Biofuels in Perspective

Biological Hydrogen Production by Anaerobic Microorganisms 207
Table 11.4 Product formation by Escherichia coli and Enterobacter
aerogenes. Products are expressed in mmoles per 100 mmoles glucose
fermented (48)
E. coli pH6.2 E. coli pH 7.8 E. aerogenes
Formate 2.4 86.0 68.4
Hydrogen 75.0 0.3 n.d.
Carbon dioxide 88.0 1.8 79.6
Acetate 36.5 38.7 51.9
Lactate 79.5 70.0 10.1
Ethanol 59.8 50.5 51.5
Succinate 10.7 14.8 13.1
2,3 butanediol 0.3 0.3 19.2
n.d., not determined
Besides hydrogen formation from formate, NADH which is formed in the conversion
of glyceraldehyde-3-phophate to 3 phosphoglycerate needs to be directed to hydrogen
formation as well. In general, mass balances show that maximally only two molecules of
hydrogen are formed per molecule of glucose. 52 However, mutant strains of Eb. cloacae
in which some pathways to alcohols had been blocked, showed hydrogen yields of up to
3.4 mol per mol of glucose. 53,54 These data indicate that in the enterobacteria NADH can
also be used for hydrogen formation.
Several studies have been performed to enhance hydrogen formation in enterobacteria.
By redirecting biochemical pathways by means of inhibitors and by creation of specific
mutants an enhanced hydrogen production was observed in Enterobacter aerogenes. 53,55,56
The hydrogen production yields and the hydrogen production rates were enhanced by
genetically modifying E. coli strains. 57,58 These authors interrupted genes involved in
lactate and succinate formation and overexpressed formate hydrogen lyase.
11.6 The Genus Clostridium
Clostridia are well-known hydrogen producers. These strictly anaerobic sporeforming
bacteria are found in environments that are rich in decaying plant materials and therefore
have the enzymatic machinery to hydrolyze polymers like cellulose, xylan, pectin,
chitin and starch. Their high hydrolytic capacity is one of the reasons that clostridia
have been investigated repeatedly for biofuel production. 59,60 In addition to hydrogen also
ethanol and butanol can be produced. The optimum growth temperature ranges from 25 to
65 ◦ C, but most are typical mesophiles with optima between 30–40 ◦ C. A few selected
species have been investigated in more detail because of the high level production of
specific end products. Cl. acetobutylicum and Cl. beyerinckii perform the so-called ABE
fermentation, producing acetone, butanol and ethanol. 61 Cl. thermocellum has been studied
predominantly for its high ethanol producing capacity combined with a high cellulose
hydrolyzing capacity. 62 Among the clostridia, Cl. thermocellum represents the one with
the highest growth temperature. A draft genome sequence of Cl. thermocellum is available
(2005) and first micro-array experiments are underway. H2 metabolism has been studied