feed rate, and type of substrate, are optimized as per the
requirement of culture used (Manish and Banerjee 2008).
The high H 2 formation at optimum pH 5–6 was reported in
various studies (Das and Veziroǧlu 2001; Bharathiraja et al.
2016; Hallenbeck et al. 2002). However, a few studies
reported between pH 6.8 and 8.0 (Hallenbeck et al. 2002;
Ren et al. 2016). Also, the formation of organic acid along
with H 2 restricts the buffering capacity of the medium
resulting in low pH. For maintaining anaerobic conditions,
N 2 plays an essential role during dark fermentation. Lin and
Lay highlighted that the optimized C/N ratio significantly
enhanced hydrogen production (Lin 2004). Moreover, the
processing parameters and selection of microorganisms used
for bioconversion of lignocellulose feedstock into H 2
majorly depend on biomass composition since the lignocellulose materials from different origins comprise different
percentages of lignin, hemicellulose, and cellulose
(Kucharska et al. 2019). If the percentage of hemicellulose is
high in lignocellulosic materials, it indicates that the cellulose is mainly produced along with glucose, xylose, arabinose, and galactose during the enzymatic hydrolysis. Higher
the percentage of hemicellulose in lignocellulosic materials,
the lower the H 2 production due to the formation of inhibitory compounds, i.e., hydroxymethylfurfural (HMF), and
furfural (Jung et al. 2015). On a laboratory scale, dark fermentation is carried in a batch mode considering the ease of
operation. However, for larger production of H 2 on the
industrial scale, a continuous stirred tank reactor (CSTR) is
required. Though several studies suggested that the efficiency of dark fermentation is significantly high (Nagarajan
et al. 2019; Ren et al. 2016; da Silva Veras et al. 2017; Patil
and Yadav 2018), the overall fermentation is an expensive
operative process (Abreu et al. 2016).
4.4 Integrated Dark and Photo-Fermentative
Sequential Fermentation
In recent studies, researchers successfully demonstrated that
by sequential coupling of dark and photo fermentation, the
productivity of the biomass conversion into hydrogen
increases many folds (Cui and Shen 2012; Wang et al.
2010). The lignocellulosic biomass can be transformed into
volatile fatty acids (VFAs: propionic acid, butyric acid, and
acetic acid) and hydrogen by the action of hydrogenase
enzymes during the dark fermentation process. These VFAs
are then expended in photo fermentation as a substrate and
further converted to hydrogen. Thus, a two-step, sequential fermentation process facilitates the optimization of
parameters while controlling microbial growth conditions to
ease the operation. Typically, the theoretical yield of the first
step receives 2 mol H 2 along with 1 mol of acetate and/or
1 mol H 2 along with 1 mol of butyrate forming due to
incomplete degradation of organic substrates during dark
fermentation. In the second step (photo fermentation), the
dark-fermentative (acetic and butyric acid) intermediates can
be wholly degraded into CO 2 and H 2 by photosynthetic
organisms with a theoretical yield of 4 mol H 2 along with
1 mol of acetate and 10 mol H 2 along with 1 mol of butyrate. In this way, the overall process’s efficiency increases by
utilizing integrating dark and photo fermentation (Eqs. 11
and 12).
CH 3 COOH þ 2H 2 O ! 4H 2 " þ 2CO 2 "
Acetate
ð11Þ
CH 3 CH 2 CH 2 COOH þ 6H 2 O ! 10H 2 " þ 4CO 2 "
Butyrate
ð12Þ
In most of the integrated studies, the dark phase’s optimal
temperature falls in the range of 31–37, and 30 °C was
maintained during the light phase (Lee et al. 2002). Some
studies highlighted the productivity of a two-stage integrated
system employing pure cultures, such as Caldicellulusiruptor
saccharolyticus and Rhodobacter capsulatus; and
Rhodobacter capsulatus hup-mutant and Rhodopseudomonas
palustris in both stages (Yadav et al. 2019). Su et al. carried out
integrated fermentation on Cassava using activated sludge for
dark fermentation followed by photo fermentation using
Rhodobacter sphaeroides and Rhodopseudomonas palustris.
The incubation period for dark and photo fermentation usually
ranged from 1 to 6 days, followed by 5–14 days of
photo-fermentative phase. The yield obtained was 2.86–
6.07 mol of H 2 per mol of hexose (2009). Zagrodnik and
Łaniecki used hybrid culture comprised Clostridium acetobutylicum and Rhodobacter sphaeroides for integrated fermentation process under repeated fed-batch conditions at
optimum pH 7. The results obtained elaborated on the
enhanced H 2 production by 2.5-fold as compared to dark
fermentation at pH > 6.5 (Zagrodnik and Łaniecki 2017). In
another integrated fermentative process, it was found that the
dark fermentation of the acid-pretreated corncob using
anaerobic mixed culture could produce 120 mL of H 2 per g of
biomass. In the second phase of photo fermentation, the
effluents of the first step were completely degraded using
photosynthetic bacteria that produced 713 mL of H 2 per g of
biomass (Yang et al. 2010). Fang et al. utilized co-culture of C.
butyricum and R. sphaeroides with optimal biomass ratio of
1:5.9 for the integrated fermentation process at optimum pH 7
(Fang et al. 2006). Zang et al. developed a coupled solar and
light-emitting diode illumination system and a thermal controlling system for integrated two-step fermentation of
hydrolyzed corn stover. The temperature (35 °C) and pH (4.5)
were maintained in the dark fermentation unit, whereas, in the
photo fermentation unit, pH 7.0 and temperature 30 °C were
70
P. D. Patil et al.
requirement of culture used (Manish and Banerjee 2008).
