7 Factors Affecting Hydrogen Production
in Dark Fermentation
7.1 Feedstock
Organic feedstocks play a major role in the production of
biohydrogen from dark fermentation methods. Glucose and
sucrose rich feedstock are model substrates for biohydrogen
production (Ghimire et al. 2015). Still, complex substrates
such as municipal solid waste, forestry and agricultural
biomasses (such as dead wood, corn stalks, wheat straw, and
rice straw) and wastages from food processing industries
(e.g., cheese whey, oil mills, and animals dungs) have been
widely used in dark fermentation process to produce
hydrogen (Keskin et al. 2019; Kargi et al. 2012; Mohammadi et al. 2011; Chen et al. 2012).
7.2 pH
pH is a major factor that regulates the enzymatic functions
thereby affecting the metabolic pathway of organisms to
produce hydrogen (Ghimire et al. 2015). In the dark fermentation process, several facultative organisms have been
used to produce hydrogen via glycolysis (Tao et al. 2007).
This enzymatic pathway of the hydrogen production is
highly sensitive to the pH. Tao et al. (2007) reported that
maximum hydrogen yield at medium pH level (pH = 6).
Thus, pH level significantly affects the hydrogenase enzyme
activity. If the medium concentration becomes acidic, pH
level gets reduced which directly shifts enzymatic metabolism towards the conversion of acid into alcohol. At the
lower pH level, hydrogen yield decreases sharply due to the
production of acidic metabolites such as carboxylic acid,
acetic acid, and formic acid. Similarly, Zagrodnik and
Laniecki (2015) reported the reduction of the production of
H 2 with increasing pH level.
7.3 Temperature
Temperature regulates the bacterial growth, rate of biohydrogen production, and microbial metabolisms in anaerobic
fermentation processes. The selection of optimal temperature
and organisms used for the fermentation process depends on
feedstock types. Due to the complexities of the lignocellulosic biomass, there is considerable variation in operating
temperature. Thus, optimal temperature selection is important based on bacteria/organisms used during fermentation.
Organisms (anaerobic bacteria) that have been used for dark
fermentation are classified into different groups (such as
psychrophiles,
mesophiles,
thermophiles,
extreme
thermophiles, and hyperthermophiles) based on the optimal
temperature in which particular organism perform higher
microbial activities and also accelerate the bioconversion
rate of feedstocks (Levin et al. 2004; Alvarado-Cuevas et al.
2015; Boileau et al. 2016). Among them, mesophilic condition (temperature range: 25–45 °C, e.g. Clostridium saccharobutylicum) is the most favorable temperature range for
the fermentative biohydrogen production (Li and Fang
2007). In contrast, thermophilic (45–65 °C) and
extreme-thermophilic (65–80 °C) bacteria can perform
effectively during fermentation of the diversified feedstock
such as buffalo manure, cheese whey, and sludge (Ghimire
et al. 2015; Verhaart et al. 2010; Pakarinen et al. 2008).
However,
biohydrogen
production
from
extreme-thermophilic conditions requires higher energy
input (Hallenbeck 2005).
7.4 Hydrogen Partial Pressure (HPP)
HPP is a pressure created by hydrogen gas inside the reactor
system (Hawkes et al. 2007). When hydrogen started to
accumulate inside the reactor, the partial pressure of
hydrogen increases and subsequently decreases the production of hydrogen. Consequently, metabolic pathway of
hydrogen production shifts and starts to the accumulated
other byproducts such as ethanol, acetone, and lactic acid,
(Ghimire et al. 2015; Hawkes et al. 2007). Lee et al. (2012)
reported that reduction of the partial pressure during the
hydrogen metabolism in dark fermentation increases the
production of H 2 .
7.5 Hydraulic Retention Time
Hydraulic retention time (or fermentation time or hydraulic
loading) is the average number of time (days) that a feedstock remains in a storage unit (digester/bioreactor).
Hydraulic retention time is calculated by dividing bioreactor
volume (gallons) by the feed volume (gal/day) (Kim et al.
2013). Higher hydrogen production is highly correlated with
shorter retention time (Zhang et al. 2013).
