Renewable and Sustainable Energy Reviews 117 (2020) 109484

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Renewable and Sustainable Energy Reviews

journal homepage: http://www.elsevier.com/locate/rser

Dark fermentative biohydrogen production from lignocellulosic biomass:

Technological challenges and future prospects
Juliana Ferreira Soares *, Ta
�ssia Carla Confortin, Izelmar Todero, Fla
�vio Dias Mayer,
Marcio Antonio Mazutti
Department of Chemical Engineering, Federal University of Santa Maria, UFSM, Roraima Avenue, Camobi, Santa Maria, 1000, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Biohydrogen is a promising low-carbon energy vector because its high energetic density, and emerging tech­
Renewable energy nologies has been studied aiming achieving higher efficiency and competitive H2 production, as is the case of
Biohydrogen dark fermentation. The objective of this paper is to review dark fermentative biohydrogen production from
Dark fermentation
lignocellulosic biomass, presenting insights of biomass pretreatment methods, influential factors in dark
Agricultural biomass
Agroindustrial biomass
fermentation, and environmental and economic aspects. Rice, corn, and wheat residues have been the main
Lignocellulosic biomass pretreatment lignocellulosic sources studied, and biohydrogen production ranged from 12 to 7019 mL H2/L. This wide vari­
Inoculum pretreatment ation is due to the source of lignocellulosic and its pretreatment method, the source and treatment conditions of
the inoculum, and the operational conditions of dark fermentation. Acid hydrolysis has been the most applied
method to breakdown the complex structure of lignocellulosic biomass, and enzymatic hydrolysis has been used
in sequence to improve the process. Moreover, additives (mainly metal materials) have been studied to enhance
dark fermentation and lignocellulosic biomass pretreatment. Heat-treated mixed culture is the main used source
of inoculum – 100 � C for 30 min is the most usual condition. Temperature, pH, and hydraulic retention time
(HRT) have also high influence in the biohydrogen production and yield. Mesophilic temperatures (around
37 � C), pH near 7.0, and HRT of 72 h, are recurrent parameters of dark biohydrogen fermentation. Finally, most
studies focused on laboratory scale, which suggest advanced studies on a large scale, and alternatives to improve
lignocellulosic biomass pretreatment and biohydrogen production is necessary to make this technology efficient,
economical and sustainable.

1. Introduction
Global energy demand depends mainly on fossil fuels such as oil,
coal, and natural gas, accounting for 81% of world total primary energy
supply in 2016 [1]. Their combustion is responsible for the emission of
pollutants such as COx, NOx, SOx, CxHy, ash, particulate matter, and
other organic compounds [2], resulting in air pollution and greenhouse
gas emissions, which consequently contribute to climate change and
affect global health and wellbeing [3]. Although the use of renewable
sources has increased in the last years, there is still a long way to
decreasing environmental impacts from fossil fuels, because renewables
correspond to only 14% of total primary energy supply [1,4].
Therefore, hydrogen (H2) has been considered as an alternative to
fossil fuels to address the environmental and health problems associated
with their growing utilization. According to Abe et al. [5], H2 can be
produced from fossil fuels (coal, natural gas, and nuclear energy) and
from renewable sources (biomass, hydro, wave, wind, solar, and
geothermal energy). Today 50% of H2 is produced from natural gas
(steam methane reforming), 30% from cracking oil products, 18% from
coal gasification, and the remainder from electrolysis of water [6]. As
can be seen, H2 is produced mainly from fossil fuels, which require a
high-energy cost and are not environmentally friendly. In this way, the
biological production of biohydrogen has significant advantages over
these methods because it occurs at low temperatures and atmospheric
pressure, theoretically requiring less energy intensity.
Dark fermentation, photo-fermentation, hybrid system, direct

Abbreviations: VS, volatile solids; VSS, volatile suspended solids; TSS, total suspended solids; COD, chemical oxygen demand; TRS, total reducing sugar; TVS, total
volatile solid; 5-HMF, 5-hydroxymethylfurfural; na, not available; LHW, liquid hot water; SCW, subcritical water; SE, steam explosion; HRT, hydraulic retention time;
UASB, up-flow anaerobic sludge blanket; SMR, steam methane reforming; DF, dark fermentation; ER, energy recovery; MEC, microbial electrolysis cell.
* Corresponding author.
E-mail address: eng.juferreira@gmail.com (J.F. Soares).

https://doi.org/10.1016/j.rser.2019.109484
Received 26 January 2019; Received in revised form 1 October 2019; Accepted 8 October 2019
Available online 18 October 2019
1364-0321/© 2019 Elsevier Ltd. All rights reserved.
J.F. Soares et al.
biophotolysis, and indirect biophotolysis are all types of biological
processes used to produce biohydrogen. Dark fermentation involves
biohydrogen production by anaerobic bacteria from carbohydrate-rich
substrates in the absence of light, whereas in photo-fermentation,
photosynthetic bacteria use light energy to produce biohydrogen from
various organic acids, food processing and agricultural wastes [7,8].
Dark fermentation and photo-fermentation are complementary tech­
nologies and can be used sequentially in a hybrid system. In direct
biophotolysis solar energy is used by algae and cyanobacteria to convert
water into oxygen and biohydrogen (2H2O → 2H2 þ O2). On the other
hand, indirect biophotolysis involve separation of the biohydrogen and
oxygen evolution reactions into separate stages, coupled via carbon di­
oxide (CO2) fixation/evolution [8].
Dark fermentation present advantages when compared to other
biological processes because it is light-independent and different raw
materials can be used as a carbon source to the microorganisms, which
have both a high rate of biohydrogen production and growth rate to
supply the system [9]. Furthermore, strict and facultative anaerobic
microorganisms can be used in the form of pure cultures (e.g. Clos­
tridium, Enterobacter, Escherichia coli) and mixed cultures (e.g. anaerobic
sludge, bovine manure, organic compost). Unlike mixed cultures, pure
cultures require a sterile environment to prevent contamination, which
is difficult and expensive to achieve in industrial scale. For this reason,
mixed cultures have been preferred in scaled-up applications [10].
In addition to pure sugars (glucose, xylose, lactose, galactose, su­
crose), lignocellulosic biomass, food waste, municipal solid waste, in­
dustrial wastewater, and glycerol are some examples of raw material
used in dark fermentation. Among them, lignocellulosic biomass is an
attractive source because it is renewable, available in large quantities
and low price, and has a high carbohydrate content that can be used in
the biohydrogen fermentation process [11]. Biohydrogen production
and yield from dark fermentation are influenced by some factors, such as
the source of substrate, source of inoculum, and operational parameters
(pH, temperature, hydraulic retention time, etc.).
Therefore, the objective of this work is to review the dark fermen­
tative biohydrogen production from lignocellulosic biomass and discuss
the main influential factors of this process and the environmental and
economic aspects. Moreover, this review presents new alternatives used
to improve dark fermentative biohydrogen production, and the future
perspective of this technology, which has been little discussed in the
other reviews and essential to make this technique viable and
competitive.
2. Methods
This review focused on the dark fermentative biohydrogen produc­
tion from lignocellulosic biomass. Agricultural, agroindustrial, and
forestry wastes were selected as the main lignocellulosic biomass. These
biomasses have been the most found in literature to produce bio­
hydrogen via dark fermentation. From these scientific articles, ligno­
cellulosic biomass pretreatment methods as well as operational
conditions were analyzed. Pretreated methods were divided into phys­
ical, chemical, physical-chemical, and biological processes for discus­
sion. Factors influencing dark fermentative biohydrogen production –
inoculum source, temperature, pH, hydraulic retention time, gas partial
pressure, and fermentation end-products – were also analyzed.
Lignocellulosic biomass pretreatment methods and factors influ­
encing dark fermentation were presented in tabular form together with
biohydrogen yields and biohydrogen productions. Results from bio­
hydrogen production were converted from all papers into the same basis
(mL H2/L) whenever possible to enable comparison. Some studies do not
provide enough information to convert all the H2 yield results in a same
unit, e.g. g H2/g biomass, mL H2/g VS, mL H2/g COD initial, mol H2/mol
carbohydrate, etc. The use of additives to improve dark fermentation
was also discussed in this review. However, the searched was not limited
to dark fermentative biohydrogen production from lignocellulosic
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Renewable and Sustainable Energy Reviews 117 (2020) 109484
biomass because there are still few studies using this feedstock.
Most references used in this review were searched in the Science­
Direct database. To discuss lignocellulosic biomass pretreatment,
influential factors of dark fermentative biohydrogen production, and the
environmental and economic aspects, papers were selected from the
period 2000–2019. On the other hand, older references were used to
argue consolidated concepts. The keywords used in this review were
selected according to the objective of this study. The main keywords
were: hydrogen, biohydrogen, biohydrogen production, dark fermen­
tation, hydrogen dark fermentation, lignocellulosic biomass, lignocel­
lulosic biomass pretreatment, among others. Keywords as lignocellulosic
biomass, mixed/pure culture, inoculum, pH, temperature, hydraulic
retention time, gas partial pressure, nanomaterials, among others, were
searched simultaneously with keywords biohydrogen or hydrogen dark
fermentation or dark fermentation. All searched articles were written in
English, the main scientific language.
3. Lignocellulosic biomass for dark fermentative biohydrogen
production
Lignocellulosic biomass has been considered a potential feedstock for
biofuels production such as biohydrogen, methane, ethanol, and butanol
because it is renewable, sustainable, and available in large quantities.
Lignocellulosic biomass includes agricultural and agroindustrial resi­
dues (sugarcane bagasse, cornstalk, corn stover, corn cobs, corn bran,
wheat straw, sorghum rusk, sorghum leaves, sorghum stover, rice straw,
rice bran, rice husk, oat straw, etc.), forestry waste, wood, grass, and
others. Lignocellulosic biomass is the most abundant raw material and
has a complex structure mainly composed of three polymers (cellulose,
hemicellulose, and lignin), besides ash and extractives [12,13].
Cellulose – the main structural component in the plant cell – is a
natural high molecular polymer composed of glucose units, with cello­
biose as the basic coupling unit [14]. Hemicellulose is the second most
abundant polymer in lignocellulose biomass, which provides a connec­
tion between lignin and cellulose. Different from cellulose, hemicellu­
lose has a heterogeneous and amorphous structure. It is a short and
highly branched polymer of pentoses (xylose, rhamnose, and arabinose),
hexoses (glucose, mannose, and galactose) and uronic acids
(4-O-methylglucuronic, D-glucuronic, and D-galactoronic acids) [14,
15]. Lignin is the third most abundant polymer in nature. It is a complex,
large molecular structure containing crosslinked aromatic polymers of
phenolic monomers (coumaryl alcohol, coniferyl alcohol, and sinapyl
alcohol). Lignin confers a rigid, impermeable resistance to microbial
attack and oxidative stress in the lignocellulosic biomass [15].
Table 1 presents the chemical composition of major lignocellulosic
biomass used in biohydrogen production. Rice, corn, and wheat residues
have been the most studied substrates. As can be seen in Table 1, the
chemical composition of lignocellulosic biomass varies considerably.
Lignocellulosic biomass present cellulose 11–46%, hemicellulose
6–42%, and lignin 1–40%. According to Muharja et al. [16], hemicel­
lulose decomposition rate is faster than cellulose because hemicellulose
has an amorphous molecular structure whereas cellulose is crystalline.
Biohydrogen yield, production rate and the overall economy of the
process are mainly dependent on the carbohydrate content of biomass,
bioavailability, and biodegradation rate [17].
Biohydrogen production from lignocellulosic biomass has been
extensively studied in a laboratory scale. Table 1 also shows the bio­
hydrogen production and biohydrogen yield obtained from different
lignocellulosic biomasses via dark fermentation. Biohydrogen produc­
tion has ranged from 12 to 7019 mL H2/L. The higher the lignin content
in biomass the more difficult is the direct conversion to biohydrogen. As
can be seen in Table 1, the highest biohydrogen production (7019 mL
H2/L) was obtained from sugarcane bagasse, which presented only 8.3%
of lignin. On the other hand, the lowest production (12 mL H2/L) was
obtained from cashew apple bagasse, which had four times more lignin
(35.26%). Although lignin content has a negative influence on the
J.F. Soares et al. Renewable and Sustainable Energy Reviews 117 (2020) 109484

Table 1
Chemical composition of different lignocellulosic biomass used in dark fermentative biohydrogen production.
Lignocellulosic Biomass Cellulose (%) Hemicellulose (%) Lignin (%) H2 production H2 yield Reference

Rice straw na na na 212.9–270.1 mL H2/L 40.04 mL H2/g VS removed [18]

Rice strawa 28.2 17.5 14.6 na 28 mL H2/g VS [20]
Rice straw na na na na 8.61–14.67 NmL H2/g VS [21]
Rice straw na na na 446.5–771.0 mL H2/L 1.89–4.39 mL H2/g biomass [22]
Rice husk na na na 92.75–139.65 mL H2/L ~31 mL H2/g VS removed [18]
Rice huska 40.26 12.54 25.40 2543–2758 mL H2/L 1.81–1.96 mol H2/mol total sugar [19]
Rice huska 40.26 12.54 25.40 na 320.6–473.1 mL H2/g biomass [23]
Rice huska 29 29 24 29.26 mL H2 5.9 mL H2/g biomass [24]
Rice bran na na na 173.65–252.40 mL H2/L 38.6 mL H2/g VS rem [18]
Rice bran na na na 969–2873 mL H2/L 38.52–117.24 mL H2/g sugar [25]
Rice bran na na na 545 mL H2/L 266.4 mL H2/g biomass [26]
Corn stovera 37.6 21.5 19.1 108.5 mmol H2/L na [27]
Corn stover 38.53 22.79 10.73 na 36.08 mL H2/g biomass [28]
Cornstalkb 34.45 27.55 21.81 na 126.22 mL H2/g biomass [29]
Corn cobs na na na na 5–141 mL H2/g COD initial [30]
Corn bran residueb 10.54 40.57 1.06 1025.19–2648.41 mL H2/L 176–338.91 mL H2/g VS [31]
Wheat straw na na na na 147.7–317.6 mL H2/g sugar [32]
Wheat strawa 44.45 19.23 5.78 0–4629.09 mL H2/L 0–269.2 mL H2/g TRS [33]
Wheat powder na na na 140–4500 mL H2/L d 20–646 mL H2/g total sugar [34]
Wheat powder na na na 773.33–1754.76 mL H2/L d 0.6–1.6 mol H2/mol total sugar [35]
Sorghum rusk na na na 186–1117 mL H2/L 1.05 mol H2/mol reducing sugar [36]
Waste sorghum leavesa 28.56 29.18 3.94 369 mL H2/L 213.14 mL H2/g sugar [37]
Sweet sorghum stovera 37.42 20.43 16.28 4020.1 mL H2/L na [38]
Sugarcane bagassea 38.1 21.2 8.3 4229–7019 mL H2/L na [39]
Sugarcane bagassea 31.4 41.6 24.8 0.33–24.06 mmol H2/L na [40]
Sugarcane leaf wastea 40.45 33.14 5.85 127.69 mL H2/L 18.6 ml H2/g sugar [41]
Oat Straw na na na na 2.9 mol H2/mol hexose consumed [42]
Oat strawa 34.8 26.7 8.7 643.25–1374.13 mL H2/L 1.10–2.39 mol H2/mol reducing sugars [43]
Pine tree wooda 39.48 22.10 37.11 1797–1916 mL H2/L 1.28–1.36 mol H2/mol total sugar [19]
Pine tree wood pelleta 39.48 22.1 37.11 na 0.31–1.27 mol H2/mol sugar [44]
Spent mushroom composta 31.2 6.9 14.0 168.8–337.6 mL H2/L 0.21–0.42 mmol H2/g TVS [45]
Spent mushroom composta 24.36 15.82 22.20 25.93–49.62 mmol H2/L na [46]
Empty palm fruit buncha 38.31 11.22 39.82 1512–1559 mL H2/L 1.08–1.11 mol H2/mol total sugar [19]
Oil palm empty fruit bruncha 38.31 11.22 9.37 na 169.53–286.98 mL H2/g biomass [47]
De-oiled Jatropha wastea 14.1 24.2 30.4 270.0–996.7 mL H2/L 5.4–20.0 mL H2/g VS [48]
Cashew apple bagassea 20.56 10.17 35.26 12.0–302.4 mL H2/L 0.08–1.89 mL H2/g biomass [49]
Peanut shella 46.1 5.6 27.8 na 6.4–39.9 mL H2/g biomass [50]
Waste paper na na na 336.8–859.9 mL H2/L 15.31–139.97 mL H2/g sugar [51]
a
Composition presented in %wt on dry basis.
b
Composition presented in %wt on wet basis.

