i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 6 ( 2 0 2 1 ) 1 1 2 0 1 e1 1 2 1 6

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Biohythane production from organic waste: Recent

advancements, technical bottlenecks and prospects

Sabarathinam Shanmugam a,b, Thangavel Mathimani c, Eldon R. Rene d,

V. Edwin Geo e, A. Arun f, Kathirvel Brindhadevi*,g,
Arivalagan Pugazhendhi h
a
Key Laboratory of Low-grade Energy Utilization Technologies and Systems, Chongqing University, Ministry of
Education, Chongqing, 400044, China
b
Institute of Engineering Thermophysics, Chongqing University, Chongqing, 400044, China
c
Department of Energy and Environment, National Institute of Technology, Tiruchirappalli, Tamil Nadu, 620015,
India
d
Department of Water Supply, Sanitation and Environmental Engineering, IHE Delft Institute for Water Education,
2601 DA Delft, the Netherlands
e
Green Vehicle Technology Research Centre, Department of Automobile Engineering, SRM Institute of Science and
Technology, Kattankulathur, Chennai, 603203, India
f
Department of Microbiology, Alagappa University, Karaikudi, Tamil Nadu, India
g
Institute of Research and Development, Duy Tan University, Da Nang, 550000, Vietnam
h
Innovative Green Product Synthesis and Renewable Environment Development Research Group, Faculty of
Environment and Labour Safety, Ton Duc Thang University, Ho Chi Minh City, Vietnam

highlights graphical abstract

The potential of biohythane as a

promising alternative fuel is
discussed.

Biohythane production from
various organic wastes is
analyzed.

Reaction mechanism of two-stage
anaerobic digestion is explored.

Potential microbes involved in
biohythane production are
presented.

Technical hurdles to be overcome
in biohythane production are
highlighted.

article info abstract

Article history: The availability of fossil fuels is a major factor that determines the economy of a country.
Received 6 April 2020 However, possible exhaustion of fossil fuel deposits as well as increased pollution, and
Received in revised form other adverse effects on the environment has prompted us to search for alternative fuels.

* Corresponding author.
E-mail addresses: kathirvelbrindhadevi@duytan.edu.vn (K. Brindhadevi), arivalagan.pugazhendhi@tdtu.edu.vn (A. Pugazhendhi).
https://doi.org/10.1016/j.ijhydene.2020.10.132
0360-3199/© 2020 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
11202 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 6 ( 2 0 2 1 ) 1 1 2 0 1 e1 1 2 1 6

12 October 2020 This resulted in the development of hythane, a blend of hydrogen with methane, at con-
Accepted 16 October 2020 centrations of 10%e30%. The breakdown of organic substrates using sequential dark
Available online 24 November 2020 fermentation (DF) and anaerobic digestion (AD) leads to biohythane production. The
quality and quantity of biohythane can be improved by altering the following aspects:
Keywords: selection, development, and/or genetic engineering of suitable microbial consortium; the
Biohythane use of cheap, appropriate substrates; improved design of bioreactors; and the imple-
Anaerobic digestion mentation of two-stage fermentation system. This review focusses on the mechanism of
Organic waste biohythane production and the different aspects involved in increasing both its production
Fossil fuels rate and quality. A comparative study has also been done to demonstrate the superiority of
Microbial consortium biohythane over other biofuels.
© 2020 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.

