Chem Eng Technol - 2020 - Sarangi - Biohydrogen Production Through Dark Fermentation

Review 601
Prakash K. Sarangi1,*
Biohydrogen Production Through Dark
Sonil Nanda2
Fermentation
Waste organic biomass is regarded as the most suitable renewable source for con-
version to produce biofuels and biochemicals. Owing to its high-energy potential
and abundancy, lignocellulosic biomass can be utilized to produce alternative
energy in the form of gaseous and liquid biofuels. Microbial conversion of waste
biomass is the most successful technology for the generation of biohydrogen
through dark fermentation. Different biological hydrogen production technologies
along with process parameters are described in this review paper with the focus
on dark fermentation. The production of biohydrogen from various substrates is
summarized along with the integrated mode of dark fermentation and photofer-
mentation. Hydrogen generation through biological water-gas shift reaction is also
highlighted.
Keywords: Biohydrogen production, Biomass, Dark fermentation, Microorganisms,

Photofermentation
Received: August 15, 2019; revised: October 16, 2019; accepted: January 07, 2020
DOI: 10.1002/ceat.201900452
1 Introduction anaerobic digestion and fermentation) pathways [8]. Waste

organic biomass or lignocellulosic materials (e.g., agricultural
The major concern about the utilization of global energy is crop residues and woody biomass), food waste, and animal
focused towards renewable and ecofriendly energy sources to manure have gained attention for their potential to produce
replace the exhausting fossil fuels. In the present day, the hydrogen and other hydrocarbon biofuels [9–14].
worldwide focus is on environmental sustainability with clean Hydrogen is an excellent energy carrier and energy vector
energy carriers [1–3]. The traditional sources for the commer- that has found more applications as a gaseous biofuel. It can be
cial production of energy carriers and various chemical-based utilized as a direct fuel, converted to hydrocarbon fuels or elec-
raw materials are exclusively dependent on fossil fuels such as tricity through fuels cells [1, 15, 16]. The use of hydrogen is
petroleum, diesel, coal, and natural gas. Approximately 80 % of possible directly in combustion engines or towards electricity
total energy around the world is completely dependent on fossil generation with the fuel cell technologies [17]. Hydrogen is
fuels [4]. The use of fossil fuels not only increases greenhouse expected to have about 11 % of the total renewable energy share
gas emissions leading to global warming but also releases sever- of 36 % by 2025 and up to 34 % of the total renewable energy
al harmful particulate pollutants, which cause environmental share of 69 % by 2050 [4].
degradation. The interest on hydrogen generation and utilization is gain-
The demand for global primary energy is expected to be ing momentum worldwide as the fuel of the future. Hydrogen
600–1000 EJ by 2050 [5]. However, diversification towards the has the calorific value (higher heating value) of 141 MJ kg–1,
production of fuels and chemicals from renewable sources as a which is the highest of all the known commercial fuels. Consid-
substitute to fossil fuels is one of the biggest challenges across ering the lower heating value (120 MJ kg–1), 1 kg of hydrogen is
the globe [6]. The current world population is 7.7 billion, which equivalent to about 2.75 kg of gasoline [18]. Moreover, hydro-
is estimated to increase by 2.4 billion in the next three decades. gen is an environmentally friendly fuel because its combustion
With the rise in population, the per capita consumption of produces water and heat energy. The fuel properties of hydro-
energy also tends to escalate. Therefore, there is a direct corre- gen and other hydrocarbon fuels are summarized in Tab. 1.
lation between the population growth, energy resource, and
energy utilization (Fig. 1). –
Over the years, there has been significant research on renew- 1
Dr. Prakash K. Sarangi
able energy sources including solar, wind, tidal, geothermal, sarangi77@yahoo.co.in
and organic biomass. Among all, organic waste biomass is the Directorate of Research, Central Agricultural University, Imphal, Man-
only source that can provide biofuels with high energy content ipur, India.
to potentially substitute fossil fuels [7]. Biofuels can be pro- 2
Dr. Sonil Nanda
duced from organic wastes through thermochemical (e.g., Department of Chemical and Biological Engineering, University of
pyrolysis, liquefaction, and gasification) and biochemical (e.g., Saskatchewan, Saskatoon, Saskatchewan, Canada.
Chem. Eng. Technol. 2020, 43, No. 4, 601–612 ª 2020 WILEY-VCH Verlag GmbH & Co. KGaA www.cet-journal.com
15214125, 2020, 4, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ceat.201900452 by Readcube (Labtiva Inc.), Wiley Online Library on [07/11/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Review 602
2 Hydrogen Production Technologies

