isomerase from Thermoanaerobacter ethanolicus was introduced in Clostridium
thermocellum, making the recombinant strain able to ferment simultaneously
xylose, glucose, cellobiose and cellulose [78].
Another strategy that has been studied to improve H 2 production yields is the
development of integrated processes such as combining dark fermentation with
photo-fermentation or with a microbial electrochemical cell (MEC) [69, 74, 79, 80].
In these integrated processes, the by-products produced during dark fermentation
are utilized as substrate by the photo-fermentative organisms, in the case of
photo-fermentation, or by electroactive organisms in MEC, which increases the H 2
yield. Integration of a MEC with dark fermentation was shown to be an efficient
solution to improve H 2 production and substrate bioconversion [79, 80]. A substrate
conversion of 90% could be achieved using this hybrid system [79]. Nevertheless,
the combination of dark and photo-fermentations is more widely studied [64, 69,
71]. In fact, the integrated approach of dark-photo-fermentation has many advantages and allows the maximum conversion of substrate to H 2 (12 mol H 2 /mol
glucose) [64, 81]. The integration of dark and photo-fermentation processes can be
carried out in sequential two-stage or in single stage (co-culture) systems. The
two-stage system has been applied to produce H 2 from several organic wastes [64,
69, 71]. An example is the two-stage process applied for BioH 2 production from
palm oil mill wastewater, which allowed the increase of H 2 production from 0.8 to
3 ml H 2 /ml waste. In this process, the fermentative bacterium Clostridium butyricum and the photo-fermentative organism Rhodopseudomonas palustris were used
[82]. The integrated dark-photo-fermentation was also applied for production of H 2
from molasses with a H 2 yield of 5.65 mol H 2 /mol hexose [83]. High H 2 production
yield was also attained from beet molasses (13.7 mol/mol hexose) when a two-stage
sequential dark-photo-fermentation was applied [81]. The single stage co-culture
reactors provide an alternative solution to maximize H 2 yield in a cost-effective
approach [64]. A co-culture of Clostridium acetobutylicum and Rhodobacter
sphaeroides was used in a fed-batch process to enhance the H 2 production from
starch [84]. This process allowed to reach a H 2 yield of 2.62 mol/mol hexose. In
addition, a higher H 2 yield (5.2 mol/mol glucose) was obtained from starch by a
co-culture Clostridium butyricum and Rhodobacter sphaeroides in a repeated batch
process [85].
Concerning whole-cell immobilization, it was found that immobilized cells are
more effective for hydrogen production than free cells [86]. Immobilization has
several advantages as it can help to reduce by-product inhibition, protect the
microorganism against the adverse impacts of organic wastes and prevent biomass
washout. Clostridium butyricum immobilized in polyethylene glycol was used for
H 2 production from palm oil mill effluent reaching the maximum hydrogen yield of
0.35 L/H 2 /g COD [87]. Clostridium sp. immobilized in fungal mycelia pellets was
used for H 2 production from corn stalk hydrolysate [85]. This strategy led to a
maximum H 2 production of 14 mmol H 2 /L/h which is 2.6 times higher than when
free cells were used. The hydrogen production yield was also improved (3.5 mol
H 2 /mol lactose) when the fermentative organism Enterobacter aerogenes was
immobilized in calcium alginate and used to produce H 2 from cheese whey [88].
Biological Production of Hydrogen
261
thermocellum, making the recombinant strain able to ferment simultaneously
xylose, glucose, cellobiose and cellulose [78].
Another strategy that has been studied to improve H 2 production yields is the
development of integrated processes such as combining dark fermentation with
photo-fermentation or with a microbial electrochemical cell (MEC) [69, 74, 79, 80].
In these integrated processes, the by-products produced during dark fermentation
are utilized as substrate by the photo-fermentative organisms, in the case of
photo-fermentation, or by electroactive organisms in MEC, which increases the H 2
yield. Integration of a MEC with dark fermentation was shown to be an efficient
solution to improve H 2 production and substrate bioconversion [79, 80]. A substrate
conversion of 90% could be achieved using this hybrid system [79]. Nevertheless,
the combination of dark and photo-fermentations is more widely studied [64, 69,
71]. In fact, the integrated approach of dark-photo-fermentation has many advantages and allows the maximum conversion of substrate to H 2 (12 mol H 2 /mol
glucose) [64, 81]. The integration of dark and photo-fermentation processes can be
carried out in sequential two-stage or in single stage (co-culture) systems. The
two-stage system has been applied to produce H 2 from several organic wastes [64,
69, 71]. An example is the two-stage process applied for BioH 2 production from
palm oil mill wastewater, which allowed the increase of H 2 production from 0.8 to
3 ml H 2 /ml waste. In this process, the fermentative bacterium Clostridium butyricum and the photo-fermentative organism Rhodopseudomonas palustris were used
[82]. The integrated dark-photo-fermentation was also applied for production of H 2
from molasses with a H 2 yield of 5.65 mol H 2 /mol hexose [83]. High H 2 production
yield was also attained from beet molasses (13.7 mol/mol hexose) when a two-stage
sequential dark-photo-fermentation was applied [81]. The single stage co-culture
reactors provide an alternative solution to maximize H 2 yield in a cost-effective
approach [64]. A co-culture of Clostridium acetobutylicum and Rhodobacter
sphaeroides was used in a fed-batch process to enhance the H 2 production from
starch [84]. This process allowed to reach a H 2 yield of 2.62 mol/mol hexose. In
addition, a higher H 2 yield (5.2 mol/mol glucose) was obtained from starch by a
co-culture Clostridium butyricum and Rhodobacter sphaeroides in a repeated batch
process [85].
Concerning whole-cell immobilization, it was found that immobilized cells are
more effective for hydrogen production than free cells [86]. Immobilization has
several advantages as it can help to reduce by-product inhibition, protect the
microorganism against the adverse impacts of organic wastes and prevent biomass
washout. Clostridium butyricum immobilized in polyethylene glycol was used for
H 2 production from palm oil mill effluent reaching the maximum hydrogen yield of
0.35 L/H 2 /g COD [87]. Clostridium sp. immobilized in fungal mycelia pellets was
used for H 2 production from corn stalk hydrolysate [85]. This strategy led to a
maximum H 2 production of 14 mmol H 2 /L/h which is 2.6 times higher than when
free cells were used. The hydrogen production yield was also improved (3.5 mol
H 2 /mol lactose) when the fermentative organism Enterobacter aerogenes was
immobilized in calcium alginate and used to produce H 2 from cheese whey [88].
Biological Production of Hydrogen
261