used and show the highest H 2 production efficiency [66, 72]. Studies have shown
that Clostridium sp. are able to produce the maximum H 2 yield of 4 mol H 2 /mol
hexose, while 2 mol H 2 /mol hexose is achieved by Enterobacter species [74].
Moreover, some Clostridium species like Clostridium thermocellum are able to
effectively break down cellulose in a single-step fermentation process [75], which
make these microorganisms potential biocatalysts to produce H 2 from lignocellulosic biomass.
Clostridium species produce H 2 fermentatively through the glycolytic pathway
[72]. Organic molecules, mainly sugars, are oxidized originating pyruvate, ATP and
NADH. Pyruvate is subsequently converted into acetyl-CoA and CO 2 through the
pyruvate-ferredoxin oxidoreductase, which also catalyzes the reduction of ferredoxin. The reduced ferredoxin is used by the electron bifurcation or monomeric
Fd-dependent FeFe-hydrogenase to reduce protons to H 2 . Furthermore, H 2 can be
also produced by the oxidation of NADH produced by glycolysis through electron
bifurcation. The acetyl-coA produced is also used in several lateral metabolic
pathways originating acetate or butyrate. Since the regeneration of NAD
+ is
required for the glycolysis process, several other by-products namely ethanol and
butanol can be also formed [72]. Therefore, the H 2 production yield is dependent on
the pathway involved. In the case of hydrogen production by Enterobacter spp.,
this process is also performed through the glycolytic pathway, but in these
microorganisms, H 2 can be also produced from formate. In Enterobacter spp., the
pyruvate is not only converted to acetyl-CoA but can also originate formate that is
further oxidized into H 2 and CO 2 by the formate-hydrogen lyase [66, 69].
Although high H 2 production is possible by dark fermentation, this process has
two mains drawbacks: low substrate conversion to H 2 and accumulation of
by-products [65]. Theoretically, the conversion of 1 mol glucose will give 12 mol
H 2 . However, according with the fermentative pathway, the maximum H 2 yield is
restricted to 4 mol H 2 /mol glucose for acetate-type fermentation, 2 mol H 2 /mol
glucose for butyrate and ethanol-type fermentation and 1 mol H 2 /mol glucose for
propionate-type fermentation [64, 66]. When organic wastes are used as substrate,
the BioH 2 yield rarely exceeds 2 mol H 2 /mol hexose [64, 66]. Therefore, several
strategies have been investigated to enhance the H 2 yield, namely metabolic
engineering, development of integrated processes, whole-cell immobilization and
incorporation of metal additives in the bioprocess [64, 65, 69, 74].
Metabolic engineering strategies reported to improve the H 2 production yield
include the construction of innovative metabolic routes and/or enhancing the main
metabolic pathway by overexpressing hydrogen-producing genes and knockout of
competitive pathways [65, 69, 76, 77]. A synthetic pyruvate:H 2 pathway with
additional co-expression of the Bacillus subtilis AmyE was constructed in E. coli
allowing the recombinant strain to became able to produce H 2 from potato starch
[76]. In addition, overexpressing the enzymes directly involved in hydrogen production also resulted in increase in the H 2 production [77]. It was reported the
overexpression of a gene encoding the HydA hydrogenase in Clostridium paraputricum enhanced an increase in the H 2 yield from 1.4 to 2.4 mol H 2 /mol glucose
from rich synthetic wastewaters [77]. Furthermore, the gene coding for a xylose
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M. Martins et al.
that Clostridium sp. are able to produce the maximum H 2 yield of 4 mol H 2 /mol
hexose, while 2 mol H 2 /mol hexose is achieved by Enterobacter species [74].
Moreover, some Clostridium species like Clostridium thermocellum are able to
effectively break down cellulose in a single-step fermentation process [75], which
make these microorganisms potential biocatalysts to produce H 2 from lignocellulosic biomass.
Clostridium species produce H 2 fermentatively through the glycolytic pathway
[72]. Organic molecules, mainly sugars, are oxidized originating pyruvate, ATP and
NADH. Pyruvate is subsequently converted into acetyl-CoA and CO 2 through the
pyruvate-ferredoxin oxidoreductase, which also catalyzes the reduction of ferredoxin. The reduced ferredoxin is used by the electron bifurcation or monomeric
Fd-dependent FeFe-hydrogenase to reduce protons to H 2 . Furthermore, H 2 can be
also produced by the oxidation of NADH produced by glycolysis through electron
bifurcation. The acetyl-coA produced is also used in several lateral metabolic
pathways originating acetate or butyrate. Since the regeneration of NAD
+ is
required for the glycolysis process, several other by-products namely ethanol and
butanol can be also formed [72]. Therefore, the H 2 production yield is dependent on
the pathway involved. In the case of hydrogen production by Enterobacter spp.,
this process is also performed through the glycolytic pathway, but in these
microorganisms, H 2 can be also produced from formate. In Enterobacter spp., the
pyruvate is not only converted to acetyl-CoA but can also originate formate that is
further oxidized into H 2 and CO 2 by the formate-hydrogen lyase [66, 69].
Although high H 2 production is possible by dark fermentation, this process has
two mains drawbacks: low substrate conversion to H 2 and accumulation of
by-products [65]. Theoretically, the conversion of 1 mol glucose will give 12 mol
H 2 . However, according with the fermentative pathway, the maximum H 2 yield is
restricted to 4 mol H 2 /mol glucose for acetate-type fermentation, 2 mol H 2 /mol
glucose for butyrate and ethanol-type fermentation and 1 mol H 2 /mol glucose for
propionate-type fermentation [64, 66]. When organic wastes are used as substrate,
the BioH 2 yield rarely exceeds 2 mol H 2 /mol hexose [64, 66]. Therefore, several
strategies have been investigated to enhance the H 2 yield, namely metabolic
engineering, development of integrated processes, whole-cell immobilization and
incorporation of metal additives in the bioprocess [64, 65, 69, 74].
Metabolic engineering strategies reported to improve the H 2 production yield
include the construction of innovative metabolic routes and/or enhancing the main
metabolic pathway by overexpressing hydrogen-producing genes and knockout of
competitive pathways [65, 69, 76, 77]. A synthetic pyruvate:H 2 pathway with
additional co-expression of the Bacillus subtilis AmyE was constructed in E. coli
allowing the recombinant strain to became able to produce H 2 from potato starch
[76]. In addition, overexpressing the enzymes directly involved in hydrogen production also resulted in increase in the H 2 production [77]. It was reported the
overexpression of a gene encoding the HydA hydrogenase in Clostridium paraputricum enhanced an increase in the H 2 yield from 1.4 to 2.4 mol H 2 /mol glucose
from rich synthetic wastewaters [77]. Furthermore, the gene coding for a xylose
260
M. Martins et al.