Presently, the industrial-scale hydrogen generation is mainly
dependent on natural gas reforming and other conversion
routes of fossil-based products, which are relatively cost-effi-
cient yet polluting [20]. Other methods for hydrogen produc-
tion include water electrolysis, photocatalysis, thermochemical
processes (e.g., gasification and pyrolysis), biological processes
(dark fermentation and photofermentation), photo-electro-
chemical processes, and photochemical methods [15, 16, 21].
Different technologies employed for hydrogen production
Figure 1. Correlation between population growth, energy along with sources are illustrated in Fig. 2.
resources, and energy utilization.
The direct usage of hydrogen as a fuel has several

safety limitations, which are related to explosive-
ness when reacted with oxygen under uncontrolled
conditions. Because of the low boiling point,
hydrogen storage is also difficult as far as other
fuels are concerned [16]. Therefore, hydrogen stor-
age in specialized vessels with insulated pressure is
preferred. The storage of hydrogen is made possi-
ble safely in form of metal hydrides [19].
Apart from being used as an energy carrier,
hydrogen has other promising applications, such as
aniline synthesis from nitrobenzene, synthesis of
methanol, hydrogenation of fats, and ammonia
synthesis [15]. This review paper describes the pro-
duction technologies of biohydrogen with special
focus on dark fermentation. The advanced technol-
ogies and utilization of different microorganisms
along with the challenges and future possibilities of
dark fermentation are thoroughly portrayed.
Figure 2. Comparison of different technologies for hydrogen production.
Table 1. Fuel properties of hydrogen and other hydrocarbons.
Characteristics Hydrogen Methane Gasoline Butanol Ethanol Methanol
Formula H2 CH4 H, C4–C12 C4H9OH CH3CH2OH CH3OH

–1
Molecular weight [kg kmol ] 2.02 16.04 100–105 74.12 46.07 32.04
Boiling point [C] –253 –162 32–210 118 78 65
Auto-ignition temperature [C] 585 540 280 343 365 435

–1
Energy density [MJ kg ] 120–142 50–55.5 44.5 33.1 26.9 19.6
Air/fuel ratio 34.3 17.19 14.6 11.2 9.0 6.5
Research octane number > 130 120 91–99 96 129 136
Motor octane number – 120 81–89 78 102 104
Viscosity at 25 C [mPa s] 0.009 0.011 0.6 2.573 1.074 0.544

–1
Higher heating value [kJ g ] 141.9 55.5 47.5 37.3 29.7 20
Flash point [C] < –253 –188 –43 28.9 13 11
Review 603
Various biomass-to-gas (BTG) conversion technologies can membrane to the cathode side and an outer external circuit.
be implemented for hydrogen generation based on the type of Inside the cathode, electrons and protons are chemically
precursor used. Thermochemical conversion is generally reduced to form hydrogen gas in the presence of a catalyst
explored when fossil fuels are used, whereas alternative tech- [35]. This technology is like a microbial fuel cell consisting of
nologies like gasification, fermentation or electrolysis can be double compartments of anode and cathode, which are sepa-
employed for renewable sources to produce hydrogen. Com- rated by a proton exchange membrane (PEM).
pared to steam reforming technologies, lignocellulosic biomass, Another way of biohydrogen production is photofermenta-
petrochemical feedstocks, and sewage sludge with high organic tion, which is accomplished by the help of photosynthetic bac-
content can be directly converted to hydrogen through thermo- teria that utilize nitrogenase enzyme in the presence of a light
chemical methods, especially gasification (also including source. Purple non-sulfur bacteria with the nitrogenase enzyme
hydrothermal gasification). However, thermochemical BTG system facilitate the photofermentation process. These bacteria
technologies have a faster rate of conversion but are less selec- can utilize reduced organic acids as the basic carbon source and
tive and require high temperature inputs compared to the com- sunlight to release molecular hydrogen by means of the nitroge-
mercial steam reforming technologies [22–24]. nase enzyme system [36]. The implementation of light-harvest-
The electrochemical methods are extensively used to gener- ing pigments like chlorophylls, phycobilins, and carotenoids
ate hydrogen through the electrolytic conversion of water. Such help in harvesting solar energy, thereby dissociating water into
methods are expensive due to high maintenance costs relating electrons, protons, and oxygen during photofermentation
to the highly corrosive reaction environments. On the other (Fig. 3). The catalytic function of nitrogenase aids the reaction
hand, biochemical methods involving microorganisms and of protons and electrons along with nitrogen and adenosine tri-
their enzymes seem to be suitable for feedstocks with high phosphate (ATP) to generate ammonia, hydrogen, adenosine
water content and less crystalline cellulose. Biological hydrogen triphosphate (ADP), and inorganic phosphates (Pi) [37]. Light
production technologies are more selective towards hydrogen energy and biomass by the assistance of the bacterial photosys-
but have low productivities and long reaction times. However, tem produce two electrons and four ATP molecules, thereby
among all methods, biochemical hydrogen-producing technol- generating hydrogen using the nitrogenase enzyme system.
ogies are environmentally friendly and less energy-intensive,
which makes them the key topic of this review.
3 Biochemical Methods for Hydrogen