biohydrogen production, other factors also influence biohydrogen pro­
duction, including carbohydrate content of lignocellulosic biomass,
lignocellulosic biomass pretreatment, operational conditions, etc.
The negative effect of high lignin content was also demonstrated by
Sattar et al. [18] and Gonzales et al. [19]. Sattar et al. [18] studied the
biohydrogen production potential from rice straw, rice husk and rice
bran. The results showed a production of 4258 mL H2 from rice straw,
1855 mL H2 from rice husk and 3473 mL H2 from rice bran on meso­
philic conditions. According to the authors, the lignin content of rice
husk was double than rice straw resulting in a lower yield. Gonzales
et al. [19] used empty palm fruit bunch, rice husk, and pine tree wood as
feedstock resulting in 0.96 mol H2/mol sugar, 1.25 mol H2/mol sugar
and 0.99 mol H2/mol sugar, respectively. Lignin content of empty palm
fruit bunch and pine tree wood bran were almost double than rice husk,
which may explain its higher yield.
3.1. Different pretreatments of lignocellulosic biomass for dark
fermentative biohydrogen production
Pretreatment of lignocellulosic biomass is a crucial factor in the
production of biohydrogen through dark fermentation, because of the
complex structure of cellulose, hemicellulose, and lignin. Pretreatment
aims to break this recalcitrant heteropolymeric structure and increase
the amount of monomeric sugars needed for H2-producing microor­
ganisms. Fig. 1 shows the main products obtained in the breaking of
cellulose and hemicellulose fraction from pretreatment. As can be seen,
the breakdown of cellulose results in glucose, cellobiose, and 5-hydrox­
ymethylfurfural (5-HMF). And the breakdown of hemicellulose results in
xylose, arabinose, and furfural. According to Moodley e Kana [41], in
the dark fermentative biohydrogen production, glucose is more easily
consumed by microorganisms than xylose, which begins to be

Fig. 1. The main products obtained in the lignocellulosic biomass pretreatment.

3
J.F. Soares et al.
metabolized when the glucose concentration approaches 0%.
The presence of inhibitors as 5-HMF and furfural in the dark
fermentation can negatively affect H2-producing microorganisms.
Mun ~ oz-Pa
�ez et al. [52] observed in individual treatments that concen­
trations of furfural and 5-HMF up to 1.0 g/L and 0.09 g/L, respectively,
did not inhibit biohydrogen production, but 0.19 g/L of 5-HMF
decreased biohydrogen production. However, the authors identified
that furfural/5-HMF mixtures inhibited the biohydrogen production.
Mixtures with 0.10 g/L of furfural and 0.02 g/L of 5-HMF, for example,
inhibited biohydrogen production by 11%. On the other hand, Nasr et al.
[30] revealed that 0.21–1.09 g/L of furfural and 0.05–0.14 g/L of 5-HMF
had no impact on biohydrogen production rates and yields. In general,
the inhibitory effect of furfural and 5-HMF will depend on both con­
centration and type of microorganisms present in the fermentation.
According to Agbor et al. [53], the pretreatment process should have
a low capital, low operational cost, effectiveness on a wide range and
loading of lignocellulosic biomass and result in the recovery of most the
lignocellulosic components. Pretreatments can be classified into phys­
ical (grinding, milling, chipping, cutting and shearing), chemical (acids,
alkalis, organic solvents, ionic liquids, etc.), physical-chemical (liquid
hot water, steam explosion, ammonia fiber expansion, etc.) and bio­
logical (fungal treatment, enzymatic hydrolysis, etc.) methods. The
combination of these techniques is generally more effective in increasing
biomass digestibility [53]. Table 2 shows the pretreatment methods of
lignocellulosic biomass used to biohydrogen production. As can be seen,
acid hydrolysis has been the most widely used method, and to improve
this process, the enzymatic hydrolysis has been used in the sequence.
Fig. 2 presents a scheme of the main lignocellulosic biomass pretreat­
ment used for dark fermentative biohydrogen production. Considering
the data in Table 2, 44.1% of works used a physical pretreatment
(milling, grinding) followed by acid hydrolysis, 17.6% used physical
pretreatment followed by acid hydrolysis and enzymatic hydrolysis, and
14.7% used only physical pretreatment (Fig. 2).
3.1.1. Physical pretreatment
Grinding and milling are the most commonly used physical pre­
treatment and generally precede chemical, physico-chemical, and bio­
logical treatments. Grinding and milling reduces the size of
lignocellulosic biomass and increase the accessible surface area to the
reaction [57]. This method has been applied to corn stover [27], wheat
straw [33], oat straw [43], rice straw [22,24,54,58], waste paper [51],
waste sorghum leaves [37], sugarcane bagasse [39], sweet sorghum
[38], among others. Tosuner et al. showed that biohydrogen production
yields increased with decreasing of rice husk particle sizes (<2000 μm,
<300 μm and <74 μm). However, these physical methods are not able to
breakdown cellulose and hemicellulose fraction, being necessary
another pretreatment in the sequence to improve biohydrogen produc­
tion. As can be seen in Table 2, biohydrogen production from rice straw,
for example, was enhanced almost three times when the physical
method (grinding) was followed by chemical (acid hydrolysis) and
biological (enzymatic hydrolysis) methods [18,22].
3.1.2. Physical-chemical pretreatment
Among physical-chemical pretreatments, liquid hot water (LHW),
subcritical water (SCW), and steam explosion (SE) are promising
methods to break down the recalcitrant structure of the lignocellulosic
biomass. These processes can also be applied before enzymatic hydro­
lysis. However, unlike liquid hot water, the steam explosion process
releases high concentrations of degradation compounds (furfural, 5-
hydroxymethylfurfural, acetic and formic acid and phenolic com­
pounds) that can inhibit enzymes and fermentative microorganisms
[59]. Cara et al. [60], investigated how liquid hot water and steam ex­
plosion pretreatments influenced on enzymatic hydrolysis of olive tree
pruning biomass. In the LHW-pretreatment were recovered 0.028 g
glucose/g raw material and 0.013 g hemicellulosic sugars/g raw mate­
rial, and in SE-pretreatment were 0.054 g glucose/g raw material and
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Renewable and Sustainable Energy Reviews 117 (2020) 109484
0.054 g hemicellulosic sugars/g raw material. The highest yield of
enzymatic hydrolysis with LHW-pretreated materials was 76.2% and
with SE-pretreated materials was 62.9%. Muharja et al. [16] combined
the green process of SCW and enzymatic hydrolysis for biohydrogen
fermentation from coconut husk. The maximum total biohydrogen yield
was 0.279 mol/mol sugar consumed, which is lower than the other
works. According to the authors, enzymatic hydrolysis was found to
contribute significantly to the total sugar obtained from coconut husk.
3.1.3. Chemical pretreatment
Chemical methods refer to the treatment of biomass with chemical
reagents such as acids, alkalis, organic solvents, ionic liquids, metal
chlorides and plasma [57]. Among these, acid pretreatment is one of the
most effective pretreatment used for lignocellulosic biomass. This pre­
treatment involves the use of concentrated or dilute acids, mainly sul­
furic acid (H2SO4) and hydrochloric acid (HCl), to break down the rigid
structure of lignocellulosic material [61], releasing sugars, inhibitory
compounds, and precipitating lignin [33]. In this process, the hemicel­
lulose is hydrolyzed fraction, while the cellulose and lignin fractions
remain almost unchanged [62].
Hu et al. [63], Gonzales et al. [44], Gokfiliz and Karapinar [64], Eker
and Sarp [51], Azman et al. [65], Azman et al. [25] and Lopez-Hidalgo
et al. [33] are some examples in which it was applied dilute acid pre­
treatment (with sulfuric acid) on lignocellulosic biomass for bio­
hydrogen production; sugarcane bagasse, pine tree wood pellet, waste
wheat powder, waste paper, palm kernel cake, rice bran and wheat
straw, respectively. Rorke and Kana [37] and Moodley e Kana [41]
compared three acid pretreatments (HCl, H2SO4, and HNO3) for xylose
and glucose recovery from sorghum residue and sugarcane leaf waste,
respectively. In both studies HCl provided the highest recovery of
sugars, 52.02 g/L of xylose and 18.42 g/L of glucose from sorghum
residue and 78 g/L of xylose and 11.48 g/L of glucose from sugarcane
leaf waste. Although H2SO4 is the most used, Vasconcelos et al. [66]
have shown that acid hydrolysis with H3PO4 results in low amounts of
inhibitors, and the H3PO4 remaining in the hydrolysate is at adequate
levels for supplying phosphorous requirement for the subsequent
fermentation process.
In addition to the use of acid, operational conditions (acid concen­
tration, reaction time, and temperature) also influence the sugar
released from acid pretreatment. Sen et al. [22] studied the effect of HCl
concentration between 0.1 to 3.0 M in the xylose and glucose recovery
from rice straw. Results showed that higher concentration of HCl
influenced the release of glucose but not xylose. The maximum xylose
and glucose concentrations were obtained with 1.0 M and 3.0 M,
respectively. Moodley and Kana [41] analyzed the influence of acid
concentration (0.5–5.0%, v/v), temperature (60–100 � C) and reaction
time (60–240 min) in the xylose and glucose recovery of sugarcane leaf
waste, using HCl, H2SO4, and HNO3. HCl and H2SO4 pretreatment has
the best result with 5% of acid, 150 min and 100 � C, while HNO3 pre­
treatment have the best result with 2.75% of acid, 240 min and 100 � C.
Acid concentration also affects dark fermentative H2 production.
Wang et al. [29] and Saratale et al. [36] evaluated the acid concentra­
tion of cornstalk wastes (0.2–1.2% HCl) and sorghum husk (0.1–0.4%
H2SO4) pretreatment, respectively, in the biohydrogen production. The
maximum biohydrogen production was obtained when was used corn­
stalk wastes treated with 0.6% of HCl and sorghum husk treated with
0.2% of H2SO4. Although high concentrations of acid can result in higher
released sugars from lignocellulosic biomasses, this is not favorable to
dark fermentative biohydrogen production, since it might inhibit
H2-producing bacteria.
Inhibitory compounds, such as furfural, 5-hydroxymethylfurfural (5-
HMF) and acetic acid, are also produced in the acid pretreatment of
lignocellulosic biomass [33,62]. Temperature, time and acid concen­
tration are parameters that can influence the generation of fermentation
inhibitors [67]. Besides, pretreated biomass fractions can contain formic
and levulinic acids from 5-HMF via acid-catalyzed thermo-chemical
J.F. Soares et al. Renewable and Sustainable Energy Reviews 117 (2020) 109484

Table 2
Summary of the latest studies performed on pretreatment methods of lignocellulosic biomass for dark fermentative biohydrogen production.
Lignocellulosic Lignocellulosic biomass pretreatment H2 production H2 yield Reference
biomass

Rice straw Physical: Milling na 28 mL H2/g VS [20]

Physical-chemical: Hydrothermal (150 � C and 210 � C)
Rice straw Physical: Cutting na 8.61–14.67 NmL H2/g VS [21]
Rice straw Physical: Grinding 446.5–771.0 mL H2/L 1.89–4.39 mL H2/g biomass [22]
Chemical: Acid hydrolysis (HCl 1.0–3.0 M)
Biological: Enzymatic hydrolysis (Cellulase 1% v/v from Trichoderma reesei
ATCC 26921)
Rice straw Physical: Grinding 212.9–270.1 mL H2/L 40.04 mL H2/g VS rem [18]
Rice straw Physical: Cutting na 0.3–15.4 mL H2/g VS rem [54]
Chemical: Alkaline pre-treatment (NaOH 4 and 8% w/v)
Rice husk Physical: Grinding 92.75–139.65 mL H2/L ~31 mL H2/g VS rem [18]
Rice husk Physical: Milling na 320.6–473.1 mL H2/g [23]
Chemical: Acid hydrolysis (H2SO4 5% w/v) biomass
Biological: Enzymatic hydrolysis (commercial enzyme)
Rice husk Physical: Grinding 29.26 mL H2 5.9 mL H2/g biomass [24]
Rice bran Chemical: Acid hydrolysis (18 M H2SO4 1% v/v) 969–2873 mL H2/L 38.52–117.24 mL H2/g sugar [25]
Corn stover Physical: Milling 108.5 mmol H2/L na [27]
Chemical: Alkali (2% NaOH)
Biological: Enzymatic hydrolysis (commercial cellulase)
Corn stover Physical: Grinding na 36.08 mL H2/g biomass [28]
Biological: Enzymatic hydrolysis (commercial cellulase)
Cornstalk Physical: Grinding na 126.22 mL H2/g biomass [29]
Chemical: Acid (HCl 0.2–1.2 wt%)
Biological: Enzymatic hydrolysis (Trichoderma viride)
Corn cobs Chemical: Dilute Acid Pretreatment na 5–141 mL H2/g COD initial [30]
Physical-chemical: High Pressure Autohydrolysis
Corn bran residue Physical: Milling 1025.19–2648.41 mL 176–338.91 mL H2/g VS [31]
Chemical: Calcined-lime mud from papermaking H2/L
Wheat straw Physical-chemical: Hydrothermal na 147.7–317.6 mL H2/g sugar [32]
Wheat straw Physical: Milling 0–4629.09 mL H2/L 0–269.2 mL H2/g TRS [33]
Chemical: Acid hydrolysis (H2SO4 0.75% v/v)
Wheat powder Physical: Grinding 140–4500 mL H2/L d 20–646 mL H2/g total sugar [34]
Chemical: Acid hydrolysis (H2SO4 pH 2.0)
Wheat powder Physical: Grinding 773.33–1754.76 mL H2/ 0.6–1.6 mol H2/mol total [35]
Chemical: Acid hydrolysis (H2SO4 pH 2.0) Ld sugar
Sorghum rusk Physical: Milling 186–1117 mL H2/L 1.05 mol H2/mol reducing [36]
Chemical: Acid (H2SO4 0.1–0.4% w/v) sugar
Biological: Enzymatic hydrolysis (Phanerochaete chrysosporium MTCC 787,
isolated actinomycetes Streptomyces sp. MDS, and Nocardiopsis sp. KNU)
Waste sorghum Physical: Milling 369 mL H2/L 213.14 mL H2/g sugar [37]
leaves Chemical: Acid hydrolysis (HCl, H2SO4 and HNO3 – 1.0, 3.5 or 6.0% v/v)
Sweet sorghum stover Physical: Milling 4020.1 mL H2/L na [38]
Biological: Delignification by the laccase from T. asperellum and Enzymatic
hydrolysis (commercial enzyme)
Sugarcane bagasse Physical: Milling 4229–7019 mL H2/L na [39]
Chemical: Acid hydrolysis (H2SO4 0.5% v/v)
Sugarcane bagasse Physical: Milling 121.7–402.0 mL H2/L 0.092–1.211 mol H2/mol [55]
Chemical: Acid hydrolysis (H2SO4 0.64% v/v) glucose
Sugarcane bagasse Physical: Milling 0.33–24.06 mmol H2/L na [40]
Physical-chemical: Autoclave
Sugarcane leaf waste Physical: Milling 127.69 mL H2/L 18.6 ml H2/g sugar [41]
Chemical: Acid hydrolysis (HCl, H2SO4, and HNO3 - 0.5, 2.75, 5.0% (v/v))
Oat straw Physical: Milling na 2.9 mol H2/mol hexose [42]
Chemical: Acid hydrolysis (HCl 2% v/v) consumed
Oat straw Physical: Milling 643.25–1374.13 mL H2/ 1.10–2.39 mol H2/mol [43]
Chemical: Acid hydrolysis (HCl) and Alkaline hydrolysis (KOH/NaClO2/KOH or L reducing sugars
NaOH/H2O2)
Biological: Enzymatic hydrolysis (commercial enzyme)
Pine tree wood pellet Physical: Milling na 0.31–1.27 mol H2/mol sugar [44]
Chemical: Acid hydrolysis (H2SO4 5% w/v)
Oil palm empty fruit Physical: Milling na 169.53–286.98 mL H2/g [47]
brunch Chemical: Acid hydrolysis (H2SO4 6% v/v) biomass
Biological: Enzymatic hydrolysis (commercial enzyme)
Cashew apple bagasse Physical: Grinding 12–302.4 mL H2/L 0.08–1.89 mL H2/g biomass [49]
Chemical: Alkaline hydrogen peroxide; Acid hydrolysis (H2SO4 0.6 M)
Biological: Enzymatic hydrolysis (commercial enzyme)
Peanut shells Physical: Grinding na 6.4–39.9 mL H2/g biomass [50]
Waste paper Physical: Grinding 336.8–859.9 mL H2/L 15.31–139.97 mL H2/g sugar [51]
Chemical: Acid hydrolysis (H2SO4)
Coconut husk Chemical: Subcritical water pretreatment na 0.104–0.175 mol H2/mol [16]
Biological: Enzymatic hydrolysis (cellulase from Aspergillus niger and endo-1,4- reducing sugar
β-xylanase from Trichoderma longibrachiatum)
Brewery spent grains Chemical: Acid hydrolysis; Alkaline hydrolysis. 52–92 mL H2/L 100–175 mL H2/g culture dry [56]
weight

