theory: it is what makes it possible to understand why a digester produces less, why it acidifies or why a fibrous substrate barely performs. This article walks through all four with their reactants, products, microorganisms and relative speeds.

It all builds on the overall process described in what anaerobic digestion is.

Overview: why divide the process into four phases

Complex organic matter (proteins, carbohydrates, lipids) does not turn into methane in one jump. No methanogenic archaeon can eat a cellulose molecule or a protein: they only use very simple substrates (acetate, hydrogen, CO₂).

That is why the process is organised as a trophic chain: each microbial group transforms the product of the previous one into something simpler, all the way to methane. Dividing it into four phases (hydrolysis, acidogenesis, acetogenesis, methanogenesis) makes it possible to locate where the process fails when something goes wrong.

Phase 1 · Hydrolysis: breakdown of macromolecules

In hydrolysis, hydrolytic bacteria secrete extracellular enzymes (cellulases, proteases, lipases) that break macromolecules into their monomers: carbohydrates into sugars, proteins into amino acids and lipids into fatty acids and glycerol.

It is an extracellular and relatively slow phase. For lignocellulosic substrates (straw, fibrous manure, plant residues), hydrolysis is the rate-limiting phase of the whole process: if the fibre does not break down, the rest of the chain is left without food. That is why many pre-treatments (mechanical, thermal, enzymatic) act precisely here. It tends to be the limiting stage of the overall biodigestion process when the substrate has a high lignocellulosic content.

Phase 2 · Acidogenesis: production of VFAs

The monomers released in hydrolysis are fermented by acidogenic bacteria, which produce volatile fatty acids (VFAs) (acetate, propionate, butyrate), together with hydrogen, CO₂ and alcohols.

It is the fastest phase of all: acidogenic bacteria grow quickly (up to 10 g COD/g cells·day). That speed imbalance is key: if acidogenesis races ahead and the following phases cannot keep up, VFAs accumulate, the pH falls and the digester acidifies.

The typical products are split at around acetate (60-70%), propionate (15-25%) and butyrate (5-15%).

Phase 3 · Acetogenesis: production of acetate and hydrogen

Acetogenic bacteria convert the longer VFAs (propionate, butyrate) and other intermediates into acetate, hydrogen and CO₂, which are the direct substrates of the methanogens.

This phase has a thermodynamic particularity: the oxidation of propionate and butyrate is only favourable if the partial pressure of hydrogen is kept very low. That is why the acetogens depend on the methanogens removing the H₂ continuously. That obligate cooperation is syntrophy, and it is one of the most fragile balances in the digester.

Phase 4 · Methanogenesis: production of methane

In the final phase, methanogenic archaea produce the methane through two routes: the acetoclastic route (from acetate, around 70% of the methane) and the hydrogenotrophic route (from H₂ and CO₂, around 30%).

They are the slowest and most sensitive microorganisms in the system: they grow slowly, they are strictly anaerobic and they are affected by pH, temperature, ammonia and H₂S. For soluble, easily degradable substrates, methanogenesis is the rate-limiting phase. It is covered in detail in the post on methanogenesis.

Relative speeds and the rate-limiting phase by substrate

As anticipated earlier, there is no single slow phase: it depends on the substrate.

• In fibrous or lignocellulosic substrates (straw, solid manure), hydrolysis is in charge: breaking down the fibre is what takes the effort.
• In soluble, easily degradable substrates (sugars, wheys, fats), hydrolysis is fast and the bottleneck moves to methanogenesis, which cannot keep up.

Identifying the rate-limiting phase of your substrate is what points to the solution: pre-treatment if it is hydrolysis, or more retention time and fine control if it is methanogenesis.

Operational implications: why each phase matters for control

The practical value of this scheme is diagnostic. An imbalance between phases translates into measurable signals:

• If acidogenesis runs ahead of methanogenesis, VFAs accumulate and the FOS/TAC ratio rises: the digester is acidifying.
• If propionate in particular rises, it usually points to a problem in acetogenesis (high H₂, overload).
• A fall in methane production with low VFAs suggests the bottleneck is further upstream, in hydrolysis.

Overall, a well-balanced process with a biodegradable substrate converts 90-95% of the incoming COD into methane. The microbiology of the digester explains which communities sustain each phase.

Phase	Microorganisms	Substrate → product	Speed
Hydrolysis	Hydrolytic bacteria	Macromolecules → monomers	Slow (limits fibrous substrates)
Acidogenesis	Acidogenic bacteria	Monomers → VFAs, H₂, CO₂	Very fast
Acetogenesis	Acetogenic bacteria	VFAs → acetate, H₂, CO₂	Medium (depends on H₂)
Methanogenesis	Methanogenic archaea	Acetate / H₂+CO₂ → CH₄	Slow (limits soluble substrates)

Frequently asked questions about the phases of anaerobic digestion

What are the 4 phases of anaerobic digestion?

In order: hydrolysis (breakdown of macromolecules into monomers), acidogenesis (fermentation of those monomers into volatile fatty acids, hydrogen and CO₂), acetogenesis (conversion of the VFAs into acetate, hydrogen and CO₂) and methanogenesis (production of methane from acetate or from hydrogen and CO₂). Each phase is carried out by a different microbial group and depends on the previous one.

What is the rate-limiting phase of anaerobic digestion?

It depends on the substrate. In fibrous or lignocellulosic substrates (straw, solid manure, plant residues) the rate-limiting phase is hydrolysis: breaking down the fibre is the slowest part. In soluble, easily degradable substrates (sugars, fats, wheys), hydrolysis is fast and the bottleneck moves to methanogenesis, which is the phase with the slowest microorganisms.

What are VFAs and why do they matter?

VFAs (volatile fatty acids) are the short-chain organic acids (acetate, propionate, butyrate) produced by acidogenesis. They matter because they are the central intermediate of the process: if the later phases do not consume them at the same rate they are produced, they accumulate, the pH falls and the digester acidifies. That is why their concentration, and the FOS/TAC ratio, is one of the most widely used stability indicators.

How long does each phase take?

There is no fixed time, but very different speeds. Acidogenesis is the fastest (the bacteria involved grow very quickly), while fibre hydrolysis and methanogenesis are slow and set the overall pace. In practice, that pace is governed by the hydraulic retention time (HRT) of the digester, which usually ranges from a few days (soluble substrates) to 20-40 days or more (fibrous substrates).

Smallops and the balance between phases

When a digester loses performance, there is almost always one phase out of sync. At Smallops we locate which one with an Operational Excellence Diagnosis and adjust feeding and control to rebalance them.

References and standards

Batstone, D.J. et al. (2002). The IWA Anaerobic Digestion Model No. 1 (ADM1). Water Science and Technology, 45 (10), 65-73.

Gerardi, M.H. (2003). The Microbiology of Anaerobic Digesters. John Wiley & Sons.

Vavilin, V.A. et al. (2008). Hydrolysis kinetics in anaerobic degradation of particulate organic material. Waste Management, 28 (6), 939-951. → doi.org/10.1016/j.wasman.2007.03.028

Pavlostathis, S.G. & Giraldo-Gomez, E. (1991). Kinetics of anaerobic treatment. Critical Reviews in Environmental Control, 21 (5-6), 411-490.