Monitoring the anaerobic digestion process

by Harry Michael Falk

33. Deublein, D. & Steinhauser, A. Biogas from Waste and Renewable Resources. (John
Wiley & Sons Inc, Apr. 2008).

Basics of anaerobic digestion

The anaerobic fermentation is a biological process involving many substeps, in
which the organic carbon is converted to its most oxidized form, CO 2, and the most
reduced form, CH4. The process of biogas production can be divided in four stages,
whereas the first and the second as well as the third and fourth stage are linked closely
with each other. Depending on the degradability of the substrate, di"erent stages may
be
responsible for their limited decomposition [33].
Hydrolysis
In this first step organic substrates are disassembled by extracellular bacterial
enzymes into oligomers and monomers performed by facultative or obligatorily
anaerobic bacterial such as Clostridium, Bacillus and Pseudomonas. Carbohydrate
cleavage by cellulases, xylanases or amylases result in simple sugars and normally
takes place within a few hours; proteins are degraded by peptidases to single amino
acids
and
(oligo-)peptides,
and energy-rich and energy-rich lipids are decomposed by lipases into glycerol and
fatty acids, normally within a few days. The biodegradation of other macro molecules
like lignocellulose or lignin is very slow and incompletely [33].
In the first phase (the hydrolysis), undissolved compounds, like cellulose, proteins, and fats are
cracked into monomers (water- soluble fragments) by exoenzymes (hydrolase) of facultative and
obligatorily anaerobic bacteria. Actually, the covalent bonds are split in a chemical reaction with water
(Figure 2.2 ). The hydrolysis of carbohydrates takes place within a few hours, the hydrolysis of proteins
and lipids within few days. Lignocellulose and lignin are degraded only slowly and incompletely. The
facultative anaerobic microorganisms take the oxygen dissolved in the water and thus cause the low
redox potential necessary for obligatorily anaerobic microorganisms.

Acidogenesis
The second phase of the first stage is the acidogenesis, named after the formation
of volatile fatty acids. Monomers, the end products of the hydrolysis step, are taken up
by di"erent facultative and obligatorily anaerobic bacteria such as Bifidobacterium spp.
Selenomonas spp. and Flavobacterium spp. and further degraded to acetic acid,
propionic acid, butyric acid, alcohols, hydrogen and carbon dioxide. The formation of
the end products is related with the partial hydrogen pressure; if the H 2 partial pressure
is increasing, fewer reduced compounds like acetate are formed [33].
The monomers formed in the hydrolytic phase are taken up by different facultativeand obligatorily
anaerobic bacteria and are degraded in the second, the acidogenic phase, to short- chain organic acids,

C1 C5 molecules (e.g., butyric acid, propionic acid, acetate, acetic acid), alcohols, hydrogen, and carbon
dioxide. The concentration of the intermediately formed hydrogen ions affects the kind of the
products of fermentation. The higher the partial pressure of the hydrogen, the fewer reduced
compounds, like acetate, are formed.
The pathways of degradation are as follows:
a.

Carbohydrates:
Formation of propionic acid by propioni bacterium via the succinate pathway and the acrylic
pathway
Formation of butyric acid (butyric acid pathway) above all by clostridium
Acetic acid 2-hydroxy butyrate trans-2-butenic acid butyric acid butanol (Figure 2.4 )

b. Fatty acids:
These are degraded e.g. from acetobacter by - oxidation. Therefore the fatty acid is bound on
Coenzyme A and then oxidizes stepwise, as with each step two C atoms are separated, which are
set free as acetate.
b. Amino acids:
These are degraded by the Stickland reaction by Clostridium botulinum taking two amino acids at
the same time one as hydrogen donor, the other as acceptor in coupling to acetate, ammonia, and
CO2. During splitting of cysteine, hydrogen sulfide is released.