Production
The biochemical production of hydrogen is recognized to be
beneficial when the issues relating to the environment and
energy requirements are concerned [25]. The types of microor-
ganism and feedstocks used have great roles in the biological
conversion of biomass to hydrogen. The biochemical hydrogen
production technologies broadly include photolysis, dark fer-
mentation, photofermentation, and microbial electrolysis cells.
Lignocellulosic biomass is regarded as the potential resources
for biohydrogen production due to its low-cost availability, Figure 3. Schematic diagram of the photofermentation process
for biohydrogen formation.
geographical abundance, renewable nature, and greater
amounts of carbohydrate contents [26]. Several authors have
extensively reported the fermentative production of biohydro- Dark fermentation operates in the absence of sunlight by
gen from waste biomass [26–31]. Prior to fermentation, ligno- utilizing microbial resources to produce hydrogen from waste
cellulosic biomass and most other organic wastes require biomass. It is advantageous over photofermentation in being
certain pretreatments involving acids, alkalis, and enzymes to inexpensive, requiring less maintenance, no requirement of
depolymerize lignin and release cellulose and hemicellulose as luminescence or light source, and smaller bioreactor necessi-
the fermentable sugars [32, 33]. ties. In the complete absence of light, anaerobic bacteria like
The production of hydrogen by microbial electrolysis cells Enterobacter, Bacillus, and Clostridium convert cellulosic sub-
(MECs) are considered as a new method for harnessing energy strates to produce hydrogen [38]. On the other hand, syngas
and protons utilizing microbial resources through the conver- rich in carbon monoxide (CO) released from gasification of
sion of organic matter. It possesses a remarkable potential for biomass is regarded as a valuable reactant towards the conver-
the generation of hydrogen as well as other value-added chemi- sion to hydrogen [39, 40].
cals such as methane, formic acid, and hydrogen peroxide Although having toxicity effects, CO is regarded as a new
using wastewater as the feedstock, thereby aiding in environ- and potential source to produce hydrogen through biological
mental remediation [34]. In this method, the oxidation of water-gas shift reaction as defined in Eq. (1):
organic matter is accomplished by releasing protons (H+) and
electrons (e–), which are then transferred through the CO þ H2 O fi CO2 þ H2 DG0 ¼ 20:1 kJ mol1 (1)
Review 604
During the biological water-gas shift reaction, the production Photolysis of water into biohydrogen is also accomplished by
of hydrogen through the reaction of water and CO is possible direct biophotolysis and indirect biophotolysis. During direct
with less energy requirements and under ambient conditions biophotolysis, solar energy is converted into chemical energy
[41]. The energy for such reaction is obtained through the elec- by the help of a photosystem (PSI and PSII) of certain algal
trochemical reactions during the transfer of electrons from CO biomass like Anabaena sp. and Chlamydomonas reinhardtii,
to water. Nevertheless, the conversion of CO to H2 requires the which later supports the cracking of water to biohydrogen
action of two types of enzymes, such as carbon monoxide (Eq. (2)). On the other hand, in the indirect method of biopho-
dehydrogenase (CODH) and CODH-dependent hydrogenase tolysis, the catalytic action of hydrogenase and nitrogenase
[42]. The carbon monoxide dehydrogenase enzyme converts enzymes helps in the production of biohydrogen from water
CO to CO2 through oxidation. On the other hand, the second (Eq. (3)).
enzyme reduces protons to hydrogen by the help of previously
released electrons in the oxidation step. Purple non-sulfur pho- 2H2 O þ light fi 2H2 þ O2 (2)
tosynthetic bacteria and anaerobic hydrogenogenic bacteria are
involved in the biological waste-gas shift reaction in the absence 6H2 O þ 6CO2 fi C6 H12 O6 þ 6O2 (3)
of a light source and under simple growth conditions [38].
Hydrogen generation through biological water-gas shift reac-
C6 H12 O6 þ 6H2 O fi 12H2 þ 6CO2 (4)
tion from CO was studied by many workers using Rhodopseu-
domonas palustris PT, Caldicellulosiruptor saccharolyticus, and
During photofermentation, the production of biohydrogen
Petrobacter succinatimandens [39, 43–45]. Kumar et al. [46]
occurs in the absence of oxygen but in the presence of light by
compared a single microorganism and a consortium of micro-
the help of photoheterotrophic bacteria. This process is deter-
organisms as far their CO conversion to H2 is concerned. The
mined with the activity of photosynthetic bacteria in the pres-
consortium like anaerobic granular sludge biomass is suitable
ence of ATP-dependent nitrogenase (Eq. (5)):
to overcome the inhibitory effects, thereby facilitating a higher
yield of hydrogen [47]. Fan et al. [48] studied the effects of sub- CH3 COOH þ 2H2 O fi 2 CO2 þ 4H2 (5)
strate concentration and product inhibition. Analyzing the
kinetics of the biological water-gas shift process can shed more Another way for production of biohydrogen is a two-stage
light in understanding and enhancing the hydrogen yields [49]. dark and photofermentation process, which seems to be more
There is increasing interest towards production of biohydro- beneficial than dark and photofermentation operating sepa-
gen through dark fermentation by microbial communities. rately. As both the processes are employed to produce bio-
Many microbial species are involved in conversion of waste hydrogen, the organic acids produced during initial dark
biomass to hydrogen through the dark fermentation process, fermentation can be employed as the substrate producing bio-
which belong to mainly mesophilic and thermophilic groups hydrogen and CO2 by using photoheterotrophic bacteria. By
(Fig. 4). These microorganisms are either obligate or facultative such a technology, the yield of biohydrogen can be enhanced
anaerobes. Instead of utilizing a pure culture, the biocatalytic theoretically up to 12 mol H2 per mole of hexose sugar, which
action of mixed consortia provides efficient conversion of is much more than an individual process. The two-stage fer-
organic wastes to biohydrogen. mentation involves the following reaction combining both dark
and photofermentation:
Stage 1 (dark fermentation by certain anaerobic
bacteria):
C6 H12 O6 þ 2H2 O fi 2CH3 COOH þ 2CO2 þ 4H2