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J.F. Soares et al.
Fig. 2. The main sequences of lignocellulosic biomass pretreatment used in dark fermentative biohydrogen production.
degradation of polysaccharides. The concentration of the formed acids
depends strongly on the feedstock composition and pretreatment con­
ditions. Undissociated form of levulinic and formic acids in the
fermentation media can diffuse into the cells at under acidic conditions
and can deteriorate the biocatalyst activity since these compounds
present some toxicity [68].
According to some studies, the concentration of furfural, 5-HMF and
acetic acid varied from 0.03 to 8.23 g/L, 0.09–1.59 g/L and 2.61–7.70 g/
L, respectively [23,30,33,43,44,47,69]. In the fermentation stage, the
presence of these compounds can block or hinder the process, since they
can affect the microbial growth in several ways [33,62]. However, ac­
cording to Nasr et al. [30], the furfural concentration of 0.21–1.09 g/L
and 5-HMF below 0.14 g/L have no impact on the yield and rate of
biohydrogen production using mixed culture as inoculum source.
3.1.4. Biological pretreatment
Biological pretreatment is based on the delignification of lignocel­
lulose from microorganisms or enzymes. Fungi are the most effective
microorganisms for delignification because of their unique ligninolytic
system [57]. They can produce enzymes to degrade lignin, hemi­
celluloses, and polyphenols present in the biomass [70]. Shanmugam
et al. [38] evaluated laccase from Trichoderma asperellum on delignifi­
cation of sweet sorghum stover biomass for biohydrogen production.
Results showed an optimal lignin removal of 76.93% leading to a bio­
hydrogen production (402.01 mL) 3.26 times higher than the control
without enzymatic pretreatment.
Saratale et al. [36] evaluated the potential of isolated actinomycetes
(Streptomyces sp. and Nocardiopsis sp.) and fungi (Phanerochaete chrys­
osporium) grown on agricultural wastes (sorghum husk, rice husk, Vigna
mungo harvesting waste, soybean straw, and waste tea powder) for
cellulase and xylanase production under solid state fermentation con­
ditions. Maximal secretion of enzymes was observed with Phanerochaete
chrysosporium using soybean straw. On the other hand, there are also
studies that use commercial enzyme (Celluclast, Novozymes, etc.) on
lignocellulosic biomass pretreatments [16,23,43,71].
Acid pretreatment can be followed by enzymatic hydrolysis,
improving enzyme accessibility to cellulose for higher yield of
fermentable sugars. As an example, studies from Arreola-Vargas et al.
[43], Saratale et al. [36], Sen et al. [22], Gonzales e Kim [23], and
Gonzales [47], where the oat straw, sorghum husk, rice straw, rice husk,
and empty fruit brunch from oil palm were used, respectively, for dark
fermentative biohydrogen production. According to Gonzales et al. [47],
enzymatic hydrolysis enhanced the sugar concentration of dilute acid
hydrolysis by 20% and biohydrogen production rate by 22%. Although
acid treatment followed by enzymatic hydrolysis improve sugars re­
covery from lignocellulosic material, the use of enzymes on a commer­
cial scale is the main factor contributing to high pretreatment costs [37].
3.1.5. Comparative of lignocellulosic biomass pretreatment
Physical pretreatments have been used in most of the studies to
reduce the size of lignocellulosic biomass used for biohydrogen pro­
duction. Although these methods are not able to breakdown cellulose
and hemicellulose fraction, they increase the accessible surface area to
subsequent processes. Liquid hot water, subcritical water, and steam
6
Renewable and Sustainable Energy Reviews 117 (2020) 109484
explosion are promising physical-chemical pretreatments to breakdown
the recalcitrant structure of lignocellulosic biomass to increase the
accessibility of cellulose and hemicellulose for subsequent acid or
enzymatic hydrolysis. These processes do not require chemicals. How­
ever, high pressures and temperatures are needed, which can make the
process more expensive on larger scale.
According to the results presented in Table 2, dilute acid hydrolysis is
the most commonly used method to pretreat lignocellulosic biomass for
dark fermentative biohydrogen production. Acid hydrolysis is a simple
process, has low cost, and results in high yield. On the other hand, the
resulting hydrolysate must be neutralized prior to dark fermentation,
and inhibitors of H2-producing microorganism (furfural, 5-HMF, acid
acetic) are produced. However, acid hydrolysis conditions can be opti­
mized to produce a low amount of inhibitor, below the concentration
supported by H2-producing microorganisms. Acid hydrolysis has also
been followed by enzymatic hydrolysis in many works to improve sugars
recovery. Enzymatic hydrolysis, with commercial enzyme, is the main
biological method applied to lignocellulosic biomass. The drawbacks
include the high cost of enzyme and the high retention time of the
process.
4. Factors influencing dark fermentative biohydrogen
production
Biohydrogen production via dark fermentation is a very complex
process and influenced by many factors such as inoculum, temperature,
pH, hydraulic retention time (HRT), gas partial pressure, and fermen­
tation end-products, which are discussed below. Table 3 shows the
inoculum source and main operational parameters (temperature, pH,
and HRT) of dark fermentation. In general, it can be observed that mixed
culture has been the main used source of inoculum. Moreover, meso­
philic temperatures (around 37 � C) and pH near 7.0 are recurrent
operational parameters. HRT effects seem to be less studied than pH and
temperature. As can be seen in Table 3, HRT ¼ 72 h is the most used.
4.1. Inoculum
Many fermentative bacteria have been used to produce biohydrogen,
including strict anaerobes (Clostridium) [25], facultative anaerobes
(Enterobacter, Escherichia coli, Rhodopseudomonas, Citrobacter) [16,33,
72–74] and aerobic bacteria (Bacillus) [75]. Among these microorgan­
isms, the bacteria of the genus Clostridium are the most widely studied
Facultative anaerobic bacteria produce up to 2 mol of H2 per mole of
glucose, while strict anaerobes produce 4 mol per mole of glucose [76].
Strict anaerobic microorganisms are extremely sensitive to oxygen. A
very small amount of oxygen in the fermentation medium completely
inhibits H2-producing activities. On the other hand, facultative anaer­
obes consume oxygen fast, recovering the anaerobic condition in the
medium immediately. Thus, biohydrogen production by facultative
anaerobes is considered more cost-effective than strict anaerobes [77].
Strict or facultative anaerobic microorganisms to biohydrogen pro­
duction may be from pure cultures (Clostridium, Enterobacter, Escherichia
coli) or mixed cultures (anaerobic sludge, bovine manure, organic
compost, urban solid waste, and soil). Pure cultures through isolation
J.F. Soares et al. Renewable and Sustainable Energy Reviews 117 (2020) 109484

Table 3
Inoculum source and operational parameters of dark fermentative biohydrogen production from lignocellulosic biomass.
Lignocellulosic Inoculum T (� C) Initial pH HRT H2 production H2 yield Reference
Biomass (h)

Rice straw Mixed culture (Anaerobic sludge from a 35 7.0 72 na 28 mL H2/g VS [20]
sewage treatment center)

Rice straw Mixed culture (Activated sewage sludge from 35 4.0–5.5 72 na 8.61–14.67 NmL H2/g [21]
a wastewater treatment plant) VS

Rice straw Mixed culture (Sludge from a bamboo park) 37 5.5 – 446.5–771.0 mL H2/L 1.89–4.39 mL H2/g [22]
biomass
Rice straw Mixed culture (Sludge from a settling 37 and 55 7.5 – 212.9–270.1 mL H2/L 40.04 mL H2/g VS rem [18]
channel)

Rice straw Mixed culture (Anaerobic sludge from an 55 6.5 – na 0.3–15.4 mL H2/g VS [54]
anaerobic digestion plant of a dairy farm;
waste activated sludge from a municipal
wastewater treatment plant)

Rice husk Mixed culture (Sludge from a settling 37 and 55 7.5 – 92.75–139.65 mL H2/ ~31 mL H2/g VS rem [18]
channel) L

Rice husk Mixed culture (Anaerobic sludge from a 35 7.0–7.5 – na 320.6–473.1 mL H2/g [23]
reactor treating brewery wastewater) biomass

Rice husk Clostridium termitidis ATCC-21846 and 37 7.5 – 29.26 mL H2 5.9 mL H2/g biomass [24]
Clostridium intestinale ATCC-BAA 1027
Rice bran Clostridium acetobutylicum YM1 30–38 5.5–6.5 – 969–2873 mL H2/L 38.52–117.24 mL H2/ [25]
g sugar
Rice bran Mixed culture (Sludge from a settling 37 and 55 7.0 – 173.65–252.4 mL H2/ 38.6 mL H2/g VS rem [18]
channel) L

Rice bran Enterobacter ludwigii 37 6.0 – 545 mL H2/L 266.4 mL H2/g [26]
biomass
Corn stover Thermoanaerobacterium 60 7.0 – 108.5 mmol H2/L na [27]
thermosaccharolyticum W16
Corn stover Enterobacter aerogenes 35 6.5 72 na 36.08 mL H2/g [28]
biomass
Cornstalk Mixed culture (Anaerobic sludge from a 36 7.0 – na 126.22 mL H2/g [29]
natural river) biomass

Corn cobs Mixed culture (Anaerobic sludge from a Mesophilic 5.5 – na 5–141 mL H2/g COD [30]
wastewater treatment plant) initial

Corn bran residue Mixed culture (Sludge from a municipal Thermophilic 6.6–8.5 – 1025.19–2648.41 mL 176–338.91 mL H2/g [31]
wastewater treatment plant) H2/L VS

Wheat straw Mixed culture (Enriched hydrogenogenic 70 – 72 na 147.7–317.6 mL H2/g [32]

culture from a lab scale CSTR fed with xylose) sugar

Wheat straw Escherichia coli WDHL 28–46 5.5–7.5 – 0–4629.09 mL H2/L 0–269.2 mL H2/g TRS [33]
Wheat powder Mixed culture (Anaerobic sludge from a 37 5.0–6.0 1, 2, 3, 140–4500 mL H2/L d 20–646 mL H2/g total [34]
wastewater treatment plant) 4.5, 6, sugar
8
Wheat powder Mixed culture (Anaerobic sludge from a 37 6.0 2, 4, 6, 773.33–1754.76 mL 0.6–1.6 mol H2/mol [35]
wastewater treatment plant of a baker yeast 10, 13 H2/L d total sugar
company)

Sorghum rusk Clostridium beijerinckii KCTC 1785 30–45 6.0–7.5 24 186–1117 mL H2/L 1.05 mol H2/mol [36]
reducing sugar
Waste sorghum Mixed culture (Anaerobic sludge from a 37.5 4.0–7.0 24–96 369 mL H2/L 213.14 mL H2/g sugar [37]
leaves wastewater treatment plant)

Sweet sorghum Mixed culture (Sewage sludge seed) 37 7.0 84 4020.1 mL H2/L na [38]
stover
Sugarcane Mixed culture (Seed sludge from a hydrogen 37 6.0 – 4229–7019 mL H2/L na [39]
bagasse pilot)

Sugarcane Mixed culture (Anaerobic sludge from a 30 3.0–9.0 – 121.7–402.0 mL H2/L 0.092–1.211 mol H2/ [55]
bagasse domestic wastewater treatment plant) mol glucose

Sugarcane Mixed culture (Bacterial consortium from 37 5.0–7.0 – 0.33–24.06 mmol H2/ na [40]
bagasse anaerobic hydrogen-producing bioreactor) L

Sugarcane leaf 37 6.5 72 127.69 mL H2/L 18.6 ml H2/g sugar [41]

waste
(continued on next page)

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J.F. Soares et al. Renewable and Sustainable Energy Reviews 117 (2020) 109484

Table 3 (continued )
Lignocellulosic Inoculum T (� C) Initial pH HRT H2 production H2 yield Reference
Biomass (h)

Mixed culture (Anaerobic sludge from a

wastewater treatment plant)

Oat Straw Mixed culture (Anaerobic sludge from an 28 5.5 6–24 na 2.9 mol H2/mol [42]
UASB reactor located at a malt industry; hexose consumed
anaerobic sludge from an UASB reactor
located at a confectionary factory)

Oat straw Mixed culture (Anaerobic sludge from an 35 7.0 – 643.25–1374.13 mL 1.10–2.39 mol H2/mol [43]
UASB reactor located at a confectionery) H2/L reducing sugars

Pine tree wood Mixed culture (Anaerobic sludge from an 35 7.0–7.5 – na 0.31–1.27 mol H2/mol [44]
pellet UASB reactor treating brewery wastewater) sugar

Spent mushroom Mixed culture (sewage sludge from a 55 Uncontrolled – 168.8–337.6 mL H2/L 0.21–0.42 mmol H2/g [45]
compost municipal sewage treatment plant; cow dung; TVS
pig slurry)

Oil palm empty Mixed culture (Anaerobic sludge from an 35 7.0–7.5 72 na 169.53–286.98 mL [47]
fruit brunch UASB reactor) H2/g biomass

De-oiled Mixed culture (Sludge from a municipal 38–72 4.8–8.2 – 270.0–996.7 mL H2/L 5.4–20.0 mL H2/g VS [48]
Jatropha waste wastewater treatment plant)

Cashew apple Clostridium roseum ATCC 17,797 38 5.5 – 12–302.4 mL H2/L 0.08–1.89 mL H2/g [49]
bagasse biomass
Peanut shells Clostridium guangxiense 35 5.5–8.0 – na 6.4–39.9 mL H2/g [50]
ZGM211T biomass
Waste paper Mixed culture (Anaerobic sludge from a – 6.8 – 336.8–859.9 mL H2/L 15.31–139.97 mL H2/ [51]
treatment plant of the baker yeast company) g sugar

Coconut husk Enterobacter aerogenes NBRC 13534 37 7.0 48 na 0.104–0.175 mol H2/ [16]
mol reducing sugar
Brewery spent Escherichia coli 37 7.5 – 52–92 mL H2/L 100–175 mL H2/g [56]
grains culture dry weight

na: not available.