Acetogenesis
In this step long chain fatty acids are reduced to acetic acid (C 2) and hydrogen (H2)
by acid-forming bacteria like Acetobacterium spp., Sporomusa spp. and Ruminococcus
spp. Under standard conditions, these biochemical reaction are endergonic (see table
1.2). For the degradation propionic acid, !G 0!= 76.2 kJ/mol are needed. At very low
concentrations of H2, however, the acetate formation by oxidation of the long chain
fatty acids is thermodynamically possible. The problem, that acetogenic bacteria are
obligatory H2 producers on the other hand is solved by living with the methanogenic
bacteria syntrophically, a process termed "interspecies hydrogen transfer" [24].
The methanogenic bacteria remove the H 2, which is formed by the acetogenic
bacteria and therefore lowering the hydrogen partial pressure [33]. Figure 1.5 presents
the influence of the hydrogen partial pressure on the free energy the bacteria can gain
in the acetogenesis and methane formation from carbon dioxide and hydrogen. Clearly,
all thereactions are thermodynamically favorable only in a small window
The products from the acidogenic phase serve as substrate for other bacteria, those of the
acetogenic phase. The acetogenic reactions (Table 2.1 ) are endergonic. With the degradation of
propionic acid are needed Gf= + 76.11 kJ mol 1, and with the degradation of ethanol Gf= + 9.6 kJ mol
1.4

In the acetogenic phase, homoacetogenic microorganisms constantly reduce exergonic H 2 and CO2 to
acetic acid.

Acetogenic bacteria are obligatory H2 producers. The acetate formation by oxidation of long- chain
fatty acids (e.g., propionic or butyric acid) runs on its own and is thus thermodynamically possible only

with very low hydrogen partial pressure. Acetogenic bacteria can get the energy necessary for their
survival and growth, therefore, only at very low H2 concentration.
Acetogenic and methane- producing microorganisms must therefore live in symbiosis. Methanogenic
organisms can survive only with higher hydrogen partial pressure. They constantly remove the products
of metabolism of the acetogenic bacteria from the substrate and so keep the hydrogen partial
pressure, pH2, at a low level suitable for the acetogenic bacteria.
When the hydrogen partial pressure is low, H 2, CO2, and acetate are predominantly formed by the
acetogenic bacteria. When the hydrogen partial pressure is higher, predominantly butyric, capronic,
propionic, and valeric acids and ethanol are formed. From these products, the methanogenic
microorganisms can process only acetate, H2, and CO2.
About 30% of the entire CH 4 production in the anaerobic sludge can be attributed to the reduction
of CO2 by H2, but only 5 6% of the entire methane formation can be attributed to the dissolved
hydrogen. This is to be explained by the interspecies hydrogen transfer by which the hydrogen moves
directly from the acetogenic microorganisms to the methanogenics, without being dissolved in the
substrate.
The anaerobic conversion of fatty acids and alcohols goes energetically at the expense of the
methanogenics, where these, however, in return, receive the substrates (H 2, CO2, acetic acid) needed
for growth from the acetogenic bacteria.
The acetogenic phase limits the rate of degradation in the final stage. From the quantity and the
composition of the biogas, a conclusion can be drawn about the activity of the acetogenic bacteria.
At the same time, organic nitrogen and sulfur compounds can be mineralized to hydrogenic sulfur
by producing ammonia. The reduction of sulfate follows for example the stoichiometric equations below
Sulfate - reducing bacteria such as Desulfovibrio, Desulfuromonas, Desulfobulbus, Desulfobacter,
Desulfococcus, Desulfosarcina, Desulfonema and Desulfotomaculum participate in the process, which
uses the energy released by the exergonic reaction.