(6)
Stage 2 (photofermentation by photoheterotro-

phic bacteria):
CH3 COOH þ 2H2 O fi 2CO2 þ 4H2 (7)
4 Dark Fermentation Process

The two important methods for biohydrogen pro-
duction include photofermentation and dark fer-
mentation, both of which utilize microbial resour-
ces. In the presence of light, the production of
hydrogen is accomplished by various photosyn-
Figure 4. Microbial communities involved in biohydrogen production through thetic microorganisms such as protists and bacteria
dark fermentation. [50]. On the other hand, dark fermentation
Review 605
operates in the complete absence of light by hydrogen-generat-

ing microorganisms like facultative anaerobes and obligate
anaerobes. Compared to photofermentation, dark fermentation
is regarded as the most promising method for hydrogen pro-
duction. During fermentation, microorganisms, mainly bacte-
ria, convert organic waste matter to produce hydrogen. As
mentioned earlier, dark fermentation requires organic sub-
strates including lignocellulosic biomass. Other raw materials
having a high fraction of carbohydrates such as sugar and
starch-containing crops, organic residues from municipal solid
wastes and wastewater from food industries and industrial ef-
fluents can also be potential feedstocks for dark fermentation
[51]. Fig. 5 illustrates the different steps during a typical dark
fermentation process.
During dark fermentation, the efficiency for biohydrogen
generation is greatly affected by biomass pretreatment meth-
Figure 6. Formation of products during dark fermentation.
ods, sugar contents in the substrate, and microorganisms used
[52, 53]. Depending on the metabolic pathway of the microor-
ganisms and initial sugar concentration in the fermentation 7C6 H12 O6 þ 6H2 O fi 24H2 þ 6CH3 CH2 CH2 COOH þ 18CO2
medium, the theoretical yield of hydrogen can be estimated.
(9)
The three thermodynamically favored dark fermentation meta-
bolic pathways for the conversion of organic substrates to bio-
hydrogen are: (i) hexose to acetic acid, (ii) hexose to butyric C6 H12 O6 þ 4H2 O fi 2CH3 COO þ 2HCO þ
3 þ 4H þ 4H2
acid, and (iii) acetate to ethanol [54, 55]. Acetate-propionate (10)
pathways have also been reported, but they are not promising
for hydrogen production [56]. In contrast to the acetate-buty-
C6 H12 O6 þ 12H2 O fi 6HCO
3 þ 12H2 þ 6H
þ
(11)
rate metabolic pathway, more stability is detected in the ace-
tate-ethanol pathway for hydrogen production [55, 56].
Due to positive standard Gibbs free energy, no microorgan-
During dark fermentation, a mixed gas containing H2 and
ism or its consortium has been reported that leads to complete
CO2 is produced with other trace gases such as CH4, CO, and
bioconversion of the substrate during dark fermentation to
H2S based on the type of microorganisms and substrate
hydrogen. Nevertheless, the highest theoretical yields of biohy-
[57–60]. Through the glycolytic pathways, bacteria can convert
drogen are detected with acetate as the end product. On the
glucose to pyruvic acid by simultaneously producing ATP
other hand, the mixture of acetic acid and butyric acid as the
from ADP and NADH. Pyruvic acid is further converted to
end products can also fetch maximum hydrogen generation
CO2 and H2 by the help of pyruvate ferredoxin oxidoreductase
despite other fermentation products like alcohols and lactic
and hydrogenase (Fig. 6). The conversion of pyruvate to
acid in which the concentration of hydrogen is detected in low
acetyl-CoA and subsequently to acetate, butyrate, and ethanol
level.
can determine the production level of biohydrogen. The
Tab. 2 summarizes a few notable microorganisms and the
ratio of butyrate-to-acetate can decide the availability of hydro-
organic substrates used for hydrogen production form dark fer-
gen from glucose. Hydrogen can be produced from acetic
mentation. Hydrogen generation by bacterial communities
acid (Eq. (8)) and butyric acid (Eq. (9)) [61, 62]. Hydrogen
includes anaerobes, e.g., Clostridia, methanogenic bacteria, and
production from glucose is also represented in Eq. (10) and
archaea, facultative anaerobes, e.g., Enterobacter, Escherichia
Eq. (11).
coli, and Citrobacter, and some aerobes, e.g., Alcaligenes and
C6 H12 O6 þ 2H2 O fi 4H2 þ 2CH3 COOH þ 2CO2 (8) Bacillus. Some other bacterial species helping in hydrogen pro-
duction belong to the group of Bacillaceae, Gram-positive coc-
ci, e.g., Micrococcaceae and Peptococcaceae, Gram-positive
asporogenous rod-shaped bacteria, e.g., Lactobacil-
lus, Gram-negative facultative anaerobic rod-
shaped bacteria, e.g., Enterobacteria and Vibriona-
ceae, and anaerobic cocci, e.g., Veillonellaceae.
4.1 Utilization of By-products from Dark