and strain procurement have been extensively used for investigations on
biohydrogen production. Nevertheless, pure cultures are highly sus­
ceptible to contaminants, for the reason which aseptic conditions are
essential. Mixed cultures are suitable for full-scale industrial applica­
tions. Mixed cultures maintenance and regulation need minimal up­
stream processing and features low operations and maintenance costs
with involvement in simpler control systems for running the entire
production pathway [78].
Mixed cultures have been preferred over pure cultures, as they pre­
sent low operational costs (savings in asepsis), simplicity of operation
and easier control, and a broader source of raw material [79,80].
Moreover, according to Mun ~ oz-Paez et al. [52] research, mixed culture
showed the ability to completely degrade furfural and partially 5-HMF
in dark fermentative biohydrogen production. However, mixed cul­
tures can contain H2-consuming species or non-hydrogen consuming
species [81], which makes necessary a pretreatment of these cultures to
deactivate H2-consuming microorganisms and activate H2-producers
(mainly Clostridium sp.).
In the literature, there are studies using inoculum from municipal
wastewater treatment plants [48,55,82], anaerobic digestion plant of a
dairy farm [17,54], a sludge pit at a palm oil mill plantation [83], UASB
reactor from a confectionery factory [43], among others. Some works
also use aerobic sludge for biohydrogen production such as Ghimire
et al. [54], Alemahdi et al. [21] and Yin and Wang [84]. Kumar et al.
[48] observed that Clostridium sp. was majorly present under the optimal
conditions for biohydrogen production.
A recent strategy that has been used to intensify the dark fermen­
tative biohydrogen production is the bioaugmentation that consists of
co-inoculation of one or two selected cultures together with indigenous
microorganisms. This strategy has resulted in a considerable increase in
biohydrogen production [85]. Kumar et al. [85] presented a review
paper about bioaugmentation in conventional dark fermentative bio­
hydrogen production. Bioaugmented processes have some advantages,
such as i) accelerating reactor start-up; ii) protecting the indigenous
microflora e.g. from organic loading shocks; ii) increasing the substrate
degrading capacity; and iv) enhancing the yield and formation rate of
the target product.
Biohydrogen production from lignocellulosic biomass is influenced
by the H2-producing microorganism, which depends on the inoculum
source and its pretreatment method. Table 4 shows the inoculum source
and inoculum treatment for dark fermentative biohydrogen production.
Pretreatment of anaerobic mixed cultures used to biohydrogen pro­
duction is performed to inhibit the activity of methanogenic bacteria
and to activate H2-producing bacteria during the fermentation process.
In the literature, there are studies using thermal, chemical, aeration and
microwave pretreatments, under different operating conditions. Ac­
cording to Wang and Wan [86], inoculum source, pretreatment method,
the specific condition of each pretreatment method and substrates
source lead to a disagreement on the optimal pretreatment method for
enriching H2-producing bacteria. However, heat treatment has been the
most used, since it is an easy and practical method for the enrichment of
H2-producing microorganisms in the mixed cultures and inhibit
H2-consumers [80,86]. Furthermore, Kumar et al. [48] demonstrated
that heat treatment was the most efficient method in comparison with
chemical, base and acid pretreatments of inoculum to produce bio­
hydrogen from de-oiled Jatropha waste. Besides, the authors identified
not only biohydrogen but also methane from chemical, base and acid
pretreatments.
As shown in Table 4, temperature and time conditions used in the
heat process range from 70 to 121 � C and from 0.17 to 24 h, resulting in
different production and yield of biohydrogen. According to Alemahdi
et al. [21] the variation of time has a more significant effect than

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J.F. Soares et al. Renewable and Sustainable Energy Reviews 117 (2020) 109484

Table 4
Inoculum source and inoculum treatment in dark fermentative biohydrogen production.
Lignocellulosic Inoculum Inoculum Treatment H2 production H2 yield Reference
Biomass treatment conditions

Rice straw Mixed culture (Sludge from a settling channel) Heat 100 � C for 212.9–270.1 mL H2/L 40.04 mL H2/g VS rem [18]
30 min
Rice straw Mixed culture (Anaerobic sludge from a sewage No – na 28 mL H2/g VS [20]
treatment center) treatment
Rice straw Mixed culture (Activated sewage sludge from a Heat 80–100 � C na 8.61–14.67 NmL H2/g [21]
wastewater treatment plant) 30–60 min VS
Rice straw Mixed culture (Sludge from a local bamboo park) Heat 95–100 � C 446.5–771.0 mL H2/L 1.89–4.39 mL H2/g [22]
for 60 min biomass
Rice straw Mixed culture (Anaerobic sludge from an anaerobic Heat 105 � C for na 0.3–15.4 mL H2/g VS [54]
digestion plant of a dairy farm; waste activated sludge 90 min
from a municipal wastewater treatment plant)
Rice husk Mixed culture (Sludge from a settling channel) Heat 100 � C for 92.75–139.65 mL H2/L ~31 mL H2/g VS rem [18]
30 min
Rice husk Mixed culture (Anaerobic sludge from a reactor treating Heat 90 � C for na 320.6–473.1 mL H2/g [23]
brewery wastewater) 30 min biomass
Rice bran Mixed culture (Sludge from a settling channel) Heat 100 � C for 173.65–252.4 mL H2/L 38.6 mL H2/g VS rem [18]
30 min
Rice bran Clostridium acetobutylicum YM1 – – 969–2873 mL H2/L 38.52–117.24 mL H2/g [25]
sugar
Rice bran Enterobacter ludwigii – – 545 mL H2/L 266.4 mL H2/g biomass [26]
Corn stover Thermoanaerobacterium thermosaccharolyticum W16 – – 108.5 mmol H2/L na [27]
Corn stover Enterobacter aerogenes – – na 36.08 mL H2/g biomass [28]
Cornstalk Mixed culture (Anaerobic sludge from a natural river) Heat 100 � C for na 126.22 mL H2/g [29]
15 min biomass
Corn cobs Mixed culture (Anaerobic sludge from a wastewater Heat 70 � C for na 5–141 mL H2/g COD [30]
treatment plant) 30 min initial
Corn bran residue Mixed culture (Sludge from a municipal wastewater Heat 80 � C for 1025.19–2648.41 mL 176–338.91 mL H2/g [31]
treatment plant) 30 min H2/L VS
Wheat straw Mixed culture (Enriched hydrogenogenic culture from a – – na 147.7–317.6 mL H2/g [32]
lab scale CSTR fed with xylose) sugar
Wheat straw Escherichia coli WDHL – – 0–4629.09 mL H2/L 0–269.2 mL H2/g TRS [33]
Wheat powder Mixed culture (Anaerobic sludge from wastewater Heat 100 � C for 140–4500 mL H2/L d 20–646 mL H2/g total [34]
treatment plant of baker yeast company) 5h sugar
Wheat powder Mixed culture (Anaerobic sludge from wastewater Heat 100 � C for 773.33–1754.76 mL 0.6–1.6 mol H2/mol [35]
treatment plant of baker yeast company) 5h H2/L d total sugar
Sorghum rusk Clostridium beijerinckii KCTC 1785 – – 186–1117 mL H2/L 1.05 mol H2/mol [36]
reducing sugar
Waste sorghum Mixed culture (Anaerobic sludge from a wastewater Heat 121 � C for 369 mL H2/L 213.14 mL H2/g sugar [37]
leaves treatment plant) 10 min

Sugarcane bagasse Mixed culture (Seed sludge from a hydrogen pilot) Heat 94 � C for 4229–7019 mL H2/L na [39]
60 min
Sugarcane bagasse Mixed culture (Anaerobic sludge from a domestic Heat 70 � C for 121.7–402.0 mL H2/L 0.092–1.211 mol H2/ [55]
wastewater treatment plant) 30 min mol glucose
Sugarcane bagasse Mixed culture (Bacterial consortium from anaerobic – – 0.33–24.06 mmol H2/L na [40]
hydrogen-producing bioreactor)
Sugarcane leaf Mixed culture (Anaerobic sludge from a wastewater Heat 121 � C for 127.69 mL H2/L 18.6 ml H2/g sugar [41]
waste treatment plant) 10 min
Oat straw Mixed culture (Anaerobic sludge from an UASB reactor Heat 100 � C for na 2.9 mol H2/mol hexose [42]
located at a malt industry; anaerobic sludge from an 30 min consumed
UASB reactor located at a confectionary factory)
Oat straw Mixed culture (Anaerobic sludge from an UASB reactor Heat 104 � C for 643.25–1374.13 mL 1.10–2.39 mol H2/mol [43]
located at a confectionery factory) 24 h H2/L reducing sugars
Pine tree wood Mixed culture (Anaerobic sludge from an UASB reactor Heat 90 � C for na 0.31–1.27 mol H2/mol [44]
pellet treating brewery wastewater in South Korea) 30 min sugar
Spent mushroom Mixed culture (sewage sludge from a municipal sewage Heat 100 � C for 168.8–337.6 mL H2/L 0.21–0.42 mmol H2/g [45]
compost treatment plant; cow dung; pig slurry) 45 min TVS
Spent mushroom Clostridium thermocellum DSM 1313 – – 25.93–49.62 mmol H2/ na [46]
compost L
Oil palm empty Mixed culture (Anaerobic sludge from an UASB reactor) Heat 90 � C for na 169.53–286.98 mL H2/ [47]
fruit brunch 30 min g biomass
Cashew apple Clostridium roseum ATCC 17,797 – – 12–302.4 mL H2/L 0.08–1.89 mL H2/g [49]
bagasse biomass
T
Peanut shells Clostridium guangxiense ZGM211 – – na 6.4–39.9 mL H2/g [50]
biomass
Waste paper Mixed culture (Anaerobic sludge from the treatment Heat 100 � C for 336.8–859.9 mL H2/L 15.31–139.97 mL H2/g [51]
plant of the baker yeast company) 2h sugar
Brewery spent Escherichia coli – – 52–92 mL H2/L 100–175 mL H2/g [56]
grains culture dry weight

