to increase growth while removing inhibitory compounds
(Zuroff and Curtis 2012). Studies evidently suggested that
the co-culture increases the yield of H 2 , reduces the time of
fermentation, and results in better performance than
mix-culture and pure culture (Nagarajan et al. 2019;
Pachapur et al. 2015). In the co-culture system, cellulolytic
clostridia, i.e., C. cellulolyticum with C. acetobutylicum, has
shown enhanced hydrogen production from biomass compared to pure culture (Salimi and Mahadevan 2013). In
another co-culture system, Citrobacter amalonaticus, a
hydrogen-producing anaerobic bacterium along with C.
cellulolyticum have produced a high yield of hydrogen from
corn stover, i.e., 51.9 L H 2 /kg total solids (Zhang et al.
2016). The co-culture of the thermophilic bacterium has also
been used for the production of hydrogen from biomass. C.
thermocellum is a well-known thermophilic cellulolytic
clostridia, which has the capability of hydrolyzing both
hemicellulose and cellulose (Akinosho et al. 2014). However, it also generates ethanol as a by-product and lowers the
overall yield of H 2 . Islam et al. have used the co-culture of
C. thermocellum and C. thermosaccharolyticum for bioconversion of sweet sorghum stalks to H 2 . At 55 °C and
5 g/L stalks, the H 2 yield of 5.1 mmol H 2 /g substrates along
with 1.05 g/L butyric acid and 1.27 g/L lactic acid was
achieved (Islam et al. 2017). Several other co-culture systems that have been used for the H 2 production from the
lignocellulose are depicted in Table 1.
5 Key Factors Affecting the Hydrogen
Generation
Despite high craving for newer, potential green bio-fuel from
biomass waste, the research is constrained to the laboratory
level. The H 2 production from lignocellulosic biomass on the
industrial scale should be accelerated for sustainable development. For the effective large-scale production of H 2 , the understanding of key factors affecting the process is essential. The
vital key factors influencing the H 2 generation are as follows:
Inoculum Type
Generally, the pure culture of Clostridium and Enterobacter
species has been used in the bioconversion of lignocellulose
to hydrogen. The major disadvantage of the use of pure
inoculum is the maintenance of energy-intensive sterile
conditions during the isolation of strain and fermentation
phase, and thus, it increases the operation cost of the process.
Apart from that, the isolation of strain is time-consuming
and complicated. Hence, the employment of mixed inoculums, such as anaerobically digested sludge, municipal solid
waste, soil, cattle dung compost, sewage sludge, etc., are
preferable (Baskar et al. 2012). Further, it was illustrated that
the co-culture of E. coli (dark fermentative) with R.
sphaeroides (photo-fermentative) effectively enhanced the
production of H 2 (Trchounian et al. 2017).
Temperature
The temperature is a crucial factor since it affects the rate of
substrate utilization, substrate hydrolysis, the maximum
growth rate, H 2 partial pressure, the formation of byproducts,
and the activity of hydrogenase. Mostly the ideal temperature
for dark fermentative hydrogen production was found to be
around 37 °C. Most experimental studies were conducted at
mesophilic temperatures (25–40 °C) to produce H 2 . The
mesophilic temperature condition accelerates the cellulose
degradation rate, which leads to an increase in hydrogen
production. Recently, few studies illustrated that conducting
dark fermentation at thermophilic conditions (mesophilic
thermophilic 40–65 °C or hyperthermophilic >80 °C)
enhanced hydrogen production. However, the use of thermophilic conditions may increase the cost of operation
(Baskar et al. 2012). The degradation of cellulose becomes
sluggish at temperatures below 25 °C or above 50 °C. In
photo fermentation, an increase in temperature in the range of
10–35 °C using solar energy enhances the activity of nitrogenase and proteins responsible for the cell growth resulting
in boosted H 2 production. However, a variation in operating
temperature may cause bacteria to spend their energy to adapt
to the change in temperature results in a reduction in the
hydrogen production efficiency (Tiwari et al. 2020;
Aguilar-Reynosa et al. 2017; Kumar et al. 2009).
pH
The acidic or alkaline nature of reaction mass is a crucial
factor affecting the rate of hydrogen production and formation of by-product. For the efficient activity of the
microorganism, the optimal pH was found to be ranging 5.0–
7.5 (Trchounian et al. 2017; Yadav et al. 2019; Li and Fang
2009). At a higher initial pH, hydrogen production decreased
due to the formation of propionate and ethanol (Gabrielyan
et al. 2015). The low pH reduces the activity of iron-bearing
hydrogenase enzymes, resulting in a decline in hydrogen
yield (Ren et al. 2009).