Fermentation
The percentage of biomass conversion leading to

Figure 5. Schematic diagram of the dark fermentation process for biohydrogen poor yield is a major bottleneck hampering the com-
formation. mercial value of biohydrogen [82]. Standardization
Review 606
Table 2. Microorganisms employed in dark fermentation for biohydrogen production.
Microorganisms Substrates Hydrogen yield References
Caldicellulosiruptor saccharolyticus DSM 8903 Hydrolyzed potato peels 3.4 mol mol–1 Mars et al. [63]
–1
Clostridium butyricum Sugarcane bagasse hydrolysate 1.73 mol mol Pattra et al. [64]
–1
Clostridium saccharoperbutylacetonicum Cheese whey 7.89 mmol g Ferchichi et al. [65]
–1
Clostridium thermocellum Wood fibers 1.47 mol mol Levin et al. [66]
–1
Clostridium thermocellum ATCC 27405 Sugarcane bagasse 109.6 mL g Tian et al. [67]
–1
Clostridium thermocellum 7072 Corn stalk 1.2 mol mol Cheng and Liu [68]
–1
Clostridium thermolacticum DSM 2910 Lactose 1.5 mol mol Collet et al. [69]
–1
Clostridium thermopalmarium DSM 5974 and Cellulose 1.36 mol mol Geng et al. [70]
Clostridium thermocellum DSM 1237
Enterobacter aerogenes Glycerol 172.9 mL g–1 Chookaew et al. [71]

–1
Enterobacter aerogenes strain HO-39 Arabinose 120.9 mL g Ren et al. [72]
–1
Enterobacter aerogenes strain HO-39 Fructose 121.9 mL g Oh et al. [73]
–1
Enterobacter aerogenes strain HO-39 Galactose 118.2 mL g Ren et al. [72]
–1
Enterobacter aerogenes strain HO-39 Glucose 124.5 mL g Yokoi et al. [74]
–1
Enterobacter aerogenes strain HO-39 Lactose 37.8 mL g Syahrial and Nomura [75]
–1
Enterobacter aerogenes strain HO-39 Maltose 140.7 mL g Hendriks and Zeeman [76]
–1
Enterobacter aerogenes strain HO-39 Mannitol 206.8 mL g Ren et al. [72]
–1
Enterobacter aerogenes strain HO-39 Mannose 121.9 mL g Ren et al. [72]
–1
Enterobacter aerogenes strain HO-39 Rhamnose 69.7 mL g Ren et al. [72]
–1
Enterobacter aerogenes strain HO-39 Sucrose 109.4 mL g Han et al. [77]
–1
Enterobacter aerogenes strain HO-39 Xylose 117.9 mL g Quéméneur et al. [78]
–1
Thermoanaerobacterium thermosaccharolyticum W16 Corn stover 2.24 mol mol Cao et al. [79]
–1
Thermotoga neapolitana Rice straw 68.2 mL g Nguyen et al. [80]
–1
Rumicococcus albus Sweet sorghum residues 2.59 mol mol Ntaikou et al. [81]
–1
Thermotoga neapolitana DSM 4349 Hydrolyzed potato peels 3.3 mol mol Mars et al. [63]
of various process parameters along with suitable feedstock, the released organic acids, thereby producing hydrogen and
microorganisms, and bioreactor design has a strong influence on methane as the end products through several methods (Fig. 7).
hydrogen productivity. The yield of hydrogen has never been The potential of purple non-sulfur bacteria is considered due
achieved above 4 moles per hexose molecule through the dark to their ability to convert dark fermentation by-products like
fermentation process because a maximum yield of 33 % (on sug- organic acids to hydrogen.
ars) is the theoretical biohydrogen production [83]. After the dark fermentation, the products containing organic
The release of many by-products from dark fermentation acids have been investigated by many researchers using the
can be the potential substrates for subsequent conversion to integrated mode like dark fermentation and photofermentation
hydrogen by other methods, which, on the other hand, can lead (DF-PF) [51, 84, 85]. The production of various valued-added
to the complete conversion of biomass with an enhanced platform chemicals through dark fermentation attracts atten-
hydrogen production. Hence, the adoption of integrated pro- tion towards higher yields of hydrogen through the integrated
cesses for utilizing by-products, especially organic acids, is a DF-PF pathway. Such method not only provides complete con-
promising approach to accomplish near-complete conversion version of biomass but also supplies hydrogen in a fruitful
of the organic biomass and reducing waste generation. In such manner towards sustainability. Such integration of both dark
consolidated bioconversion systems for hydrogen generation, fermentation and photofermentation is defined in Eqs. (12)
the first stage comprises of dark fermentation, which converts and (13). Depending on the different parameters, various prod-
carbohydrates to organic acids, and the second stage utilizes ucts can also be generated from such integrated pathways
Review 607
process parameters during the up-