8 Conclusion
Lignocellulosic biomass has been extensively used for biohydrogen production. It consists of biopolymer components
such as cellulose, hemicellulose, and lignin. Glucose and
xylose are the final products after the appropriate pretreatment of hemicellulose or lignocellulosic biomass. Different
pretreatment methods, for example, physical, chemical, and
276
J. R. Khatiwada et al.
in Dark Fermentation
7.1 Feedstock
Organic feedstocks play a major role in the production of
biohydrogen from dark fermentation methods. Glucose and
sucrose rich feedstock are model substrates for biohydrogen
production (Ghimire et al. 2015). Still, complex substrates
such as municipal solid waste, forestry and agricultural
biomasses (such as dead wood, corn stalks, wheat straw, and
rice straw) and wastages from food processing industries
(e.g., cheese whey, oil mills, and animals dungs) have been
widely used in dark fermentation process to produce
hydrogen (Keskin et al. 2019; Kargi et al. 2012; Mohammadi et al. 2011; Chen et al. 2012).
7.2 pH
pH is a major factor that regulates the enzymatic functions
thereby affecting the metabolic pathway of organisms to
produce hydrogen (Ghimire et al. 2015). In the dark fermentation process, several facultative organisms have been
used to produce hydrogen via glycolysis (Tao et al. 2007).
This enzymatic pathway of the hydrogen production is
highly sensitive to the pH. Tao et al. (2007) reported that
maximum hydrogen yield at medium pH level (pH = 6).
Thus, pH level significantly affects the hydrogenase enzyme
activity. If the medium concentration becomes acidic, pH
level gets reduced which directly shifts enzymatic metabolism towards the conversion of acid into alcohol. At the
lower pH level, hydrogen yield decreases sharply due to the
production of acidic metabolites such as carboxylic acid,
acetic acid, and formic acid. Similarly, Zagrodnik and
Laniecki (2015) reported the reduction of the production of
H 2 with increasing pH level.
7.3 Temperature
Temperature regulates the bacterial growth, rate of biohydrogen production, and microbial metabolisms in anaerobic
fermentation processes. The selection of optimal temperature
and organisms used for the fermentation process depends on
feedstock types. Due to the complexities of the lignocellulosic biomass, there is considerable variation in operating
temperature. Thus, optimal temperature selection is important based on bacteria/organisms used during fermentation.
Organisms (anaerobic bacteria) that have been used for dark
fermentation are classified into different groups (such as
psychrophiles,
mesophiles,
thermophiles,
extreme
thermophiles, and hyperthermophiles) based on the optimal
temperature in which particular organism perform higher
microbial activities and also accelerate the bioconversion
rate of feedstocks (Levin et al. 2004; Alvarado-Cuevas et al.
2015; Boileau et al. 2016). Among them, mesophilic condition (temperature range: 25–45 °C, e.g. Clostridium saccharobutylicum) is the most favorable temperature range for
the fermentative biohydrogen production (Li and Fang
2007). In contrast, thermophilic (45–65 °C) and
extreme-thermophilic (65–80 °C) bacteria can perform
effectively during fermentation of the diversified feedstock
such as buffalo manure, cheese whey, and sludge (Ghimire
et al. 2015; Verhaart et al. 2010; Pakarinen et al. 2008).
However,
biohydrogen
production
from
extreme-thermophilic conditions requires higher energy
input (Hallenbeck 2005).
7.4 Hydrogen Partial Pressure (HPP)
HPP is a pressure created by hydrogen gas inside the reactor
system (Hawkes et al. 2007). When hydrogen started to
accumulate inside the reactor, the partial pressure of
hydrogen increases and subsequently decreases the production of hydrogen. Consequently, metabolic pathway of
hydrogen production shifts and starts to the accumulated
other byproducts such as ethanol, acetone, and lactic acid,
(Ghimire et al. 2015; Hawkes et al. 2007). Lee et al. (2012)
reported that reduction of the partial pressure during the
hydrogen metabolism in dark fermentation increases the
production of H 2 .
7.5 Hydraulic Retention Time
Hydraulic retention time (or fermentation time or hydraulic
loading) is the average number of time (days) that a feedstock remains in a storage unit (digester/bioreactor).
Hydraulic retention time is calculated by dividing bioreactor
volume (gallons) by the feed volume (gal/day) (Kim et al.
2013). Higher hydrogen production is highly correlated with
shorter retention time (Zhang et al. 2013).
8 Conclusion
Lignocellulosic biomass has been extensively used for biohydrogen production. It consists of biopolymer components
such as cellulose, hemicellulose, and lignin. Glucose and
xylose are the final products after the appropriate pretreatment of hemicellulose or lignocellulosic biomass. Different
pretreatment methods, for example, physical, chemical, and
276
J. R. Khatiwada et al.