1.4.4. Methanogenesis
The final step of reducing the organic intermediates to methane is the methanogenesis,
an exergonic reaction, which only takes place under strictly anaerobic conditions. The
responsible bacteria all belong to the archaea family, such as Methanococci spp.,
Methanobacteria spp. and Methanomicrobia spp. Central to the anaerobic fermentation
and methane generation this methanogenic bacteria are very sensitive to all sort of
process disturbances, especially pH-fluctuations and oxygen. Due to their very low
growth rate, the whole process of anaerobic digestion is optimized for these bacteria.
Not all methanogenic species can use all available substrates. These can be divided
into three major groups and their methane forming reactions can be found in table 1.3:
CO2 type: CO2, HCOO Methyl-type: CH3OH, CH3NH3, (CH3)2NH2+,(CH3)3NH+, CH3SH, (CH3)2S

 Acetate type: CH3COO

The energy yield for the bacteria is varying with the biochemical reactions. The direct
reduction of CO2 and H2 gains up to - 136 kJ / mol and can be done by all methanogenic
species. In contrast, the comproportionation of acetate only yields - 31 kJ / mol.
Interestingly, only 27 % - 30 % of the methane arises from the reduction pathway, while
70 % are generated by the combined reduction and oxidation of acetate [33].

1.5. Process parameters

The previous section clarified, that the di"erent bacteria have a different optimum for
different process parameters. The first stage of hydrolysis and acidogenesis has a pH
optimum of 3 - 4 and can also have aerobic conditions, in contrast to methanogenesis,
which has to be strictly anaerobic. It is important to inhibit fluctuations like rapid
substrate changes or temperature shifts, because this can lead to a deficit of the gas
production. With a two-stage plant, it is possible to optimize the two stages of the
biochemical reactions. In a one-stage plant, the process must be optimized for the
methanogenic bacteria, because of their low growth rate and higher sensitivity to
environmental factors [33]. The following sections will give a short overview of the
di"erent parameters that can be either set to an optimal range or should be monitored
closely
for
an
early
detection of problems during the biochemical breakdown of the biomass to methane.
1.5.1. Temperature
For every biological process, the temperature is one of the most important factors.
Dependent on the microorganisms optimum, di"erent temperature levels are used for
the fermentation process:
1. psyrchophilic at a temperature range between 15 C and 25 C
2. mesophilic at a temperature range between 25 C and 45 C
3. thermophilic at a temperature range between 45 C and 70 C
At a higher temperature, the biogas yield is increased (thermophilic microorganisms),
but meanwhile the susceptibility to errors is also heightened. If the process
temperature is low, the biogas yield will decrease as well. Most biogas plants will
therefore be run at a mesophilic temperature range with temperatures around 37 C to
40 C to ensure a balanced compromise between biogas yield and process stability. In
two-stage systems, the first hydrolysis step and the methane digester can be operated
at di"erent temperatures, although this varies individually depending on the used
substrate.
1.5.2. pH value
The pH optimum of the methanogenic bacteria is at pH = 6.7 - 7.5. If it decreases to pH
< 6.5, a positive feedback leads to a further decrease, because the activity of the
methanogenic bacteria is inhibited and thus the volatile fatty acids in the process
cannot be oxidized. With rising concentrations, the pH sinks even more and the process
will come to a halt. In the fermentation process, however, two bu"er systems ensure a
pH in the optimal range. A too strong acidification is avoided by the carbon dioxide /
hydrogen carbonate / carbonate bu"er system, which is created by the equilibrium
between dissolved carbon dioxide and hydrocarbonic acid (pK a = 6.35):
CO2 H2CO3 H+ + HCO3- 2 H+ + 2 CO32At pH 4, the bu"er equilibrium is shifted to free carbon dioxide, at pH 13 all carbon
dioxide is bound as carbonate in the system. For monitoring the process, a rise of the

carbon dioxide percentage in the biogas can be an indicator for a process disturbance
[33]. A second system, the ammonia-ammonium bu"er inhibits a too weak acidification
(pKa = 9.25):
NH3 + H2O NH4+ + OH
NH3 + H+ NH4+
Although these bu"er systems equilibrate the pH, both can still be overloaded with e.g.
a too high organic load of easy degradable carbohydrates (starch powder, potato
wastewater), which can lead to a rapid increase of the volatile fatty acids.