stream and downstream processing as
well as fermentation are concerned.
Such parameters are temperature, pH,
hydraulic retention time, volatile fatty
acids, partial pressure of H2/CO2, and
inorganic content, which affects the an-
aerobic hydrogen fermentation process.
The bacterial growth and metabolic
activities as well as hydrogen produc-
tion rate are directly dependent on the
process temperature. Hydrogen genera-
tion through dark fermentation in-
cludes various ranges of bacteria like
mesophilic range (25–40 C), thermo-
philic range (40–65 C), extreme
thermophilic range (65–80 C), and hy-
perthermophilic range (> 80 C). In
general, dark fermentation processes
Figure 7. Integration of dark fermentation with other processes towards H2 and CH4 produc-
tion. are suitable at a temperature range of
35–55 C. Biohydrogen production is
enhanced by bacterial species under
based on the nature of substrates as indicated in Eqs. (14)–(16) high thermophilic conditions when compared to mesophilic
[86, 87]. conditions [92]. A maximum hydrogen yield of 4 moles per
Dark fermentation: mole glucose is detected at extremely thermophilic condition
(70 C) [93]. On the other hand, the production of hydrogen is
C6 H12 O6 þ 2H2 O fi 2CH3 COOH þ 2CO2 þ 4H2 (12) reduced to 1 mole under mesophilic and less than 2 moles
under thermophilic conditions per mole glucose.
Photofermentation: A high range of temperature during fermentation also
restricts the growth of pathogenic bacteria [94]. At extremely
Light energy
CH3 COOH þ 2H2 O ! 4H2 þ 2CO2 (13) high temperature, there is less chance of contamination with
methanogens and solventogens as well. The enhancement of
Lactic acid: tolerance to an increasing range of hydrogen partial pressures
has been detected in extremely thermophilic bacterial species,
C3 H6 O3 þ 3H2 O fi 6H2 þ 3CO2 (14) thereby shifting the metabolic process towards the non-hydro-
gen-generating pathways such as solvent production [95].
Propionic acid: According to Hallenbeck [96], dark fermentation at high tem-
peratures is regarded thermodynamically positive for hydrogen
C3 H6 O2 þ 4H2 O fi 7H2 þ 3CO2 (15) production as the rise in temperature increases the level of
entropy, which results in the fermentation to be more energetic.
Butyric acid: Another process parameter such as pH level has also a poten-
tial effect towards enhancing biohydrogen production. The
C4 H8 O2 þ 6H2 O fi 10H2 þ 4CO2 (16)
enzymatic activity of microorganisms during the bioconversion
process works well at a specific range of pH. The pH level of
Redwood et al. [88] described different combinations of pro-
5.5 is found to be optimal for biohydrogen production as
cesses for the utilization of dark fermentation by-products.
reported by most researchers [83, 97, 98].
Special consideration is given to the following factors during
Another important parameter for biohydrogen production
the integration of DF-PF pathways such as: (i) simple adoption
by the dark fermentation process is the hydraulic retention
of photofermentation after dark fermentation, (ii) cultivation
time (HRT). In a typical continuous stirred-tank reactor
of different microorganisms for dark fermentation and photo-
(CSTR) system, short HRTs are used to clean out the methano-
fermentation in a single reactor, and (iii) separation of two fer-
gens by selecting acid-producing bacteria [99]. According to
mentation processes by a membrane [89–91].
Kim et al. [100], in a CSTR system, a short HRT of less than
3 days could facilitate hydrogen production. Sometimes, com-
bined effects of pH and HRT are also detected for hydrogen
4.2 Parameters Affecting Dark Fermentation
production. In general, during an anaerobic process, a short
HRT can result in a low pH level [73]. On the other hand,
Though the production of biohydrogen has many potentials to-
wards the mitigation of future energy crises as an alternative hydrolysis of organic wastes is also affected by the dilution rate
fuel, there are certain challenges in its production as far as the [101].
Review 608
Another factor like the partial pressure of hydrogen inside terials during the pretreatment to hydrogen production can
the bioreactor can also determine the hydrogen production. bring efficiency to the process. Nanotechnology in dark fer-
When the partial pressure decreases inside the bioreactor, it mentation is attracting attention due to its enhancement of
increases the transfer of hydrogen changing liquid to gas phase microbial activity, thereby facilitating the hydrogen-yielding
[102, 103]. There is reversible oxidation-reduction of ferredox- process.
in, which influences the activity of hydrogenase. Thus, due to Some metal cofactors in their ionic form of iron, nickel, and
increased concentration of hydrogen in the liquid phase, the potassium using nanomaterials demonstrated promising effects
oxidation of ferredoxin becomes unfavorable, thereby reducing in microbial activity [124, 125]. Various parameters like hydro-
ferredoxin [104], and finally clears the way for biohydrogen gen yield and productivity showed advanced activity with the
production. use of nanomaterials [126]. Viability of microbial cells and
Biomass hydrolysis and pretreatment influence the hydrogen improved hydrogenase activity towards hydrogen generation
production during the dark fermentation of lignocellulosic sub- using different types of nanostructured materials, metal oxide
strates [105]. Prior to fermentation, pretreatment of biomass is nanoparticles, nanocomposites, and graphene-based nanoma-
necessary to degrade the lignin, decrease the crystallinity of cel- terials can be considered as novel approaches for efficient
lulosic, and release fermentable sugars. A reduction in the crys- utilization of cellulosic biomass. Many promising properties
tallinity of cellulose increases the surface area, thereby improv- like high adsorption capacity and catalytic efficiency are
ing the separation of lignin and hemicellulose [106]. This detected in nanomaterial-assisted biofuel bioproduction [127].
makes it easier for microorganisms and their enzymes to access More research on nanotechnology can improve the efficiency
the cellulosic fibers for fermentation [107]. In addition, the in dark fermentation, thereby directing towards more hydrogen
removal of lignin is necessary because it creates a restriction for yield.
the enzymes to access the cellulose and hemicellulose. More-
over, lignin releases certain inhibitory compounds such as
furfural and hydroxymethylfurfural, which are inhibitory to 5 Outlook and Future Perspectives
microorganisms during fermentation [33]. Hence, pretreat-
ment not only provides more cellulosic sugars for fermentation The production of biohydrogen has great potential as far as the
but also results in a high yield of hydrogen through enhanced clean energy crisis is concerned. As discussed earlier, hydrogen
dark fermentation. is gaining more interest in research and practical applications
Various chemical, physical, and biological pretreatment worldwide because of its clean burning nature and high energy
methods such as acidic, alkaline, and ultrasonication methods output. The use of hydrogen satisfies one of the many goals of
have been used for enriching biohydrogen-producing bacteria the United Nations Sustainable Development Goals relating to
[108–111]. Better yields of hydrogen can be achieved through renewable energy [128]. There is an increasing interest towards
greater availability of cellulosic sugars, which is possible biohydrogen production through dark fermentation owing to
through the combination of different pretreatment methods its thriving potential in industrial use for energy utilization.
such as physical, chemical, and biological procedures [76, 112– The production of hydrogen can be accomplished by differ-
114]. Many authors have studied the applications of several ent modes like biological and thermochemical methods by uti-
pretreatment methods for fermentative hydrogen production lizing biomass as a green substrate. As far as the commercial
[115–118]. production of hydrogen is concerned in the future, biohydro-
The availability and concentrations of some nutrients have gen can be considered as a promising option compared to oth-
also a great influence on biohydrogen generation through dark er modes of hydrogen production, e.g., reforming of gaseous
fermentation because the bacterial metabolic activity largely fossil fuels. In this context, dark fermentation provides an opti-
depends on the type and concentration of essential nutrients. mal platform for biohydrogen production by utilizing micro-
Some of the essential nutrients are nitrogen, phosphate, metal organisms and waste organic residues. However, biological
ions, and other micronutrients. The nutrients not only control hydrogen production faces many challenges to be economically