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J.F. Soares et al.
variation of temperature on biohydrogen yield from rice straw. This
information demonstrates the need to determine the appropriate con­
dition for each case, since each source of inoculum has a different
composition of microorganisms. According to Table 4, heat treatment at
100 � C for 30 min is the most usual.
4.2. Temperature and pH
Temperature and pH are the most important factors in the anaerobic
fermentation process. These parameters influence H2-producing bacte­
ria and biohydrogen production. At appropriate intervals, raising the
temperature and pH may increase the ability of H2-producing bacteria to
produce biohydrogen during fermentation. However, very high levels
may decrease this capacity [86]. According to Łukajtis et al. [87], higher
biohydrogen yields are achieved with thermophilic bacteria, whereas
volumetric biohydrogen production rates are better with mesophilic
bacteria, as a result of slower proliferation and lower cell densities.
The control of pH is a strategy that has been adopted to suppress the
formation of CH4 in the bacterial sources of inoculum. Additionally, pH
regulates H2 metabolism and drifts between solventogenesis and
acidogenesis. Besides, pH would play a main role in suppressing the
methanogenic activity, which is very crucial for biohydrogen fermen­
tation [88]. Several studies have demonstrated the influence of tem­
perature and pH on the biohydrogen production via anaerobic
fermentation. According to Table 3, temperature and pH values ranged
from 28 to 72 � C and from 3.0 to 9.0, respectively, showing different
performances in the papers. However, mesophilic temperatures (around
37 � C) and pH near 7.0 are the most used conditions.
Anaerobic fermentation can be performed at mesophilic (25–40 � C),
thermophilic (40–65 � C), extreme thermophilic (65–80 � C) or hyper
thermophilic (>80 � C) temperatures [89]. Sattar et al. [18] quantified
the production of biohydrogen in mesophilic (37 � C) and thermophilic
(55 � C) conditions from rice straw, rice husk, rice bran, and rice waste.
Initial pH was set at 7.0 in rice bran and rice waste fermentations and at
7.5 for straw and husk fermentations. Results showed that the bio­
hydrogen production potential increased with an increase in tempera­
ture from 37 to 55 � C for all substrates, except for rice waste. The
average yield of biohydrogen obtained from rice crop wastes was 30.36
and 33.16 mL/g VS removed under mesophilic and thermophilic con­
ditions, respectively. Authors also observed that mesophilic pH decrease
was higher than thermophilic decrease in the rice straw, rice husk, and
rice waste fermentations, whereas rice bran gives an opposite trend. The
optimum pH range for biohydrogen production from straw, bark, and
rice bran is between 6 and 7 and in case of rice waste from pH 5.5 to pH
4.
The pH control of the medium can be carried out throughout the
fermentation process, which would make the operation economically
unfeasible due to the large use of reagents, or only at the start of the
fermentation process. The control of the initial pH has been adopted in
most of the studies [18,25,65,90]. Phowan and Danvirutai [91] inves­
tigated the effect of initial pH (5.0–8.0) on fermentative biohydrogen
production from cassava pulp hydrolysate. All the experiments were
carried out at a constant temperature of 35 � C. The initial pH of 5.5 led to
the highest cumulative volume of biohydrogen (358 mL), while the
initial pH of 8.0 gave the lowest cumulative volume of biohydrogen
(125 mL). Reddy et al. [55] studied the effect of initial pH (3.0–9.0) on
biohydrogen production from sugarcane bagasse hydrolysate, conduct­
ed at 30 � C. According to the results, when the initial pH was increased
from 3.0 to 5.0, biohydrogen yield increased from 0.391 to 0.842 mol
H2/mol glucose, respectively. However, an increase in pH to 9.0 resulted
in a drastic drop in biohydrogen yield to 0.562 mol H2/mol glucose.
Azman et al. [25] investigated the effect of initial pH (5.5–6.5) and
temperature (30–38 � C) on biohydrogen production using de-oiled rice
bran. The highest yield of biohydrogen (117.24 mL H2/g sugar
consumed) was reached with a temperature of 34 � C and pH of 5.5,
while the highest volume of cumulative biohydrogen (574.6 mL) was
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Renewable and Sustainable Energy Reviews 117 (2020) 109484
obtained with a temperature of 34 � C and pH of 6.5. Lopez-Hidalgo et al.
[33] evaluated the influence of pH (4.8–8.2) and temperature
(21.9–52.1 � C) on biohydrogen production by using wheat straw hy­
drolysate. The highest production (509.2 mL H2) and yield (269.2 mL
H2/g TRS) were obtained with pH of 6.5 and temperature of 37 � C, while
the highest production rate (25.27 mL H2/L.h) was achieved with pH of
8.2 and temperature of 37 � C.
Wongthanate et al. [90] evaluated the influence of initial pH
(4.5–7.0) and temperature (35 and 50 � C) on biohydrogen production
using wastewater from coconut milk as a substrate and anaerobic sludge
as a source of microorganisms. Results showed that the initial pH of 6.5
in mesophilic conditions (35 � C) favors biohydrogen production. The
biohydrogen production was two times higher in mesophilic (35 � C,
0.28 L H2/L) than in thermophilic (50 � C, 0.16 L H2/L) temperature.
According to the authors, the temperature can affect the activity of
H2-producing bacteria by influencing the activity of some essential en­
zymes, such as hydrogenase. After 7 days of fermentation, under 35 and
50 � C temperature conditions, the initial fermentation pH of 6.5 dropped
to 4.5. And, Kumar et al. [48] studied the effect of temperature
(45–65 � C) and initial pH (5.5–7.5) in the biohydrogen production using
de-oiled Jatropha waste as substrate. The better result (1.4 L/L.d) was
obtained with pH 6.5 and 55 � C. As can be seen, the optimal conditions
of pH and temperature are different in the mentioned studies, which can
be mainly explained by the type of substrate and source of inoculum
used.
4.3. Hydraulic retention time
Hydraulic retention time (HRT) is another operational condition of
the anaerobic fermentation process that influences microbial meta­
bolism and, consequently, biohydrogen production and final products of
fermentation. Furthermore, Bakonyi et al. [92], reported that HRT,
together with the solid retention time, is also a crucial factor for
anaerobic biohydrogen reactors performance. According to Table 3, a
wide range of HRT has been used (from 1 to 96 h). However, 72 h was
the most frequently used HRT. In the literature, there are studies that
evaluate the influence of different HRT on the biohydrogen production
process, and different results have been obtained [35,42,72,93–98].
Arriaga et al. [42], for example, identified that with decreasing HRT
from 24 to 6 h, biohydrogen production rate from oat straw hydrolysate
increased (up to 81.4 mL H2/L reactor h) but biohydrogen yield
decreased (2.9 mol H2/mol hexose consumed down to 0.2 mol H2/mol
hexose consumed).
Antonopoulo et al. [93] evaluated different hydraulic retention times
(4, 6, 8, 12 and 24 h) in biohydrogen production from sorghum extract.
Biohydrogen production rate increased when HRT decreased from 24 to
6 h (410–2550 mL H2/d) and decrease for 4 h of HRT (2180 mL H2/d),
while the highest biohydrogen yield (0.86 mol H2/mol glucose) was
obtained in 12 h. The main by-products of the fermentation were butyric
acid and acetic acid. The butyric acid concentration was higher
(5780 mg/L) for the 12 h of HRT, while the higher acetic acid concen­
tration (3480 mg/L) occurred in the 8 h. The concentration of propionic
acid increased, and lactic acid decreased with the increase of HRT,
whereas ethanol production was favored in 6 and 4 h (634 mg/L).
Kumar et al. [99] studied mesophilic biohydrogen production from
de-oiled jatropha waste in a continuous system at various HRTs ranging
from 48 to 12 h. The experimental results showed that the highest bio­
hydrogen production rate and yield were 0.9 L/L.d and 86 mL/g
reducing sugars, respectively, at 16 h HRT, with butyrate as the pre­
dominant volatile fatty acid. Kirli and Karapinar [35] investigated the
effect of HRT (2–13 h) on biohydrogen production from acid hydrolyzed
waste wheat. Experimental results indicated that biohydrogen produc­
tion increases with decreasing HRT. The highest biohydrogen produc­
tion volume and yield were 1.75 L H2/L.d and 1.6 moL H2/mol TS,
respectively, at 2 h HRT. From these results, an inconsistency in the data
can be verified. The behavior of biohydrogen yield and production rate
J.F. Soares et al.
is different for different HRT. Furthermore, better HRT was not the same
from these works.
4.4. H2 partial pressure
H2 partial pressure is a very important factor for the continuous
production of biohydrogen because production pathways are sensitive
to H2 concentrations. As H2 concentrations increase, biohydrogen pro­
duction decreases and metabolic pathways shift to other substrates, such
as ethanol, acetone, butanol, lactate, or alanine. On the other hand, as
the process temperature increases, conditions that favor reaction are less
influenced by H2 concentration [89]. Considering the negative effects on
the biohydrogen production caused by the increase of its partial pres­
sure, some methods have been studied to reduce this problem. Among
these, the most common are nitrogen gas (N2) and carbon dioxide (CO2)
sparging and vacuum [100–102].
Kim et al. [101] investigated the effect of gas sparging (internal
biogas, N2, and CO2) on fermentative biohydrogen production using
different flow rates (100–400 mL/min). Sparging with external gases
(N2 and CO2) showed higher biohydrogen yield when compared to in­
ternal biogas sparging and the control (no sparging). Among external
gases, CO2 presented higher efficiency and higher biohydrogen pro­
duction than N2. In addition, CO2 has little effect on H2-producing
bacteria and inhibitory effect on acetogenic and lactic acid-producing
microorganisms, which are competitive with H2-producers. The best
result was obtained by CO2 sparging of 300 mL/min, resulting in the
highest yield (1.68 mol H2/mol consumed hexose) and the highest
production rate (6.89 L H2/g VSS.d) of biohydrogen.
The effect of reduced pressure (by vacuum) on biohydrogen pro­
duction in a CSTR reactor was investigated by Lee et al. [102]. The
pressures were analyzed from 130 to 760 mmHg in an HRT of 12 h.
Biohydrogen production rate increased slightly (about 8%) from
0.348 mol/L.d to 0.376 mol/L.d when the pressure decreased from 760
to 380 mmHg and remained the same when the pressure was reduced
from 380 to 130 mmHg. Biohydrogen yield and biohydrogen production
efficiency presented the same tendency as biohydrogen production rate.
The highest yield and production efficiency were 3.57 mol H2/mol su­
crose and 45.1%, respectively, at a pressure of 380 mmHg.
4.5. Fermentation end-products
Biohydrogen production by fermentative bacteria produces organic
acids as end-product, which causes a decrease in pH and inhibition of
H2-producing bacteria [77]. The composition of fermentation
end-products depends on the environmental conditions of the medium
[89], with acetic (137–3480 mg/L), butyric (119–5780 mg/L) and pro­
pionic (160–1920 mg/L) acids being the major constituents [103]. Other
fermentation end-products are also produced, but in reduced pro­
portions, such as ethanol (31–634 mg/L), butanol (35–350 mg/L) and
lactic (760–1080 mg/L) acid, which contain hydrogen that has not been
released as gas [89].
In general, the production of acetic and butyric acid favors bio­
hydrogen production. When acetic acid is the final product, a theoretical
maximum of 4 mol H2/mol glucose (Equation (1)) is obtained, and when
the butyric acid is the final product, the theoretical maximum is reduced
to 2 mol H2/mol glucose (Equation (2)). Thus, the higher theoretical
biohydrogen yields are associated with acetic acid as fermentation end-
product. On the other hand, propionic acid production consumes bio­
hydrogen (Equation (3)), and the biohydrogen balance in the lactic acid
and ethanol production pathways is zero. Thus, low yields are associated
with propionic acid and reduced end-products (alcohols and lactic acid)
[89,93,104].
C6H12O6 þ 2H2O → 2CH3COOH þ 2CO2 þ 4H2 (1)
C6H12O6 → CH3CH2CH2COOH þ 2CO2 þ 2H2 (2)
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Renewable and Sustainable Energy Reviews 117 (2020) 109484
C6H12O6 þ 2H2 → 2CH3CH2COOH þ 2H2O (3)
Ghimire et al. [17] identified acetic (9.2–63.4 mM), butyric
(4.9–22.1 mM) and propionic (0.7–13.3 mM) acids as the main
by-products of six different types of waste biomass used for biohydrogen
production (buffalo manure, olive oil mill wastewater, olive pomace,
fennel waste, dried blood, potato waste and pumpkin waste). Antono­
poulo et al. [93], using sweet sorghum biomass as a source for
fermentative biohydrogen production, found butyric acid (5780 mg/L)
and acetic acid (3480 mg/L) as major end-products. In addition, propi­
onic acid, ethanol and lactic acid were also produced, but in a lesser
amount.
Butyric and acetic acids were also identified by Liu et al. [105]
(1.42–4.82 g butyric acid/L and 1.80–2.31 g acetic acid/L) and Ghimire
et al. [54] (46.4–614.1 mM butyric acid/kg VS and 462.6–775.0 mM
acetic acid/kg VS) as the main metabolites of biohydrogen production
from rice straw hydrolysate. On contrary, Han et al. [82] found as main
end-products ethanol (157.4–520.1 mg/L) and acetic acid
(137.0–394.8 mg/L) in the biohydrogen fermentation from food waste
hydrolysate, accounting for 40.9–49.4% and 35.6–42.2% of total
end-products, respectively.
5. Emerging strategies to improve dark fermentative
biohydrogen production
In recent years, studies have been developed to improve dark
fermentative biohydrogen production from the use of additives. How­
ever, there are still few studies using these additives in dark fermenta­
tive biohydrogen production from lignocellulosic biomass. According to
Yang and Wang [106], only 9.4% of researched papers from 2001 to
2017 used lignocellulosic biomass as a substrate in dark fermentation
with additives. The main used additives were metal monomers (Fe0, Au,
Ag, Ni, Pd, Cu), metal ions (Fe2þ, Ni2þ, Mg2þ, Ca2þ, Na2þ), and metal
oxides (CoO, Fe2O3, Fe3O4, NiO, and TiO2) [106]. Among these, iron is
the most used. The addition of metal into dark fermentation media fa­
cilitates intracellular electron transportation and provided the essential
nutrition for microbial growth [107]. Reddy et al. [55], for example,
evaluated the concentration of Fe2þ and magnetite nanoparticles on
biohydrogen production from sugarcane bagasse hydrolysate. The
addition of Fe2þ 200 mg/L and magnetite nanoparticles 200 mg/L
enhanced the biohydrogen yield by 62.1% and 69.6%, respectively. In
addition, high concentrations of H2-producing communities and hy­
drogenase gene were observed.
Camacho et al. [108] studied the effects of Fe0 (1 g/L and 2 g/L) in
the dynamics of biohydrogen dark fermentation from organic market
waste. The results showed a 46% increase in the amount of gas and a
greater H2/CO2 ratio in the presence of 2 g/L compared with the control
test (0 g/L). According to the authors, the action of Fe0 led to an
enhancement of key enzyme activity, increasing biohydrogen produc­
tion. Hwang et al. [109] also investigate the Fe0 addition to enhance
biohydrogen fermentation. However, glucose was used as a substrate.
The addition of Fe0 increased biohydrogen yield by up to 54%. Ac­
cording to the authors, Fe0 creates a more favorable environment for
H2-producing microorganisms by providing a buffering effect to prevent
pH drop due to produced hydroxyl ions as it dissolved. Zhang et al. [110]
and Lin et al. [111] evaluated the effect of ferric oxide/carbon nano­
particles and ferric oxide nanoparticles, respectively, on the dark
fermentative biohydrogen production from glucose. Zhang et al. [110]
obtained an increase of 33.7% in biohydrogen yield with 200 mg/L
ferric oxide/carbon nanoparticles, while Lin et al. [111] achieved only
16.96%. However, using pre-treated cassava starch as a substrate, Lin
et al. [111] reached an increase of up to 63.12%. Mishra et al. [112]