next stage to form biohythane [13] (Fig. 1). Hence, several re-
Introduction searchers have focused on the modification of existing
anaerobic-digestors for the co-generation of biohydrogen and
Industrial revolution has played a significant role in biomethane using the two-stage process [14,15].
exhausting fossil fuels thereby taking them towards the verge Choosing the appropriate substrate for biohythane pro-
of depletion [1]. The advent of rapid industrialization has also duction is a necessity. Examples of different types of organic
negatively affected the environment [2]. This has given rise to substrates include wheat straw hydrolysate, food waste,
the concept of responsible exploitation of renewable energy starch wastewater, crop starch waste, palm oil mill waste, and
sources beneficial to both humans and the environment. organic solid waste [16]. Another important aspect is the
Therefore, researchers are focusing on developing alternative, optimization of different parameters for biohythane produc-
renewable and eco-friendly fuel sources. Nowadays, various tion, such as pH, temperature, pre-treatment methods, feed-
biofuels like biodiesel, biobutanol, bioethanol, biohydrogen stock, and the ratio of substrate to inoculum [17]. Biohythane
and biomethane have been produced from various forms of was introduced in the year 1995 as transportation fuel in a
renewable sources (i.e. involving solid, liquid and gaseous project named Montreal Hythane Bus, and later, Sweden and
substrates). Biofuels are emerging as a renewable, cleaner, China carried out the Beijing Hythane Bus project demon-
and alternative source of energy for mechanized trans- strating 45% of NOx emission [18]. In recent times, USA and
portation [3]. India has started commercializing biohythane as an auto-
Among the different biofuels, biohydrogen is emerging as a mobile fuel. Companies like Toyota, Fiat and Volvo have used
clean fuel, which releases water as the only product upon them for mechanized transportation [19e21]. Therefore,
combustion, and has a high energy density of 141.9 MJ/kg, considering the recent growth in the trend of utilizing bio-
followed by that of biomethane (50e55 MJ/kg) [4,5]. Further, H2 hythane as a transportation fuel, the present review is
can act as a catalyst when combined with other gaseous bio- compiled to provide a detailed overview on the production of
fuels, which significantly improves the combustion charac- biohythane and the mechanism involved, the types of
teristics of the resultant fuel [6,7]. Thus, the co-generation of
these renewable energy sources from various organic waste
biomass is deemed a powerful strategy with several distinct
advantages such as minimized CFP, energy generation, and
waste management. Further, the co-generation of bio-
hydrogen and biomethane from organic wastes has strong
potential to bring about ~ 8e43% increment in energy recov-
ery. Moreover, they complement each other in the form of
hythane, that significantly improves its economy and effi-
ciency as an alternative fuel [8,9]. According to recent trends,
similar to biohydrogen, biohythane is a potentially promising
fuel that generates higher energy and low primary and sec-
ondary pollutants [10]. It is a blend of hydrogen (10%) and
methane (30%). The production of hythane occurs by anaer-
obic digestion (AD) of organic materials such as agricultural
waste, food waste and municipal waste [11]. Anaerobic
digestion of substrate occurs in two processes. In the first
phase, hydrogen is formed, and in the second phase, methane
is generated [12]. The two major goals during AD are the
production of hydrogen during DF, and the digestion of ef- Fig. 1 e Strategies in biohythane production using two-
fluents for the generation of methane, that combines in the stage anaerobic fermentation.
 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 6 ( 2 0 2 1 ) 1 1 2 0 1 e1 1 2 1 6 11203