the enzymatic activities but also manage the microbial growth, efficient for maximum hydrogen recovery.
thereby affecting hydrogen production. Supplementing Various constraints such as exploration of cheaper raw mate-
nutrients in a microbial fermentation medium can help in the rials, appropriate pretreatment methods, bioreactor develop-
conversion of cellulosic biomass to hydrogen by enhancing the ment, parameter standardization as well as process modeling
microbial activities [119–121]. The presence of heavy metals in and simulation can be addressed for achieving a successful dark
the fermentation medium or in the heterogeneous feedstocks, fermentation process with maximum biohydrogen recovery. In
e.g., municipal solid wastes, industrial effluents, or certain lig- addition, the combination of dark fermentation and photofer-
nocellulosic biomass, can be toxic to microorganisms and mentation can lead to biohydrogen production with higher effi-
inhibit dark fermentation. Some of such toxic heavy metals ciencies than the individual process.
include cadmium, chromium, zinc, copper, nickel, and lead Waste lignocellulosic biomass requires different pretreatment
[122, 123]. technologies for enhancing biohydrogen production through
Bioconversion of lignocellulosic biomass to hydrogen mainly dark fermentation by reducing the crystallinity of cellulosic
depends on the availability of cellulosic portions and the effi- and enhancing the access of microbial enzymes to ferment the
ciency of enzymes. The potential of nanotechnology in dark sugars leading to biohydrogen production [129]. Various pre-
fermentation for biohydrogen production is considered as one treatment processes involving physical, chemicals and biologi-
of the most emerging strategies. Supplementing some nanoma- cal agents have been adapted for biomass pretreatment prior to
Review 609
biohydrogen production [130]. In addition, pretreatment of sludge bed reactor [145], and rhomboidal reactor [146].
inoculum also has the potential towards net biohydrogen pro- Among these, the multilayered photobioreactor has shown uni-
duction to destroy the hydrogen-consuming bacteria like form light distribution of the energy source for photosynthetic
hydrogenotrophic methanogens, homoacetogens, propionate microorganisms [140]. The major problem of the gas space that
producers, and sulfate-reducing bacteria [131–133]. decreases the working volume is partially overcome in the
During dark fermentation for biohydrogen production, rhomboidal bioreactor [146].
organic substrates are converted into volatile fatty acids and The production of electricity from biohydrogen exhibited
alcohols by anaerobic bacteria. The produced metabolites may more benefits over fossil fuels as far as environmental sustain-
be focused as substrates for further conversion into hydrogen ability is concerned over life cycle assessment (LCA) studies
by purple non-sulfur bacteria through photofermentation, fur- [147]. Djomo and Blumberga [148] made a comparative study
ther supplementing the biohydrogen yield and thus lowering on the energetic and environmental impacts of hydrogen pro-
the chemical oxygen demand (COD). Hence, designing a two- duction from various biomasses like wheat straw, sweet sor-
stage fermentation process by optimization of the cultural con- ghum stalk, and steam potato peels. According to their studies,
ditions and employing competent microorganisms can be a comparable energy ratios were found to be 1.08 for wheat
possible option for greater biohydrogen production and recov- straw, 1.14 for sweet sorghum stalk, and 1.17 for steam potato
ery of metabolites. The implementation of genetically engi- peels. They also demonstrated a savings towards greenhouse
neered microorganisms with the capability of utilizing a wide gas emissions by 52–56 % and 54–57 % when compared to die-
variety of substrates, less nutrient requirement, enhanced sta- sel and steam methane reforming for hydrogen production,
bility, as well as resistance to pretreatment inhibitors and con- respectively. The experiments on LCA using a single-stage pro-
taminants is a key to achieve higher biohydrogen production cess (CH4) and two-stage process (H2/CH4) by utilizing food
through dark and photofermentation in large-scale processes. waste and wheat straw revealed that the two-stage process
New technologies such as immobilization of microorganisms, could reduce certain environmental burdens as far as carcino-
optimization of cultural conditions leading to their selection gens and ecotoxicity are concerned compared to diesel [149].
and enrichments, and modifications of bioreactors with the The overall biohydrogen production technology is largely
standardization of process parameters are other strategies for affected by cost parameters, such as bioreactor development,
efficient biohydrogen production [134]. maintenance and operational cost, cost related to feedstock
Hosseini et al. [135] reviewed the suitable carbon source for processing and overhead expenditures, which ultimately influ-
an efficient and ecofriendly biohydrogen production by isolat- ence the sustainability of the final fuel product (biohydrogen).
ing light-dependent photosynthetic bacteria. Dadak et al. [136] In this context, applications of metabolic and genetic engineer-
proposed eco-exergy computational analysis as a useful deci- ing can help in reducing the production cost for biohydrogen
sion-making tool for substrate concentration and light intensity while simultaneously increasing its yield. The yield of biohy-
during photobiological hydrogen production. For example, the drogen is directly proportional to the operational costs and the
authors indicated that sodium acetate concentration and light production rate is directly proportional to the cost of the biore-
intensity of 1 g L–1 and 1000 lux, respectively, were the stan- actor (installation, operation, and maintenance).
dardized conditions for biohydrogen production. Similar The reduction in the cost of a photobioreactor and storage
research on the optimization of process conditions (both system depends on appropriate and less expensive materials
upstream and downstream) could lead to greater biohydrogen used in the fabrication of the photobioreactors. It is estimated
production and lesser by-product formation as well as lower that hydrogen production amounting for 80 kg ac–1d–1 could be
environmental impacts during dark fermentation. possible by converting photosynthetic efficiency of algae in the
Process modeling and simulation using computational fluid biohydrogen production process [150]. With an efficiency of
dynamics (CFD) can also be useful in designing a fermentative 50 %, the cost for biohydrogen production seems to about
hydrogen production process. These tools provide an analytical 2.80 $ kg–1 [151].
framework to help identify and overcome many scale-up issues During dark fermentation, the production cost of biohydro-
before the real operation. The CFD tool can provide a rational gen can be diminished by utilizing cheaper raw materials like
approach for scaling-up, designing, and optimizing the CSTR, sludge and distillery waste. For commercial production of bio-
thereby enhancing biohydrogen production [137]. hydrogen through dark fermentation, the vital factors are the
Apart from an efficient and cost-effective pretreatment pro- cost for reactor and storage system for hydrogen. It is also
cess, the design of suitable bioreactor types along with cheaper influenced by the combination of a photoreactor with a dark
feedstock can bring great opportunities in scaling-up of bio- fermentation reactor.
hydrogen production through dark fermentation. The cost Large-scale and efficient production of biohydrogen through
parameters in the design, development, construction, and oper- dark fermentation depends on many factors and parameters
ation of suitable bioreactors play a great role in final hydrogen which is in the stage of further scaling and facing many chal-
production at a large-scale facility. The CSTR is generally used lenges. Global researches on process engineering, genetic engi-
for continuous production of biohydrogen from various sub- neering, mechanical engineering, and chemical engineering can
strates [138, 139]. Several other bioreactors investigated for bio- be focused complementary to achieve efficient and sustainable
hydrogen production include multilayered photobioreactor biohydrogen production through dark fermentation at a com-
[140], fixed-bed bioreactor [141], fluidized-bed bioreactor mercial scale.
[142], up-flow anaerobic sludge blanket bioreactor [143], con-
tinuous stirred-tank bioreactor [144], carrier-induced granular
Review 610
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Chem Eng Technol - 2020 - Sarangi - Biohydrogen Production Through Dark Fermentation