Nitrogen and Phosphate
N 2 is an essential nutrient for the growth of
hydrogen-producing microorganisms. The various nitrogen
sources are classified as inorganic and organic. Ammonia
nitrogen, ammonium chloride, and ammonium bicarbonates
are examples of inorganic nitrogen sources, while yeast
extract, steep corn liquor, and peptone are the organic
sources. The ammonia nitrogen in the concentration range of
72
P. D. Patil et al.
(Zuroff and Curtis 2012). Studies evidently suggested that
the co-culture increases the yield of H 2 , reduces the time of
fermentation, and results in better performance than
mix-culture and pure culture (Nagarajan et al. 2019;
Pachapur et al. 2015). In the co-culture system, cellulolytic
clostridia, i.e., C. cellulolyticum with C. acetobutylicum, has
shown enhanced hydrogen production from biomass compared to pure culture (Salimi and Mahadevan 2013). In
another co-culture system, Citrobacter amalonaticus, a
hydrogen-producing anaerobic bacterium along with C.
cellulolyticum have produced a high yield of hydrogen from
corn stover, i.e., 51.9 L H 2 /kg total solids (Zhang et al.
2016). The co-culture of the thermophilic bacterium has also
been used for the production of hydrogen from biomass. C.
thermocellum is a well-known thermophilic cellulolytic
clostridia, which has the capability of hydrolyzing both
hemicellulose and cellulose (Akinosho et al. 2014). However, it also generates ethanol as a by-product and lowers the
overall yield of H 2 . Islam et al. have used the co-culture of
C. thermocellum and C. thermosaccharolyticum for bioconversion of sweet sorghum stalks to H 2 . At 55 °C and
5 g/L stalks, the H 2 yield of 5.1 mmol H 2 /g substrates along
with 1.05 g/L butyric acid and 1.27 g/L lactic acid was
achieved (Islam et al. 2017). Several other co-culture systems that have been used for the H 2 production from the
lignocellulose are depicted in Table 1.
5 Key Factors Affecting the Hydrogen
Generation
Despite high craving for newer, potential green bio-fuel from
biomass waste, the research is constrained to the laboratory
level. The H 2 production from lignocellulosic biomass on the
industrial scale should be accelerated for sustainable development. For the effective large-scale production of H 2 , the understanding of key factors affecting the process is essential. The
vital key factors influencing the H 2 generation are as follows:
Inoculum Type
Generally, the pure culture of Clostridium and Enterobacter
species has been used in the bioconversion of lignocellulose
to hydrogen. The major disadvantage of the use of pure
inoculum is the maintenance of energy-intensive sterile
conditions during the isolation of strain and fermentation
phase, and thus, it increases the operation cost of the process.
Apart from that, the isolation of strain is time-consuming
and complicated. Hence, the employment of mixed inoculums, such as anaerobically digested sludge, municipal solid
waste, soil, cattle dung compost, sewage sludge, etc., are
preferable (Baskar et al. 2012). Further, it was illustrated that
the co-culture of E. coli (dark fermentative) with R.
sphaeroides (photo-fermentative) effectively enhanced the
production of H 2 (Trchounian et al. 2017).
Temperature
The temperature is a crucial factor since it affects the rate of
substrate utilization, substrate hydrolysis, the maximum
growth rate, H 2 partial pressure, the formation of byproducts,
and the activity of hydrogenase. Mostly the ideal temperature
for dark fermentative hydrogen production was found to be
around 37 °C. Most experimental studies were conducted at
mesophilic temperatures (25–40 °C) to produce H 2 . The
mesophilic temperature condition accelerates the cellulose
degradation rate, which leads to an increase in hydrogen
production. Recently, few studies illustrated that conducting
dark fermentation at thermophilic conditions (mesophilic
thermophilic 40–65 °C or hyperthermophilic >80 °C)
enhanced hydrogen production. However, the use of thermophilic conditions may increase the cost of operation
(Baskar et al. 2012). The degradation of cellulose becomes
sluggish at temperatures below 25 °C or above 50 °C. In
photo fermentation, an increase in temperature in the range of
10–35 °C using solar energy enhances the activity of nitrogenase and proteins responsible for the cell growth resulting
in boosted H 2 production. However, a variation in operating
temperature may cause bacteria to spend their energy to adapt
to the change in temperature results in a reduction in the
hydrogen production efficiency (Tiwari et al. 2020;
Aguilar-Reynosa et al. 2017; Kumar et al. 2009).
pH
The acidic or alkaline nature of reaction mass is a crucial
factor affecting the rate of hydrogen production and formation of by-product. For the efficient activity of the
microorganism, the optimal pH was found to be ranging 5.0–
7.5 (Trchounian et al. 2017; Yadav et al. 2019; Li and Fang
2009). At a higher initial pH, hydrogen production decreased
due to the formation of propionate and ethanol (Gabrielyan
et al. 2015). The low pH reduces the activity of iron-bearing
hydrogenase enzymes, resulting in a decline in hydrogen
yield (Ren et al. 2009).
Nitrogen and Phosphate
N 2 is an essential nutrient for the growth of
hydrogen-producing microorganisms. The various nitrogen
sources are classified as inorganic and organic. Ammonia
nitrogen, ammonium chloride, and ammonium bicarbonates
are examples of inorganic nitrogen sources, while yeast
extract, steep corn liquor, and peptone are the organic
sources. The ammonia nitrogen in the concentration range of
72
P. D. Patil et al.