investigated the biohydrogen production from palm oil mill effluent
with the addition of NiO and CoO nanoparticles. Results showed that the
addition of optimal concentration of NiO (1.5 mg/L) and CoO
(1.0 mg/L) nanoparticles enhances the biohydrogen yield by 1.51 and
J.F. Soares et al.
1.67 times, respectively, when compared with the control. From these
data, it is possible to observe the difference of nanoparticle effects on
biohydrogen production and its optimum concentration.
Other interesting studies were developed by Wang et al. [113], Wang
et al. [114] and Wu et al. [115]. which tested for the first time the use of
free nitrous acid, calcium peroxide, and poly aluminum chloride,
respectively, to improve biohydrogen production from dark fermenta­
tion of waste activated sludge. It was observed in these works that the
three substances caused severe inhibition to H2-consumers and pro­
moted the biodegradability of organics released, providing more
biodegradable substrates for biohydrogen production. Wang et al. [113]
identified that with an increase of nitrite from 0 to 250 mg/L, the H2
yield increased from 8.5 to 15.0 mL/g VSS at pH 5.5 and 8.1–13.0 mL/g
VSS at pH 6.0. The results of Wang et al. [114] indicated that with CaO2
increased from 0.05 to 0.25 g/g VSS, the biohydrogen yield increased
from 0.77 to 10.55 mL H2/g VSS. And the results of Wu et al. [115]
showed that with an increase of poly aluminum chloride addition from
0 to 20 mg Al/g TSS, the biohydrogen yield increased from 20.9 mL to
27.4 mL/g VSS. These researches can serve as an impulse for other
studies that use lignocellulosic biomass and mixed culture as an inoc­
ulum sources.
Considering the complex structure of lignocellulosic biomass, studies
have also been developed to improve the breakdown of cellulose and
hemicellulose fraction. Perfluoroalkylsulfonic and alkylsulfonic acid-
functionalized magnetic nanoparticles, for example, were evaluated
for Pen ~ a et al. [116] in the wheat straw hydrolysis. The results showed
an efficiency of up to 66.3% of the hemicellulose conversion. On the
other hand, Fe3O4 nanoparticles and Fe3O4/Alginate nanocomposites
[117], and MnO2 nanoparticles [118], were used to enhance enzymatic
pretreatment of lignocellulosic biomass. According to Srivastava et al.
[117], enzyme treatment with Fe3O4 nanoparticles and Fe3O4/Alginate
nanocomposites significantly enhanced cellulase production, enzyme
activities and improved thermostability. Besides, using Fe3O4/Alginate
treated enzyme 42.4 g/L of glucose was produced, while about 33.0 g/L
of glucose was produced using untreated cellulase. Cherian et al. [118]
observed that the properties of the immobilized cellulase on MnO2
nanoparticles were more stable than the free enzyme. Also, the reus­
ability of cellulase increased considerably after immobilization, retain­
ing 60% activity even after five cycles. Finally, the study of alternatives
to improve dark fermentative biohydrogen production as well as to
promoting lignocellulosic biomass pretreatment is very important to
make biohydrogen dark fermentation more efficient, economical and
sustainable on a large scale.
6. Environmental and economic aspects of dark fermentative
biohydrogen production
World demand for hydrogen is about 70 million tonnes/year, mostly
consumed in ammonia production and oil refining. Therefore, the sup­
ply of low-carbon hydrogen is necessary, because of its potential utili­
zation in vehicle, industrial feedstock, and fuel in building heating. As a
result, biological processes such as dark fermentation has been exten­
sively studied in a laboratory scale using different sources of biomass. A
study of Manish and Banerjee [120] indicated a reduction of 57% (7.3 kg
CO2) and 67% (126.3 MJ) in greenhouse gas emissions and
non-renewable energy use, respectively, in the production of 1.0 kg H2
from dark fermentation compared to steam methane reforming (SMR).
In addition, the net energy ratio of SMR is 0.64 while dark fermentation
is 1.9.
However, the net energy ratio of H2 production from SMR and dark
fermentation are 0.6 and 0.67, respectively, if a harmonized life cycle
study is considered, i.e. the life cycle from both process is on the same
basis of comparison [121]. Also, in terms of energy output, dark
fermentation has lower efficiency if compared to second-generation
ethanol, resulting in 5.9 times less energy produced from the same
feedstock (corn stover) [122]. Therefore, comparing different pathways
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Renewable and Sustainable Energy Reviews 117 (2020) 109484
to produce hydrogen is necessary, and life cycle assessment is an
important methodology used in this effort. Ochs et al. [123] demon­
strated in a LCA study that hydrogen from a combined dark and photo
fermentation have an impact 5.7 higher than SMR, most from the use of
phosphate in the fermentation process. Mehmet et al. [124] showed that
hydrogen from SMR has a GWP potential of 12.13 kg CO2eq/kg H2, while
hydrogen production combining dark fermentation (DF) and microbial
electrolysis cell (MEC) with energy recovery (ER)1 has a GWP of 9.80 kg
CO2eq/kg H2. GWP for hydrogen production from DF/MEC without
energy recovery is 19.00 kg CO2eq/kg H2. Also, hydrogen from SMR
consumes 4.0 times less water than in DF/MEC-ER process [124]. In
general, SMR proved to be a less impacting process compared to
DF/MEC-ER in 15 out of 18 mid-point impact categories [124], sug­
gesting that dark fermentation needs technological improvements in its
process efficiency in the way to be an environmentally friendly option to
displace fossil fuels.
Several articles have shown that lignocellulosic biomass is a poten­
tial source for fermentative biohydrogen production. Although the
lignocellulosic biomass is available in large amount worldwide,
exceeding 220 billion tons per year [12], studies are still limited on a
laboratory scale. Bundhoo [125] was the first to estimate the worldwide
potential of biohydrogen production from dark fermentation of 25 crop
residues. Among these biomasses, wheat and rice straws are produced in
greater amount. According to the author, it is estimated that 58,
002 Mm3/year and 34,680 Mm3/year of biohydrogen can be produced
worldwide by untreated rice straw and untreated wheat straw, respec­
tively, which corresponds to a bio-energy potential of 623 PJ/year and
373 PJ/year.
In relation to its utilization, H2 is the most dangerous gas for ex­
plosion [126]. H2 have the wider range of inflammability limit
(9.5–66.3% of inflammable gas in air) and the low temperature at which
explosion occurs (769 � C) when compared with other gases (e.g. carbon
monoxide, coal gas, methane, acetylene, etc.). On the other hand, bio­
hydrogen has many advantages when compared with fossil fuels or even
other renewables. Unlike fossil fuels, H2 combustion results in a large
amount of energy, releasing only water vapor, with no pollutants and
greenhouse gas emissions. Even in the case of incomplete combustion,
the products are water vapor and hydrogen [126]. Moreover, hydrogen
has the highest energy content of any known fuel [7], as can be seen in
Table 5.
Therefore, H2 is very attractive as a fuel considering its heating
value, although its cost has been prohibitive. The use of low-carbon
hydrogen blended in natural gas - limited to 20% of H2 for safety rea­
sons, is an effective option to reduce CO2 emissions. It is estimated that
using low-carbon hydrogen could reduce European emissions in 60 Mt
CO2 [6].Table 6 shows the costs of natural gas and different sources of
Table 5
Lower heating value of different fossil and renewable fuels.
Fuel Lower heating value (MJ/kg)
Hydrogen 120.210
Natural gas 47.141
Propane 46.296
Butane 45.277
Conventional gasoline 43.448
Low sulfur diesel 46.612
Ethanol 26.952
Biodiesel (methyl ester) 37.528
Source: Biomass Energy Databook [127].
1
Effluent from the dark fermentation process is used in a MEC to produce
hydrogen. Energy recovery is the utilization of cellulose, lignin, and biogas
from the process in a boiler to generate steam and electricity [122].
J.F. Soares et al.
biohydrogen. Natural gas is presented because is the most consumed
gaseous fuel worldwide, including utilization in vehicles, and bio­
hydrogen could be a renewable substitute for it. Data from Table 6
inform that biohydrogen is far away to be competitive with natural gas
prices, even hydrogen produced via conventional process (SMR and
electrolysis). Biohydrogen produced through dark fermentation is even
more expensive. The cost of hydrogen projected to 2025 (5.65 US$/kg)
is 42 times the price of natural gas (0.134 US$/kg), although the costs
based on energy density is 16 times higher for biohydrogen projected to
2025 (47,000 US$/106 MJ) in relation to natural gas (2840 US
$/106 MJ).
According to Ren et al. [130], the substrate used in the fermentative
process has a major influence in biohydrogen costs, indicating the ne­
cessity of utilization of low-price raw material, as is the case of ligno­
cellulosic biomass and industrial residues. Despite the low cost of
lignocellulosic biomass, its processing requires pre-treatment and pro­
cess conditions that usually are severe and more expensive. Therefore,
sugar or starch based-residues or wastewater from industry become very
attractive, as is the case presented in different studies [128,131–134].
In a study of Han et al. [132] it was found that the higher the pro­
duction scale (50 m3), the higher the feasibility. Therefore, dark
fermentative biohydrogen production requires more advanced studies
on large scale to overcome technological and economic barriers in the
way to become a viable and competitive technology. According to
Randolph and Studer [135], the projected cost of biohydrogen produc­
tion from dark fermentation of biomass (corn stover) using techniques
and strains currently in development at a laboratory scale is greater than
US$50/kg (untaxed, high production rates). However, it is expected to
drop dramatically to $5.65/kg in 2025, if assumed improvements in the
technology and high volumes are realized.
Other more economical alternative is the production of biohydrogen
– to be used to produce electricity in a fuel cell – as a by-product from
wastewater and municipal waste treatment [112,136–140], or alongside
chemicals, fertilizers, and other biofuels (ethanol, methane) [22,33,69,
133,141–143]. In the study of Bonk et al. [133], for example, volatile
fatty acids, fertilizer, and biohydrogen are produced from the organic
fraction of municipal solid waste. In this case, biohydrogen is not the
main product in the dark fermentation process. On the other hand,
Kumar et al. [144] reported the possibility of using microbial electro­
chemical systems as a post-treatment for dark fermentation in order to
harness extra biohydrogen energy and thus, improve the emerging
hydrogen energy sector. Also, Singh et al. [145] cited that dark
fermentation followed by photo fermentation is the most promising and
economic technology, because photo fermentation consumes uncon­
verted metabolites, mainly acetic acid, from dark fermentation
increasing the process efficiency.
Finally, dark fermentative biohydrogen production still has a long
way to become a competitive technology. In this regard, to obtain value-
added products (e.g. organic acids, fertilizer, ethanol, methane) in the
same process, especially as a technology used in wastewater and waste
treatment, or combine more than one processes may be alternatives to
improve techno-economic feasibility.
7. Conclusions and future prospects
Dark fermentative biohydrogen production represents a promising
area of technology development for renewable energy generation. In
this regard, many studies have been developed on a laboratory scale to
find pathways to improve the yield and rates of H2 production. Ligno­
cellulosic biomass has been considered a potential feedstock for bio­
hydrogen production since it is renewable, sustainable, and available in
large quantities. However, this type of biomass presents a complex
structure, requiring a pre-treatment before the fermentation process to
increase the amount of monomeric sugars needed for H2-producing
microorganisms. This factor has been the main challenge of process
since each pretreatment method is better adjusted to a particular
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Renewable and Sustainable Energy Reviews 117 (2020) 109484
Table 6
Price of natural gas and cost of H2 from different production technologies.
Fuel US$/kg Reference
a
Natural gas 0.134 [119]
Steam methane reforming 1.0–2.0 [119]
Electrolysis 4.0–6.0 [119]
Biohydrogen production from waste bread 14.89 [128]
Current (2015) fermentation biohydrogen 51.02 [129]
Future (2015) fermentation biohydrogen 5.65 [129]
a
Price of natural gas in US (3.0 US$/MBtu).
biomass. In this regard, some studies have been developed to enhance
the breakdown of lignocellulosic biomass using additives (e.g. metal
nanoparticles) in the pretreatment.
According to the research, mixed cultures have been the most widely
used in biohydrogen production because they present low operational
costs, simplicity of operation and easy control, and a wide source of raw
material. Heat treatment (at 100 � C for 30 min) of these cultures has
been the main method used. Inoculum source, temperature, pH, hy­
draulic retention time, gas partial pressure, and fermentation end-
products showed to be important factors that influence biohydrogen
production via dark fermentation. Temperature and pH values ranged
from 28 to 72 � C and from 3.0 to 9.0, respectively, showing different
performances in the papers. In this sense, it is extremely important to
study these factors on the fermentation process to maximize bio­
hydrogen production. The use of additives, such as iron materials, is
another alternative that has been used to improve dark fermentative
biohydrogen production.
Biohydrogen produced through dark fermentation is still very
expensive. Moreover, most researches produce biohydrogen on a labo­
ratory scale. In this regard, dark fermentative biohydrogen production
requires more advanced studies on large scale to overcome technolog­
ical and economic barriers in the way to become a viable and compet­
itive technology. The use of additives in the lignocellulosic biomass
pretreatment and in the dark fermentation and the production of value-
added compounds (e.g. organic acids, fertilizer, ethanol, methane) in the
same fermentation process may be excellent alternatives to improve the
techno-economic aspects of biohydrogen production.
Funding
This study was financed in part by the Coordenaça ~o de Aperfeiçoa­
mento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.
Declaration of competing interest
None.
Acknowledgements
The author would like to acknowledge the Coordenaça ~o de Aper­
feiçoamento de Pessoal de Nível Superior - Brasil (CAPES) for financial
assistance.
References
[1] International Energy Agency. World energy balances: overview. 2018. https://
doi.org/10.15713/ins.mmj.3.
[2] Das D, Veziroglu TN. Hydrogen production by biological processes: a survey of
literature. Int J Hydrogen Energy 2001;26:13–28. https://doi.org/10.1016/
s0360-3199(00)00058-6.
[3] Watts N, Amann M, Ayeb-Karlsson S, Belesova K, Bouley T, Boykoff M, et al. The
Lancet Countdown on health and climate change: from 25 years of inaction to a
global transformation for public health. Lancet 2017;391:581–630. https://doi.
org/10.1016/s0140-6736(17)32464-9.
[4] Watts N, Amann M, Arnell N, Ayeb-Karlsson S, Belesova K, Berry H, et al. The
2018 report of the Lancet Countdown on health and climate change: shaping the
health of nations for centuries to come. Lancet 2018;392:2479–514. https://doi.
org/10.1016/S0140-6736(18)32594-7.
J.F. Soares et al.
[5] Abe JO, Popoola API, Ajenifuja E, Popoola OM. Hydrogen energy, economy and
storage: review and recommendation. Int J Hydrogen Energy 2019. https://doi.
org/10.1016/j.ijhydene.2019.04.068.
[6] International Energy Agency. World energy outlook 2018. 2018.
[7] Bharathiraja B, Sudharsanaa T, Bharghavi A, Jayamuthunagai J,
Praveenkumar R. Biohydrogen and Biogas – an overview on feedstocks and
enhancement process. Fuel 2016;185:810–28. https://doi.org/10.1016/j.