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Keywords: Biohydrogen production, Biomass, Dark fermentation, Microorganisms,

1 Introduction anaerobic digestion and fermentation) pathways [8]. Waste

2 Hydrogen Production Technologies

The direct usage of hydrogen as a fuel has several

Table 1. Fuel properties of hydrogen and other hydrocarbons.

Characteristics Hydrogen Methane Gasoline Butanol Ethanol Methanol

Formula H2 CH4 H, C4–C12 C4H9OH CH3CH2OH CH3OH

Boiling point [C] –253 –162 32–210 118 78 65

Auto-ignition temperature [C] 585 540 280 343 365 435

Air/fuel ratio 34.3 17.19 14.6 11.2 9.0 6.5

Research octane number > 130 120 91–99 96 129 136

Motor octane number – 120 81–89 78 102 104

Viscosity at 25 C [mPa s] 0.009 0.011 0.6 2.573 1.074 0.544

Flash point [C] < –253 –188 –43 28.9 13 11

3 Biochemical Methods for Hydrogen

C6 H12 O6 þ 2H2 O ﬁ 2CH3 COOH þ 2CO2 þ 4H2

Stage 2 (photofermentation by photoheterotro-

CH3 COOH þ 2H2 O ﬁ 2CO2 þ 4H2 (7)

4 Dark Fermentation Process

operates in the complete absence of light by hydrogen-generat-

4.1 Utilization of By-products from Dark

The percentage of biomass conversion leading to

Table 2. Microorganisms employed in dark fermentation for biohydrogen production.

Microorganisms Substrates Hydrogen yield References

Enterobacter aerogenes Glycerol 172.9 mL g–1 Chookaew et al. [71]

process parameters during the up-

6 Conclusions [3] B. Abdullah, S. A. F. S. Muhammad, Z. Shokravic, S. Ismail,

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