1.3 Mixed Culture Hydrogen Dark Fermentation
Usually, the by-products (H 2 , VFAs, and alcohols) during mixed culture dark
fermentation are ultimately converted to CH 4 . In balanced mixed culture fermentation, H 2 is produced through the activity of the hydrogenase of acidogenic bacteria as
an intermediate by-product with acetate and butyrate production (Table 8.2; Eqs. 1
and 2) [59]. If H 2 -consuming microorganisms are inhibited, a maximum of 4 or
2 mol H 2 /mol glucose could be produced when acetate or butyrate (Table 8.2; Eqs. 1
and 2, respectively) is the only final product, respectively. Generally, acidogenic
bacteria prefer to produce acetate because it regenerates the reducing equivalents and
allows them to synthesize ATP [60]; however, under elevated P H 2 (>60 Pa),
acidogens produce butyrate to avoid the accumulation of inhibitory reducing
equivalents [61].
Anaerobic mixed culture contains different species of strict anaerobes and facultative anaerobic bacteria. These bacteria produce H 2 during their metabolism when
they dispose the excess electrons in the form of molecular H 2 through the activity of
hydrogenase [62]. Strict anaerobes such as Clostridia, methylotrophs, and rumen
bacteria produce H 2 as a by-product with their main product which is the VFA
[63]. The most effective H 2 -producing microorganisms in dark fermentation are the
species of Clostridia [62, 64] such as C. pasteurianum, C. butyricum, and
C. beijerinckii [65–67]. Clostridium species (Clostridiaceae) constituted 64.6% of
the clones detected in H 2 -producing mesophilic mixed cultures [68]. Because Clostridia and Bacillus species are spore formers, then heat treatment can be used to
enrich them, while methanogens are eliminated because they are not spore formers.
Other anaerobes such as Actinomyces sp. and Porphyromonas sp. have also been
detected in addition to Clostridia in H 2 -producing cultures [69]. Species of facultative anaerobes such as Enterobacteriaceae [70], Citrobacter [71], Lactobacillaceae,
Bacillaceae [72], and Klebsiella [68] have been detected in H 2 -producing anaerobic
mixed cultures. Generally, facultative bacteria produce less quantities of H 2 than
strict anaerobes.
1.3.1 Pure Versus Mixed Culture for H 2 Production
Examples of pure culture strains that were used in H 2 production include Klebsiella
sp. TR17 [73], Clostridium pasteurianum [74], Thermotoga [75], Escherichia [76],
Rhodopseudomonas palustris [77], and Enterobacter aerogenes EB-06
[78, 79]. However, economic H 2 production requires a cheap and sustainable
process. The pure culture cannot be used to treat “waste” and generate H 2 because
the substrate (i.e., the waste) itself is a major source of microbial contamination.
Microbial contamination cannot be eliminated, and the strain purity cannot be
maintained. Continuous heating of the substrate (waste) to bring its temperature to
the range usually used during sterilization and to maintain it for a specific time is an
energy drain that disqualifies the process as an economically profitable investment.
332
A. Hajizadeh et al.
Usually, the by-products (H 2 , VFAs, and alcohols) during mixed culture dark
fermentation are ultimately converted to CH 4 . In balanced mixed culture fermentation, H 2 is produced through the activity of the hydrogenase of acidogenic bacteria as
an intermediate by-product with acetate and butyrate production (Table 8.2; Eqs. 1
and 2) [59]. If H 2 -consuming microorganisms are inhibited, a maximum of 4 or
2 mol H 2 /mol glucose could be produced when acetate or butyrate (Table 8.2; Eqs. 1
and 2, respectively) is the only final product, respectively. Generally, acidogenic
bacteria prefer to produce acetate because it regenerates the reducing equivalents and
allows them to synthesize ATP [60]; however, under elevated P H 2 (>60 Pa),
acidogens produce butyrate to avoid the accumulation of inhibitory reducing
equivalents [61].
Anaerobic mixed culture contains different species of strict anaerobes and facultative anaerobic bacteria. These bacteria produce H 2 during their metabolism when
they dispose the excess electrons in the form of molecular H 2 through the activity of
hydrogenase [62]. Strict anaerobes such as Clostridia, methylotrophs, and rumen
bacteria produce H 2 as a by-product with their main product which is the VFA
[63]. The most effective H 2 -producing microorganisms in dark fermentation are the
species of Clostridia [62, 64] such as C. pasteurianum, C. butyricum, and
C. beijerinckii [65–67]. Clostridium species (Clostridiaceae) constituted 64.6% of
the clones detected in H 2 -producing mesophilic mixed cultures [68]. Because Clostridia and Bacillus species are spore formers, then heat treatment can be used to
enrich them, while methanogens are eliminated because they are not spore formers.
Other anaerobes such as Actinomyces sp. and Porphyromonas sp. have also been
detected in addition to Clostridia in H 2 -producing cultures [69]. Species of facultative anaerobes such as Enterobacteriaceae [70], Citrobacter [71], Lactobacillaceae,
Bacillaceae [72], and Klebsiella [68] have been detected in H 2 -producing anaerobic
mixed cultures. Generally, facultative bacteria produce less quantities of H 2 than
strict anaerobes.
1.3.1 Pure Versus Mixed Culture for H 2 Production
Examples of pure culture strains that were used in H 2 production include Klebsiella
sp. TR17 [73], Clostridium pasteurianum [74], Thermotoga [75], Escherichia [76],
Rhodopseudomonas palustris [77], and Enterobacter aerogenes EB-06
[78, 79]. However, economic H 2 production requires a cheap and sustainable
process. The pure culture cannot be used to treat “waste” and generate H 2 because
the substrate (i.e., the waste) itself is a major source of microbial contamination.
Microbial contamination cannot be eliminated, and the strain purity cannot be
maintained. Continuous heating of the substrate (waste) to bring its temperature to
the range usually used during sterilization and to maintain it for a specific time is an
energy drain that disqualifies the process as an economically profitable investment.
332
A. Hajizadeh et al.