fuel.2016.08.030.
[8] Hallenbeck PC, Benemann JR. Biological hydrogen production; Fundamentals
and limiting processes. Int J Hydrogen Energy 2002;27:1185–93. https://doi.org/
10.1016/B978-008044356-0/50008-7.
[9] Krishna RH, Krishna RH. Review of research on production methods of Hydrogen:
future fuel. Eur J Biotechnol Biosci 2013;1:84–93.
[10] Bakonyi P, Nemest� othy N, Simon V, B�elafi-Bak� o K. Review on the start-up
experiences of continuous fermentative hydrogen producing bioreactors. Renew
Sustain Energy Rev 2014;40:806–13. https://doi.org/10.1016/j.
rser.2014.08.014.
[11] Nasirian N, Almassi M, Minaei S, Widmann R. Development of a method for
biohydrogen production from wheat straw by dark fermentation. Int J Hydrogen
Energy 2011;36:411–20. https://doi.org/10.1016/j.ijhydene.2010.09.073.
[12] Kumar G, Bakonyi P, Periyasamy S, Kim SH, Nemest� othy N, B�elafi-Bak�
o K.
Lignocellulose biohydrogen: practical challenges and recent progress. Renew
Sustain Energy Rev 2015;44:728–37. https://doi.org/10.1016/j.
rser.2015.01.042.
[13] Travaini R, Martín-Ju� arez J, Lorenzo-Hernando A, Bolado-Rodríguez S.
Ozonolysis: an advantageous pretreatment for lignocellulosic biomass revisited.
Bioresour Technol 2016;199:2–12. https://doi.org/10.1016/j.
biortech.2015.08.143.
[14] Chen H. Biotechnology of lignocellulose: theory and practice. 2014. https://doi.
org/10.1007/978-94-007-6898-7.
[15] Bajpai P. Structure of lignocellulosic biomass. Pretreat. Lignocellul. Biomass
Towar. Biofuel Prod. 2016:7–12. https://doi.org/10.1007/978-3-319-67678-4_9.
[16] Muharja M, Junianti F, Ranggina D, Nurtono T, Widjaja A. An integrated green
process: subcritical water, enzymatic hydrolysis, and fermentation, for
biohydrogen production from coconut husk. Bioresour Technol 2018;249:
268–75. https://doi.org/10.1016/j.biortech.2017.10.024.
[17] Ghimire A, Frunzo L, Pontoni L, d’Antonio G, Lens PNL, Esposito G, et al. Dark
fermentation of complex waste biomass for biohydrogen production by pretreated
thermophilic anaerobic digestate. J Environ Manag 2015;152:43–8. https://doi.
org/10.1016/j.jenvman.2014.12.049.
[18] Sattar A, Arslan C, Ji C, Sattar S, Umair M, Sattar S, et al. Quantification of
temperature effect on batch production of bio-hydrogen from rice crop wastes in
an anaerobic bio reactor. Int J Hydrogen Energy 2016;41:11050–61. https://doi.
org/10.1016/j.ijhydene.2016.04.087.
[19] Gonzales RR, Sivagurunathan P, Kim SH. Effect of severity on dilute acid
pretreatment of lignocellulosic biomass and the following hydrogen fermentation.
Int J Hydrogen Energy 2016;41:21678–84. https://doi.org/10.1016/j.
ijhydene.2016.06.198.
[20] He L, Huang H, Lei Z, Liu C, Zhang Z. Enhanced hydrogen production from
anaerobic fermentation of rice straw pretreated by hydrothermal technology.
Bioresour Technol 2014;171:145–51. https://doi.org/10.1016/j.
biortech.2014.08.049.
[21] Alemahdi N, Che Man H, Abd Rahman N, Nasirian N, Yang Y. Enhanced
mesophilic bio-hydrogen production of raw rice straw and activated sewage
sludge by co-digestion. Int J Hydrogen Energy 2015;40:16033–44. https://doi.
org/10.1016/j.ijhydene.2015.08.106.
[22] Sen B, Chou YP, Wu SY, Liu CM. Pretreatment conditions of rice straw for
simultaneous hydrogen and ethanol fermentation by mixed culture. Int J
Hydrogen Energy 2016;41:4421–8. https://doi.org/10.1016/j.
ijhydene.2015.10.147.
[23] Gonzales RR, Kim SH. Dark fermentative hydrogen production following the
sequential dilute acid pretreatment and enzymatic saccharification of rice husk.
Int J Hydrogen Energy 2017;42:27577–83. https://doi.org/10.1016/j.
ijhydene.2017.08.185.
[24] Tosuner ZV, Taylan GG, Ozmıhçı
€ S. Effects of rice husk particle size on
biohydrogen production under solid state fermentation. Int J Hydrogen Energy
2018. https://doi.org/10.1016/j.ijhydene.2018.10.230.
[25] Azman NF, Abdeshahian P, Kadier A, Al-Shorgani NKN, Salih NKM, Lananan I,
et al. Biohydrogen production from de-oiled rice bran as sustainable feedstock in
fermentative process. Int J Hydrogen Energy 2016;41:145–56.
[26] Tandon M, Thakur V, Tiwari KL, Jadhav SK. Enterobacter ludwigii strain IF2SW-
B4 isolated for bio-hydrogen production from rice bran and de-oiled rice bran.
Environ Technol Innov 2018;10:345–54. https://doi.org/10.1016/j.
eti.2018.03.008.
[27] Ren N-Q, Cao G-L, Guo W-Q, Wang A-J, Zhu Y-H, Liu B, et al. Biological hydrogen
production from corn stover by moderately thermophile Thermoanaerobacterium
thermosaccharolyticum W16. Int J Hydrogen Energy 2010;35:2708–12. https://
doi.org/10.1016/j.ijhydene.2009.04.044.
[28] Zhang T, Jiang D, Zhang H, Jing Y, Tahir N, Zhang Y, et al. Comparative study on
bio-hydrogen production from corn stover: photo-fermentation, dark-
fermentation and dark-photo co-fermentation. Int J Hydrogen Energy 2019.
https://doi.org/10.1016/j.ijhydene.2019.04.170.
[29] Wang Y, Wang H, Feng X, Wang X, Huang J. Biohydrogen production from
cornstalk wastes by anaerobic fermentation with activated sludge. Int J Hydrogen
Energy 2010;35:3092–9. https://doi.org/10.1016/j.ijhydene.2009.07.024.
14
Renewable and Sustainable Energy Reviews 117 (2020) 109484
[30] Nasr N, Gupta M, Elbeshbishy E, Hafez H, El Naggar MH, Nakhla G. Biohydrogen
production from pretreated corn cobs. Int J Hydrogen Energy 2014;39:19921–7.
https://doi.org/10.1016/j.ijhydene.2014.10.004.
[31] Zhang J, Zhang J, Zang L. Thermophilic bio-hydrogen production from corn-bran
residue pretreated by calcined-lime mud from papermaking process. Bioresour
Technol 2015;198:564–70. https://doi.org/10.1016/j.biortech.2015.09.082.
[32] Kongjan P, O-Thong S, Kotay M, Min B, Angelidaki I. Biohydrogen production
from wheat straw hydrolysate by dark fermentation using extreme thermophilic
mixed culture. Biotechnol Bioeng 2010;105:899–908. https://doi.org/10.1002/
bit.22616.
[33] Lopez-Hidalgo AM, S� anchez A, De Le�
on-Rodríguez A. Simultaneous production of
bioethanol and biohydrogen by Escherichia coli WDHL using wheat straw
hydrolysate as substrate. Fuel 2017;188:19–27. https://doi.org/10.1016/j.
fuel.2016.10.022.
[34] Gorgec FK, Karapinar I. Biohydrogen production from hydrolyzed waste wheat by
dark fermentation in a continuously operated packed bed reactor: the effect of
hydraulic retention time. Int J Hydrogen Energy 2019;44:136–43. https://doi.
org/10.1016/j.ijhydene.2018.08.155.
[35] Kirli B, Karapinar I. The effect of HRT on biohydrogen production from acid
hydrolyzed waste wheat in a continuously operated packed bed reactor. Int J
Hydrogen Energy 2018;43:10678–85. https://doi.org/10.1016/j.
ijhydene.2018.01.175.
[36] Saratale GD, Kshirsagar SD, Saratale RG, Govindwar SP, Oh MK. Fermentative
hydrogen production using sorghum husk as a biomass feedstock and process
optimization. Biotechnol Bioproc Eng 2015;20:733–43. https://doi.org/10.1007/
s12257-015-0172-3.
[37] Rorke D, Gueguim Kana EB. Biohydrogen process development on waste sorghum
(Sorghum bicolor) leaves: optimization of saccharification, hydrogen production
and preliminary scale up. Int J Hydrogen Energy 2016;41:12941–52. https://doi.
org/10.1016/j.ijhydene.2016.06.112.
[38] Shanmugam S, Hari A, Ulaganathan P, Yang F, Krishnaswamy S, Wu YR. Potential
of biohydrogen generation using the delignified lignocellulosic biomass by a
newly identified thermostable laccase from Trichoderma asperellum strain
BPLMBT1. Int J Hydrogen Energy 2018;43:3618–28. https://doi.org/10.1016/j.
ijhydene.2018.01.016.
[39] Sangyoka S, Reungsang A, Lin CY. Optimization of biohydrogen production from
sugarcane bagasse by mixed cultures using a statistical method. Sustain Environ
Res 2016;26:235–42. https://doi.org/10.1016/j.serj.2016.05.001.
[40] Rabelo CABS, Soares LA, Sakamoto IK, Silva EL, Varesche MBA. Optimization of
hydrogen and organic acids productions with autochthonous and allochthonous
bacteria from sugarcane bagasse in batch reactors. J Environ Manag 2018;223:
952–63. https://doi.org/10.1016/j.jenvman.2018.07.015.
[41] Moodley P, Gueguim Kana EB. Comparative study of three optimized acid-based
pretreatments for sugar recovery from sugarcane leaf waste: a sustainable
feedstock for biohydrogen production. Eng Sci Technol an Int J 2018;21:107–16.
https://doi.org/10.1016/j.jestch.2017.11.010.
[42] Arriaga S, Rosas I, Alatriste-Mondrag�on F, Razo-Flores E. Continuous production
of hydrogen from oat straw hydrolysate in a biotrickling filter. Int J Hydrogen
Energy 2011;36:3442–9. https://doi.org/10.1016/j.ijhydene.2010.12.019.
[43] Arreola-Vargas J, Razo-Flores E, Celis LB, Alatriste-Mondrag� on F. Sequential
hydrolysis of oat straw and hydrogen production from hydrolysates: role of
hydrolysates constituents. Int J Hydrogen Energy 2015;40:10756–65. https://doi.
org/10.1016/j.ijhydene.2015.05.200.
[44] Gonzales RR, Kumar G, Sivagurunathan P, Kim SH. Enhancement of hydrogen
production by optimization of pH adjustment and separation conditions following
dilute acid pretreatment of lignocellulosic biomass. Int J Hydrogen Energy 2017;
42:27502–11. https://doi.org/10.1016/j.ijhydene.2017.05.021.
[45] Lin CY, Lay CH, Chen CC, Sen B, Sung IY. Biohydrogen production from
mushroom cultivation waste by anaerobic solid-state fermentation. J Chin Chem
Soc 2016;63:199–204. https://doi.org/10.1002/jccs.201500359.
[46] Lin HN, Wang YT, Zhu MJ. Evaluation of spent mushroom compost as a
lignocellulosic substrate for hydrogen production by Clostridium thermocellum.
Int J Hydrogen Energy 2017;42:26687–94. https://doi.org/10.1016/j.
ijhydene.2017.09.040.
[47] Gonzales RR, Kim JS, Kim SH. Optimization of dilute acid and enzymatic
hydrolysis for dark fermentative hydrogen production from the empty fruit bunch
of oil palm. Int J Hydrogen Energy 2019;44:2191–202. https://doi.org/10.1016/
j.ijhydene.2018.08.022.
[48] Kumar G, Bakony P, Sivagurunathan P, Nemest� othy N, B�
elafi-Bak�
o K, Lin C-Y.
Improved microbial conversion of de-oiled Jatropha waste into biohydrogen via
inoculum pretreatment: process optimization by experimental design approach.
Biofuel Res J 2015;5:209–14. https://doi.org/10.18331/brj2015.2.1.7.
[49] Silva JS, Mendes JS, Correia JAC, Rocha MVP, Micoli L. Cashew apple bagasse as
new feedstock for the hydrogen production using dark fermentation process.
J Biotechnol 2018;286:71–8. https://doi.org/10.1016/j.jbiotec.2018.09.004.
[50] Qi N, Hu X, Zhao X, Li L, Yang J, Zhao Y, et al. Fermentative hydrogen production
with peanut shell as supplementary substrate: effects of initial substrate, pH and
inoculation proportion. Renew Energy 2018;127:559–64.
[51] Eker S, Sarp M. Hydrogen gas production from waste paper by dark fermentation:
effects of initial substrate and biomass concentrations. Int J Hydrogen Energy
2017;42:2562–8. https://doi.org/10.1016/j.ijhydene.2016.04.020.
[52] Mu~ noz-P�aez KM, Alvarado-Michi EL, Buitr�on G, Valdez-Vazquez I. Distinct effects
of furfural, hydroxymethylfurfural and its mixtures on dark fermentation
hydrogen production and microbial structure of a mixed culture. Int J Hydrogen
Energy 2019;4:2289–97. https://doi.org/10.1016/j.ijhydene.2018.04.139.
J.F. Soares et al.
[53] Agbor VB, Cicek N, Sparling R, Berlin A, Levin DB. Biomass pretreatment:
fundamentals toward application. Biotechnol Adv 2011;29:675–85. https://doi.
org/10.1016/j.biotechadv.2011.05.005.
[54] Ghimire A, Sposito F, Frunzo L, Trably E, Escudi�e R, Pirozzi F, et al. Effects of
operational parameters on dark fermentative hydrogen production from
biodegradable complex waste biomass. Waste Manag 2016;50:55–64. https://doi.
org/10.1016/j.wasman.2016.01.044.
[55] Reddy K, Nasr M, Kumari S, Kumar S, Gupta SK, Enitan AM, et al. Biohydrogen
production from sugarcane bagasse hydrolysate: effects of pH, S/X, Fe2þ, and
magnetite nanoparticles. Environ Sci Pollut Res 2017;24:8790–804. https://doi.
org/10.1007/s11356-017-8560-1.
[56] Poladyan A, Trchounian K, Vassilian A, Trchounian A. Hydrogen production by
Escherichia coli using brewery waste: optimal pretreatment of waste and role of
different hydrogenases. Renew Energy 2018;115:931–6. https://doi.org/
10.1016/j.renene.2017.09.022.
[57] Ren NQ, Zhao L, Chen C, Guo WQ, Cao GL. A review on bioconversion of
lignocellulosic biomass to H2: key challenges and new insights. Bioresour Technol
2016;215:92–9. https://doi.org/10.1016/j.biortech.2016.03.124.
[58] Sattar A, Arslan C, Ji C, Sattar S, Mari IA, Rashid H, et al. Comparing the bio-
hydrogen production potential of pretreated rice straw co-digested with seeded
sludge using an anaerobic bioreactor under mesophilic thermophilic conditions.
Energies 2016;9. https://doi.org/10.3390/en9030198.
[59] Koppram R, Tom� as-Pej�
o E, Xiros C, Olsson L. Lignocellulosic ethanol production
at high-gravity : challenges and perspectives. Trends Biotechnol 2014;32:36–43.
[60] Cara C, Moya M, Ballesteros I, Negro MJ, Gonz� alez A, Ruiz E. Influence of solid
loading on enzymatic hydrolysis of steam exploded or liquid hot water pretreated
olive tree biomass. Process Biochem 2007;42:1003–9. https://doi.org/10.1016/j.
procbio.2007.03.012.
[61] Brodeur G, Yau E, Badal K, Collier J, Ramachandran KB, Ramakrishnan S.
Chemical and physicochemical pretreatment of lignocellulosic biomass: a review.
Enzym Res 2011;2011:787532. https://doi.org/10.4061/2011/787532.
[62] Guerra-Rodríguez E, Portilla-Rivera OM, Jarquín-Enríquez L, Ramírez JA,
V�azquez M. Acid hydrolysis of wheat straw: a kinetic study. Biomass Bioenergy
2012;36:346–55. https://doi.org/10.1016/j.biombioe.2011.11.005.
[63] Hu B Bin, Li MY, Wang YT, Zhu MJ. High-yield biohydrogen production from
non-detoxified sugarcane bagasse: fermentation strategy and mechanism. Chem
Eng J 2018;335:979–87. https://doi.org/10.1016/j.cej.2017.10.157.
[64] Gokfiliz P, Karapinar I. The effect of support particle type on thermophilic
hydrogen production by immobilized batch dark fermentation. Int J Hydrogen
Energy 2017;42:2553–61. https://doi.org/10.1016/j.ijhydene.2016.03.041.
[65] Azman NF, Abdeshahian P, Kadier A, Shukor H, Al-Shorgani NKN, Hamid AA,
et al. Utilization of palm kernel cake as a renewable feedstock for fermentative
hydrogen production. Renew Energy 2016;93:700–8. https://doi.org/10.1016/j.
renene.2016.03.046.
[66] de Vasconcelos SM, Santos AMP, Rocha GJM, Souto-Maior AM. Diluted
phosphoric acid pretreatment for production of fermentable sugars in a
sugarcane-based biorefinery. Bioresour Technol 2013;135:46–52. https://doi.
org/10.1016/j.biortech.2012.10.083.
[67] Palmqvist E, Hahn-H€ agerdal B. Fermentation of lignocellulosic hydrolysates. II:
inhibitors and mechanisms of inhibition. Bioresour Technol 2000;74:25–33.
https://doi.org/10.1016/S0960-8524(99)00161-3.
[68] Shobana S, Kumar G, Bakonyi P, Saratale GD, Al-Muhtaseb AH, Nemest� othy N,
et al. A review on the biomass pretreatment and inhibitor removal methods as
key-steps towards efficient macroalgae-based biohydrogen production. Bioresour
Technol 2017;244:1341–8. https://doi.org/10.1016/j.biortech.2017.05.172.
[69] Arreola-Vargas J, Flores-Larios A, Gonz� alez-Alvarez
� V, Corona-Gonz� alez RI,
M�endez-Acosta HO. Single and two-stage anaerobic digestion for hydrogen and
methane production from acid and enzymatic hydrolysates of Agave tequilana
bagasse. Int J Hydrogen Energy 2016;41:897–904. https://doi.org/10.1016/j.
ijhydene.2015.11.016.
[70] Behera S, Arora R, Nandhagopal N, Kumar S. Importance of chemical
pretreatment for bioconversion of lignocellulosic biomass. Renew Sustain Energy
Rev 2014;36:91–106. https://doi.org/10.1016/j.rser.2014.04.047.
[71] Sigurbjornsdottir MA, Orlygsson J. Combined hydrogen and ethanol production
from sugars and lignocellulosic biomass by Thermoanaerobacterium AK54,
isolated from hot spring. Appl Energy 2012;97:785–91. https://doi.org/10.1016/
j.apenergy.2011.11.035.
[72] Satar I, Ghasemi M, Aljlil SA, Isahak WNRW, Abdalla AM, Alam J, et al.
Production of hydrogen by Enterobacter aerogenes in an immobilized cell reactor.
Int J Hydrogen Energy 2017;42:9024–30. https://doi.org/10.1016/j.
ijhydene.2016.04.150.
[73] Oh YK, Seol EH, Lee EY, Park S. Fermentative hydrogen production by a new
chemoheterotrophic bacterium Rhodopseudomonas palustris P4. Int J Hydrogen
Energy 2002;27:1373–9. https://doi.org/10.1016/S0360-3199(02)00100-3.
[74] Oh YK, Seol EH, Kim JR, Park S. Fermentative biohydrogen production by a new
chemoheterotrophic bacterium Citrobacter sp. Y19. Int J Hydrogen Energy 2003;