The high H 2 formation at optimum pH 5–6 was reported in
various studies (Das and Veziroǧlu 2001; Bharathiraja et al.
2016; Hallenbeck et al. 2002). However, a few studies
reported between pH 6.8 and 8.0 (Hallenbeck et al. 2002;
Ren et al. 2016). Also, the formation of organic acid along
with H 2 restricts the buffering capacity of the medium
resulting in low pH. For maintaining anaerobic conditions,
N 2 plays an essential role during dark fermentation. Lin and
Lay highlighted that the optimized C/N ratio significantly
enhanced hydrogen production (Lin 2004). Moreover, the
processing parameters and selection of microorganisms used
for bioconversion of lignocellulose feedstock into H 2
majorly depend on biomass composition since the lignocellulose materials from different origins comprise different
percentages of lignin, hemicellulose, and cellulose
(Kucharska et al. 2019). If the percentage of hemicellulose is
high in lignocellulosic materials, it indicates that the cellulose is mainly produced along with glucose, xylose, arabinose, and galactose during the enzymatic hydrolysis. Higher
the percentage of hemicellulose in lignocellulosic materials,
the lower the H 2 production due to the formation of inhibitory compounds, i.e., hydroxymethylfurfural (HMF), and
furfural (Jung et al. 2015). On a laboratory scale, dark fermentation is carried in a batch mode considering the ease of
operation. However, for larger production of H 2 on the
industrial scale, a continuous stirred tank reactor (CSTR) is
required. Though several studies suggested that the efficiency of dark fermentation is significantly high (Nagarajan
et al. 2019; Ren et al. 2016; da Silva Veras et al. 2017; Patil
and Yadav 2018), the overall fermentation is an expensive
operative process (Abreu et al. 2016).
4.4 Integrated Dark and Photo-Fermentative
Sequential Fermentation
In recent studies, researchers successfully demonstrated that
by sequential coupling of dark and photo fermentation, the
productivity of the biomass conversion into hydrogen
increases many folds (Cui and Shen 2012; Wang et al.
2010). The lignocellulosic biomass can be transformed into
volatile fatty acids (VFAs: propionic acid, butyric acid, and
acetic acid) and hydrogen by the action of hydrogenase
enzymes during the dark fermentation process. These VFAs
are then expended in photo fermentation as a substrate and
further converted to hydrogen. Thus, a two-step, sequential fermentation process facilitates the optimization of
parameters while controlling microbial growth conditions to
ease the operation. Typically, the theoretical yield of the first
step receives 2 mol H 2 along with 1 mol of acetate and/or
1 mol H 2 along with 1 mol of butyrate forming due to
incomplete degradation of organic substrates during dark
fermentation. In the second step (photo fermentation), the
dark-fermentative (acetic and butyric acid) intermediates can
be wholly degraded into CO 2 and H 2 by photosynthetic
organisms with a theoretical yield of 4 mol H 2 along with
1 mol of acetate and 10 mol H 2 along with 1 mol of butyrate. In this way, the overall process’s efficiency increases by
utilizing integrating dark and photo fermentation (Eqs. 11
and 12).
CH 3 COOH þ 2H 2 O ! 4H 2 " þ 2CO 2 "
Acetate
ð11Þ
CH 3 CH 2 CH 2 COOH þ 6H 2 O ! 10H 2 " þ 4CO 2 "
Butyrate
ð12Þ
In most of the integrated studies, the dark phase’s optimal
temperature falls in the range of 31–37, and 30 °C was
maintained during the light phase (Lee et al. 2002). Some
studies highlighted the productivity of a two-stage integrated
system employing pure cultures, such as Caldicellulusiruptor
saccharolyticus and Rhodobacter capsulatus; and
Rhodobacter capsulatus hup-mutant and Rhodopseudomonas
palustris in both stages (Yadav et al. 2019). Su et al. carried out
integrated fermentation on Cassava using activated sludge for
dark fermentation followed by photo fermentation using
Rhodobacter sphaeroides and Rhodopseudomonas palustris.
The incubation period for dark and photo fermentation usually
ranged from 1 to 6 days, followed by 5–14 days of
photo-fermentative phase. The yield obtained was 2.86–
6.07 mol of H 2 per mol of hexose (2009). Zagrodnik and
Łaniecki used hybrid culture comprised Clostridium acetobutylicum and Rhodobacter sphaeroides for integrated fermentation process under repeated fed-batch conditions at
optimum pH 7. The results obtained elaborated on the
enhanced H 2 production by 2.5-fold as compared to dark
fermentation at pH > 6.5 (Zagrodnik and Łaniecki 2017). In
another integrated fermentative process, it was found that the
dark fermentation of the acid-pretreated corncob using
anaerobic mixed culture could produce 120 mL of H 2 per g of
biomass. In the second phase of photo fermentation, the
effluents of the first step were completely degraded using
photosynthetic bacteria that produced 713 mL of H 2 per g of
biomass (Yang et al. 2010). Fang et al. utilized co-culture of C.
butyricum and R. sphaeroides with optimal biomass ratio of
1:5.9 for the integrated fermentation process at optimum pH 7
(Fang et al. 2006). Zang et al. developed a coupled solar and
light-emitting diode illumination system and a thermal controlling system for integrated two-step fermentation of
hydrolyzed corn stover. The temperature (35 °C) and pH (4.5)
were maintained in the dark fermentation unit, whereas, in the
photo fermentation unit, pH 7.0 and temperature 30 °C were
70
P. D. Patil et al.