production, and feasibility of using a versatile range of organic molecules,
including organic wastes [66]. Therefore, the dark fermentation offers not only an
environmentally friendly way to produce a value-added product but can also contribute to waste recycling. This section reviews the progress of BioH 2 production
through dark fermentation processes using microorganisms as whole-cell catalysts
to produce H 2 from organic wastes and one-carbon substrates.
3.1 Biohydrogen Production from Organic Wastes
From a realistic and economical point of view, dark fermentation is the most
promising approach to produce BioH 2 from industrial wastewaters. Dark fermentation is carried out by anaerobic microorganisms (facultative or obligate) in the
absence of light. The metabolic pathways responsible for fermentative H 2 production are diverse and depend on the microorganism and substrate used. Generally, H 2 is not the only product of the fermentation process, and various other
by-products such as ethanol, acetate, propionate and butyrate will be also originated
according to the pathway involved. In dark fermentation, the electrons produced
from the oxidation of organic substrates are used to reduce protons to BioH 2 . The
reduction process is catalyzed by a hydrogenase, which can be a FeFe- or a
NiFe-hydrogenase [67, 68].
Several organic wastes were investigated as substrates for H 2 production through
dark fermentation namely distillery effluent, beverage wastewater, cheese whey,
palm oil mill wastewater and sugar-rich effluents [69]. In general, wastes contain a
complex mixture of molecules that are first hydrolyzed to hexoses, which are then
metabolized to H 2 . Lignocellulosic biomass is also an attractive substrate for
hydrogen production due to its wide availability. However, this complex material
composed by cellulose, hemicellulose and lignin, requires a pretreatment such as
enzymatic, chemical or thermochemical hydrolysis to release simple sugars that can
be readily used by most H 2 -producing microorganisms [69, 70]. Another interesting
substrate is glycerol that is a by-product of biodiesel producing industries. Glycerol
is not only an abundant and low cost substrate but it also provides more reducing
equivalents than sugars, making it an attractive raw material for the biological
production of different fuels, including H 2 [71].
For the bioconversion of complex substrates like organic wastes to H 2 , it is
essential to find the most suitable microbial catalysts. Many microorganisms have
been reported to be able to produce hydrogen through dark fermentation using
organic wastes as substrate, namely Clostridium butyricum, Clostridium pasteurianum, Enterobacter asburiae, Enterobacter aerogenes, Thermoanaerobium,
Citrobacter spp. and Bacillus spp. [66, 69, 72, 73]. Fermentative organisms can be
divided in two groups according to their catabolism: saccharolytic fermenting
bacteria which ferment the complex sugars such as oligosaccharides, cellulose and
simple sugars, and the proteolytic bacteria that hydrolyze proteins and further
ferment the amino acids [64]. Among all H 2 -producing microorganisms,
Clostridium spp., which are saccharolytic fermenting bacteria, are the most widely
Biological Production of Hydrogen
259
including organic wastes [66]. Therefore, the dark fermentation offers not only an
environmentally friendly way to produce a value-added product but can also contribute to waste recycling. This section reviews the progress of BioH 2 production
through dark fermentation processes using microorganisms as whole-cell catalysts
to produce H 2 from organic wastes and one-carbon substrates.
3.1 Biohydrogen Production from Organic Wastes
From a realistic and economical point of view, dark fermentation is the most
promising approach to produce BioH 2 from industrial wastewaters. Dark fermentation is carried out by anaerobic microorganisms (facultative or obligate) in the
absence of light. The metabolic pathways responsible for fermentative H 2 production are diverse and depend on the microorganism and substrate used. Generally, H 2 is not the only product of the fermentation process, and various other
by-products such as ethanol, acetate, propionate and butyrate will be also originated
according to the pathway involved. In dark fermentation, the electrons produced
from the oxidation of organic substrates are used to reduce protons to BioH 2 . The
reduction process is catalyzed by a hydrogenase, which can be a FeFe- or a
NiFe-hydrogenase [67, 68].
Several organic wastes were investigated as substrates for H 2 production through
dark fermentation namely distillery effluent, beverage wastewater, cheese whey,
palm oil mill wastewater and sugar-rich effluents [69]. In general, wastes contain a
complex mixture of molecules that are first hydrolyzed to hexoses, which are then
metabolized to H 2 . Lignocellulosic biomass is also an attractive substrate for
hydrogen production due to its wide availability. However, this complex material
composed by cellulose, hemicellulose and lignin, requires a pretreatment such as
enzymatic, chemical or thermochemical hydrolysis to release simple sugars that can
be readily used by most H 2 -producing microorganisms [69, 70]. Another interesting
substrate is glycerol that is a by-product of biodiesel producing industries. Glycerol
is not only an abundant and low cost substrate but it also provides more reducing
equivalents than sugars, making it an attractive raw material for the biological
production of different fuels, including H 2 [71].
For the bioconversion of complex substrates like organic wastes to H 2 , it is
essential to find the most suitable microbial catalysts. Many microorganisms have
been reported to be able to produce hydrogen through dark fermentation using
organic wastes as substrate, namely Clostridium butyricum, Clostridium pasteurianum, Enterobacter asburiae, Enterobacter aerogenes, Thermoanaerobium,
Citrobacter spp. and Bacillus spp. [66, 69, 72, 73]. Fermentative organisms can be
divided in two groups according to their catabolism: saccharolytic fermenting
bacteria which ferment the complex sugars such as oligosaccharides, cellulose and
simple sugars, and the proteolytic bacteria that hydrolyze proteins and further
ferment the amino acids [64]. Among all H 2 -producing microorganisms,
Clostridium spp., which are saccharolytic fermenting bacteria, are the most widely
Biological Production of Hydrogen
259