28:1353–9. https://doi.org/10.1016/S0360-3199(03)00024-7.
[75] Kotay SM, Das D. Microbial hydrogen production with Bacillus coagulans IIT-BT
S1 isolated from anaerobic sewage sludge. Bioresour Technol 2007;98:1183–90.
https://doi.org/10.1016/j.biortech.2006.05.009.
[76] Das D, Veziroglu TN. Advances in biological hydrogen production processes. Int J
Hydrogen Energy 2008;33:6046–57. https://doi.org/10.1016/j.
ijhydene.2008.07.098.
[77] Yokoi H, Ohkawara T, Hirose J, Hayashi S, Takasaki Y. Characteristics of
hydrogen production by aciduric Enterobacter aerogenes strain HO-39. J Ferment
Bioeng 1995;80:571–4. https://doi.org/10.1016/0922-338X(96)87733-6.
15
Renewable and Sustainable Energy Reviews 117 (2020) 109484
[78] Kumar G, Cho SK, Sivagurunathan P, Anburajan P, Mahapatra DM, Park JH, et al.
Insights into evolutionary trends in molecular biology tools in microbial
screening for biohydrogen production through dark fermentation. Int J Hydrogen
Energy 2018;43:19885–901. https://doi.org/10.1016/j.ijhydene.2018.09.040.
[79] Valdez-Vazquez I, Ríos-Leal E, Esparza-García F, Cecchi F, Poggi-Varaldo HM.
Semi-continuous solid substrate anaerobic reactors for H 2 production from
organic waste: mesophilic versus thermophilic regime. Int J Hydrogen Energy
2005;30:1383–91. https://doi.org/10.1016/j.ijhydene.2004.09.016.
[80] Li C, Fang HHP. Fermentative hydrogen production from wastewater and solid
wastes by mixed cultures. Crit Rev Environ Sci Technol 2007;37:1–39. https://
doi.org/10.1080/10643380600729071.
[81] Tapia-Venegas E, Ramirez-Morales JE, Silva-Illanes F, Toledo-Alarc� on J, Paillet F,
Escudie R, et al. Biohydrogen production by dark fermentation: scaling-up and
technologies integration for a sustainable system. Rev Environ Sci Biotechnol
2015;14:761–85. https://doi.org/10.1007/s11157-015-9383-5.
[82] Han W, Na D, Wen Y, Hong J, Feng Y, Qi N. Biohydrogen production from food
waste hydrolysate using continuous mixed immobilized sludge reactors.
Bioresour Technol 2015;180:54–8. https://doi.org/10.1016/j.
biortech.2014.12.067.
[83] Lutpi NA, Md Jahim J, Mumtaz T, Harun S, Abdul PM. Batch and continuous
thermophilic hydrogen fermentation of sucrose using anaerobic sludge from palm
oil mill effluent via immobilisation technique. Process Biochem 2016;51:
297–307. https://doi.org/10.1016/j.procbio.2015.11.031.
[84] Yin Y, Wang J. Biohydrogen production using waste activated sludge
disintegrated by gamma irradiation. Appl Energy 2015;155:434–9. https://doi.
org/10.1016/j.apenergy.2015.05.105.
[85] Kumar G, Bakonyi P, Kobayashi T, Xu KQ, Sivagurunathan P, Kim SH, et al.
Enhancement of biofuel production via microbial augmentation: the case of dark
fermentative hydrogen. Renew Sustain Energy Rev 2016;57:879–91. https://doi.
org/10.1016/j.rser.2015.12.107.
[86] Wang J, Wan W. Factors influencing fermentative hydrogen production: a review.
Int J Hydrogen Energy 2009;34:799–811. https://doi.org/10.1016/j.
ijhydene.2008.11.015.
[87] Łukajtis R, Hołowacz I, Kucharska K, Glinka M, Rybarczyk P, Przyjazny A, et al.
Hydrogen production from biomass using dark fermentation. Renew Sustain
Energy Rev 2018;91:665–94. https://doi.org/10.1016/j.rser.2018.04.043.
[88] Kumar G, Zhen G, Sivagurunathan P, Bakonyi P, Nemest� othy N, B�
elafi-Bak�o K,
et al. Biogenic H2 production from mixed microalgae biomass: impact of pH
control and methanogenic inhibitor (BESA) addition. Biofuel Res J 2016;3:470–4.
https://doi.org/10.18331/BRJ2016.3.3.6.
[89] Levin DB, Pitt L, Love M. Biohydrogen production: prospects and limitations to
practical application. Int J Hydrogen Energy 2004;29:173–85. https://doi.org/
10.1016/S0360-3199(03)00094-6.
[90] Wongthanate J, Chinnacotpong K, Khumpong M. Impacts of pH, temperature and
pretreatment method on biohydrogen production from organic wastes by sewage
microflora. Int J Energy Environ Eng 2014;5:1–6. https://doi.org/10.1007/
s40095-014-0076-6.
[91] Phowan P, Danvirutai P. Hydrogen production from cassava pulp hydrolysate by
mixed seed cultures: effects of initial pH, substrate and biomass concentrations.
Biomass Bioenergy 2014;64:1–10. https://doi.org/10.1016/j.
biombioe.2014.03.057.
[92] Bakonyi P, Nemest� othy N, Simon V, B�elafi-Bak�
o K. Fermentative hydrogen
production in anaerobic membrane bioreactors: a review. Bioresour Technol
2014;156:357–63. https://doi.org/10.1016/j.biortech.2014.01.079.
[93] Antonopoulou G, Gavala HN, Skiadas IV, Angelopoulos K, Lyberatos G. Biofuels
generation from sweet sorghum: fermentative hydrogen production and
anaerobic digestion of the remaining biomass. Bioresour Technol 2008;99:110–9.
https://doi.org/10.1016/j.biortech.2006.11.048.
[94] Buitr�on G, Carvajal C. Biohydrogen production from Tequila vinasses in an
anaerobic sequencing batch reactor: effect of initial substrate concentration,
temperature and hydraulic retention time. Bioresour Technol 2010;101:9071–7.
https://doi.org/10.1016/j.biortech.2010.06.127.
[95] Wang B, Li Y, Ren N. Biohydrogen from molasses with ethanol-type fermentation:
effect of hydraulic retention time. Int J Hydrogen Energy 2013;38:4361–7.
https://doi.org/10.1016/j.ijhydene.2013.01.120.
[96] Moreno-Andrade I, Carrillo-Reyes J, Santiago SG, Bujanos-Adame MC.
Biohydrogen from food waste in a discontinuous process: effect of HRT and
microbial community analysis. Int J Hydrogen Energy 2015;40:17246–52.
https://doi.org/10.1016/j.ijhydene.2015.04.084.
[97] Nam JY, Kim DH, Kim SH, Lee W, Shin HS, Kim HW. Harnessing dark
fermentative hydrogen from pretreated mixture of food waste and sewage sludge
under sequencing batch mode. Environ Sci Pollut Res 2016;23:7155–61. https://
doi.org/10.1007/s11356-015-4880-1.
[98] Kumar G, Sivagurunathan P, Park JH, Park JH, Park HD, Yoon JJ, et al. HRT
dependent performance and bacterial community population of granular
hydrogen-producing mixed cultures fed with galactose. Bioresour Technol 2016;
206:188–94. https://doi.org/10.1016/j.biortech.2016.01.104.
[99] Kumar G, Sivagurunathan P, Sen B, Kim SH, Lin CY. Mesophilic continuous
fermentative hydrogen production from acid pretreated de-oiled jatropha waste
hydrolysate using immobilized microorganisms. Bioresour Technol 2017;240:
137–43. https://doi.org/10.1016/j.biortech.2017.03.059.
[100] Mizuno O, Dinsdale R, Hawkes FR, Hawkes DL, Noike T. Enhancement of
hydrogen production from glucose by nitrogen gas sparging. Bioresour Technol
2000;73:59–65. https://doi.org/10.1016/s0960-8524(99)00130-3.
J.F. Soares et al.
[101] Kim DH, Han SK, Kim SH, Shin HS. Effect of gas sparging on continuous
fermentative hydrogen production. Int J Hydrogen Energy 2006;31:2158–69.
https://doi.org/10.1016/j.ijhydene.2006.02.012.
[102] Lee KS, Tseng TS, Liu YW, Hsiao YD. Enhancing the performance of dark
fermentative hydrogen production using a reduced pressure fermentation
strategy. Int J Hydrogen Energy 2012;37:15556–62. https://doi.org/10.1016/j.
ijhydene.2012.04.039.
[103] Kapdan IK, Kargi F. Bio-hydrogen production from waste materials. Enzym
Microb Technol 2006;38:569–82. https://doi.org/10.1016/j.
enzmictec.2005.09.015.
[104] Urbaniec K, Grabarczyk R. Hydrogen production from sugar beet molasses - a
techno-economic study. J Clean Prod 2014;65:324–9. https://doi.org/10.1016/j.
jclepro.2013.08.027.
[105] Liu CM, Wu SY, Chu CY, Chou YP. Biohydrogen production from rice straw
hydrolyzate in a continuously external circulating bioreactor. Int J Hydrogen
Energy 2014;39:19317–22. https://doi.org/10.1016/j.ijhydene.2014.05.175.
[106] Yang G, Wang J. Various additives for improving dark fermentative hydrogen
production: a review. Renew Sustain Energy Rev 2018;95:130–46. https://doi.
org/10.1016/j.rser.2018.07.029.
[107] Sun Y, He J, Yang G, Sun G, Sage V. A review of the enhancement of bio-hydrogen
generation by chemicals addition. Catalysts 2019;9:1–21. https://doi.org/
10.3390/catal9040353.
[108] G�omez Camacho CE, Romano FI, Ruggeri B. Macro approach analysis of dark
biohydrogen production in the presence of zero valent powered Fe� . Energy 2018;
159:525–33. https://doi.org/10.1016/j.energy.2018.06.171.
[109] Hwang Y, Sivagurunathan P, Lee MK, Yun YM, Song YC, Kim DH. Enhanced
hydrogen fermentation by zero valent iron addition. Int J Hydrogen Energy 2019;
44:3387–94. https://doi.org/10.1016/j.ijhydene.2018.06.015.
[110] Zhang J, Fan C, Zhang H, Wang Z, Zhang J, Song M. Ferric oxide/carbon
nanoparticles enhanced bio-hydrogen production from glucose. Int J Hydrogen
Energy 2018;43:8729–38. https://doi.org/10.1016/j.ijhydene.2018.03.143.
[111] Lin R, Cheng J, Ding L, Song W, Liu M, Zhou J, et al. Enhanced dark hydrogen
fermentation by addition of ferric oxide nanoparticles using Enterobacter
aerogenes. Bioresour Technol 2016;207:213–9. https://doi.org/10.1016/j.
biortech.2016.02.009.
[112] Mishra P, Thakur S, Mahapatra DM, Wahid ZA, Liu H, Singh L. Impacts of nano-
metal oxides on hydrogen production in anaerobic digestion of palm oil mill
effluent – a novel approach. Int J Hydrogen Energy 2018;43:2666–76. https://
doi.org/10.1016/j.ijhydene.2017.12.108.
[113] Wang Y, Zhao J, Wang D, Liu Y, Wang Q, Ni BJ, et al. Free nitrous acid promotes
hydrogen production from dark fermentation of waste activated sludge. Water
Res 2018;145:113–24. https://doi.org/10.1016/j.watres.2018.08.011.
[114] Wang D, Zhang D, Xu Q, Liu Y, Wang Q, Ni BJ, et al. Calcium peroxide promotes
hydrogen production from dark fermentation of waste activated sludge. Chem
Eng J 2019;355:22–32. https://doi.org/10.1016/j.cej.2018.07.192.
[115] Wu Y, Wang D, Liu X, Xu Q, Chen Y, Yang Q, et al. Effect of poly aluminum
chloride on dark fermentative hydrogen accumulation from waste activated
sludge. Water Res 2019;153:217–28. https://doi.org/10.1016/j.
watres.2019.01.016.
[116] Pe~na L, Ikenberry M, Hohn KL, Wang D. Acid-functionalized nanoparticles for
pretreatment of wheat straw. J Biomaterials Nanobiotechnol 2012;3:342–52.
https://doi.org/10.4236/jbnb.2012.33032.
[117] Srivastava N, Singh J, Ramteke PW, Mishra PK, Srivastava M. Improved
production of reducing sugars from rice straw using crude cellulase activated with
Fe3O4/Alginate nanocomposite. Bioresour Technol 2015;183:262–6. https://doi.
org/10.1016/j.biortech.2015.02.059.
[118] Cherian E, Dharmendirakumar M, Baskar G. Immobilization of cellulase onto
MnO2 nanoparticles for bioethanol production by enhanced hydrolysis of
agricultural waste. Cuihua Xuebao/Chinese J Catal 2015;36:1223–9. https://doi.
org/10.1016/S1872-2067(15)60906-8.
[119] IEA. World Energy Outlook 2018. 2018.
[120] Manish S, Banerjee R. Comparison of biohydrogen production processes. Int J
Hydrogen Energy 2008;33:279–86. https://doi.org/10.1016/j.
ijhydene.2007.07.026.
[121] Valente A, Iribarren D, Dufour J. Harmonising the cumulative energy demand of
renewable hydrogen for robust comparative life-cycle studies. J Clean Prod 2018;
175:384–93. https://doi.org/10.1016/j.jclepro.2017.12.069.
[122] Dai Q, Elgowainy A, Kelly J, Han J, Wang M. Life cycle analysis of hydrogen
production from non-fossil sources. IL: Lemont; 2016. https://doi.org/10.3303/
CET1021194.
[123] Ochs D, Wukovits W, Ahrer W. Life cycle inventory analysis of biological
hydrogen production by thermophilic and photo fermentation of potato steam
16
Renewable and Sustainable Energy Reviews 117 (2020) 109484
peels (PSP). J Clean Prod 2010;18:S88–94. https://doi.org/10.1016/j.
jclepro.2010.05.018.
[124] Mehmeti A, Angelis-Dimakis A, Arampatzis G, McPhail S, Ulgiati S. Life cycle
assessment and water footprint of hydrogen production methods: from
conventional to emerging technologies. Environments 2018;5:24. https://doi.
org/10.3390/environments5020024.
[125] Bundhoo ZMA. Potential of bio-hydrogen production from dark fermentation of
crop residues: a review. Int J Hydrogen Energy 2018. https://doi.org/10.1016/j.
ijhydene.2018.11.098.
[126] Suzuki Y. On hydrogen as fuel gas. Int J Hydrogen Energy 1982;7:227–30.
[127] Boundy B, Diegel SW, Wright L, Davis SC. Biomass energy data book. fourth ed.
Oak Ridge National Laboratory; 2011.
[128] Han W, Hu YY, Li SY, Li FF, Tang JH. Biohydrogen production from waste bread
in a continuous stirred tank reactor: a techno-economic analysis. Bioresour
Technol 2016;221:318–23. https://doi.org/10.1016/j.biortech.2016.09.055.
[129] James BD, DeSantis DA, Saur G. Final report: hydrogen production pathways cost
analysis. 2013–2016. https://doi.org/10.2172/1346418. 2016.
[130] Ren N, Guo W, Liu B, Cao G, Ding J. Biological hydrogen production by dark
fermentation: challenges and prospects towards scaled-up production. Curr Opin
Biotechnol 2011;22:365–70. https://doi.org/10.1016/j.copbio.2011.04.022.
[131] Han W, Yan Y, Gu J, Shi Y, Tang J, Li Y. Techno-economic analysis of a novel
bioprocess combining solid state fermentation and dark fermentation for H2
production from food waste. Int J Hydrogen Energy 2016;41:22619–25. https://
doi.org/10.1016/j.ijhydene.2016.09.047.
[132] Han W, Liu Z, Fang J, Huang J, Zhao H, Li Y. Techno-economic analysis of dark
fermentative hydrogen production from molasses in a continuous mixed
immobilized sludge reactor. J Clean Prod 2016;127:567–72. https://doi.org/
10.1016/j.jclepro.2016.04.055.
[133] Bonk F, Bastidas-Oyanedel JR, Schmidt JE. Converting the organic fraction of
solid waste from the city of Abu Dhabi to valuable products via dark fermentation
- economic and energy assessment. Waste Manag 2015;40:82–91. https://doi.org/
10.1016/j.wasman.2015.03.008.
[134] Hsu CW, Lin CY. Commercialization model of hydrogen production technology in
Taiwan: dark fermentation technology applications. Int J Hydrogen Energy 2016;
41:4489–97. https://doi.org/10.1016/j.ijhydene.2015.07.080.
[135] Randolph K, Studer S. Hydrogen production cost from fermentation, vol. 8; 2017.
[136] Chang PL, Hsu CW, Lin CY, Hsiung CM. Constructing a new business model for
fermentative hydrogen production from wastewater treatment. Int J Hydrogen
Energy 2011;36:13914–21. https://doi.org/10.1016/j.ijhydene.2011.03.066.
[137] Chang PL, Hsu CW. Value analysis for commercialization of fermentative
hydrogen production from biomass. Int J Hydrogen Energy 2012;37:15746–52.
https://doi.org/10.1016/j.ijhydene.2012.02.113.
[138] R�ozsenberszki T, Ko�ok L, Bakonyi P, Nemest� othy N, Logro~ no W, P�erez M, et al.
Municipal waste liquor treatment via bioelectrochemical and fermentation (H2 þ
CH4) processes: assessment of various technological sequences. Chemosphere
2017;171:692–701. https://doi.org/10.1016/j.chemosphere.2016.12.114.
[139] R�ozsenberszki T, Ko�ok L, Hutv�agner D, Nemest�othy N, B�elafi-Bak�
o K, Bakonyi P,
et al. Comparison of anaerobic degradation processes for bioenergy generation
from liquid fraction of pressed solid waste. Waste and Biomass Valorization 2015;
6:465–73. https://doi.org/10.1007/s12649-015-9379-y.
[140] Noblecourt A, Christophe G, Larroche C, Fontanille P. Hydrogen production by
dark fermentation from pre-fermented depackaging food wastes. Bioresour
Technol 2018;247:864–70. https://doi.org/10.1016/j.biortech.2017.09.199.
[141] Islam MS, Guo C, Liu CZ. Enhanced hydrogen and volatile fatty acid production
from sweet sorghum stalks by two-steps dark fermentation with dilute acid
treatment in between. Int J Hydrogen Energy 2018;43:659–66. https://doi.org/
10.1016/j.ijhydene.2017.11.059.
[142] N�athia-Neves G, Neves T de A, Berni M, Dragone G, Mussatto SI, Forster-
Carneiro T. Start-up phase of a two-stage anaerobic co-digestion process:
hydrogen and methane production from food waste and vinasse from ethanol
industry. Biofuel Res J 2018;5:813–20. https://doi.org/10.18331/BRJ2018.5.2.5.
[143] Sarkar O, Mohan SV. Deciphering acidogenic process towards biohydrogen,
biohythane, and short chain fatty acids production: multi-output optimization
strategy. Biofuel Res J 2016;3:458–69. https://doi.org/10.18331/BRJ2016.3.3.5.
[144] Kumar G, Bakonyi P, Zhen G, Sivagurunathan P, Ko� ok L, Kim SH, et al. Microbial
electrochemical systems for sustainable biohydrogen production: surveying the
experiences from a start-up viewpoint. Renew Sustain Energy Rev 2017;70:
589–97. https://doi.org/10.1016/j.rser.2016.11.107.
[145] Singh A, Sevda S, Abu Reesh IM, Vanbroekhoven K, Rathore D, Pant D.
Biohydrogen production from lignocellulosic biomass: technology and
sustainability. Energies 2015;8:13062–80. https://doi.org/10.3390/en81112357.