fermentation processes, different organic substrates
employed, parameters optimized to increase the production
rate, and the applications of biohythane as a mechanized
transportation fuel.
Biohythane
In the early 1990s, Hydrogen Component Inc. (HCI) conducted
a study using a blend of Compressed Natural Gas (CNG) and
hydrogen as fuel in vehicle engines. They termed this com-
bination of fuels as hythane. Its potential use as a vehicle fuel
has gained attention worldwide. Hydrogen and methane are
common fuels used in chemical and process industries due to
their high calorific values of 143 kJ/g and 55 kJ/g [7,22].
Hythane is a better fuel than CNG due to the presence of
hydrogen which improves its flammability [6]. The reducing
potential of H2 initiates or stimulates the combustion of CH4
and increases the burning capacity at lower temperatures.
Hythane also has several environmental benefits due to the
increased composition of hydrogen and methane than that of
carbon, thus decreasing the generation of greenhouse gases
[23].
In the present scenario, hythane is produced using a ther-
mochemical process using natural gas as substrate [19]. It is a
high energy-consuming process and it solely depends upon the
already existing fossil fuels. To avoid these drawbacks, nowa-
days, “hythane” is being replaced by “biohythane,” as organic
wastes (biowastes) are used as substrate for its production.
Organic substrates like agricultural crop waste and food waste
have been used to produce “biohythane” under anaerobic
environment [10]. The production of biohydrogen and bio-
methane by fermentation and AD of organic wastes (agricul-
tural waste, domestic waste, etc) is already under practice [24].
Combining both these (fermentation and AD) processes into a
two-stage anaerobic fermentation process will help in pro-
ducing hythane with the composition of H2 at 10%e15%, and
CH4 at 50%e55%, respectively [25]. The superiority of bio-
hythane production in comparison to the existing biogas fuels
using two-stage anaerobic fermentation technology is depic-
ted in Table 1.
Further, removing CO2 during the two-stage anaerobic
fermentation process will upgrade the technology to produce
biohythane [16]. European countries have ~ 17,000 full scale
AD plants in operation; among them, Germany has total
generation capacity of 8,293 MWeI [26]. However, less than 1%
of these plants have fully developed the two-stage AD process
due to the higher cost involved in this process [7].
Mechanism of biohythane production
Mechanism of acidogenesis
The basic principle of DF is the conversion of sugars into acetic
acid by preferred microbes, resulting in hydrogen production.
The complex sugar is hydrolysed and transferred to the
glycolytic pathway for pyruvate production. During the pro-
cess of glycolysis, ATP is used as the form of energy; mean-
while, pyruvate is converted to acetate and butyrate leading to
the release of H2 [27]. The hydrogen production mechanism
differs between facultative and obligative anaerobes (Fig. 2).
In obligate anaerobes, pyruvate-ferredoxin oxidoreductase
(PFOR) enzyme is employed to oxidize pyruvate to acetyl co-
enzyme A (acetyl-CoA). In this step, ferredoxin (Fd) is reduced,
and Fd is oxidized by (FeFe) hydrogenase enzyme leading to
the formation of H2 (Eqs. (1) and (2)).
Pyruvate þ CoA þ 2Fd(ox) / Acetyl-CoA þ 2Fd (Red) þ CO2 (1)

Table 1 e Types and properties of fuels produced using single/two-stage digestion technology.
Fuel Substrates Technology Products DG DH Theoretical Pros Cons References
(kJ/ (kJ/ energy
mol) mol) recovery
Biohydrogen Organic Photo/Dark H2 (40 206 89 41%
A range of feed-
Cleaning of gas [16,23,96,112]
Waste Fermentation e60%) stock can be used. mixture for the
CO2
Metabolites from removal of CO2.
(40e60%) fermentation can
Inhibition of
be used as added- O2-sensitive
value products. hydrogenase
enzymes.

Low yield.
Biomethane Organic Anaerobic CH4 419 137 90%
Low investment.
Longer HRT
Waste Digestion (50e60%)
Easy maintenance and operational
CO2 and operation. period
(40e50%) (20e30 days).

Lower OLR.
Biohythane Organic Two stage H2 (5 242 117 104%
Shorter HRT and
Time lag
Waste Anaerobic e10%) operation period between
fermentation CH4 (60%) (14 days). two-stage
CO2 (30%)
Higher pollutant digestion.
removal.
Accumulation of
organic acid as
intermediates.
11204 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 6 ( 2 0 2 1 ) 1 1 2 0 1 e1 1 2 1 6

Obligate anaerobes employ FeeFe hydrogenase enzyme in

the catalysis of H2 by transferring electrons from Fe to proton.
Pyruvate-ferredoxin oxidoreductase (PFOR) helps in the con-
version of pyruvate to acetyl CoA and CO2, generating
2 mol mol1 glucose, and the NADH formed during glycolysis
can be used to produce 2 mol of H2, leading to the overall
production of 4 mol of H2 [33]. An electron is transferred from
NADH to ferredoxin via NADH: ferredoxin oxidoreductase
(NFOR), and it gets self-oxidized [34]. Acetate, butyrate, lactate
or ethanol produced in the metabolic pathways by obligative
anaerobes decrease the yield of H2. Due to the lesser avail-
ability of NADH and PFOR, the yield of H2 obligate anaerobes is
very less.

Mechanism of methanogenesis
Fig. 2 e Hydrogen production in Obligate and Facultative
anaerobes. Methyl-coenzyme M reductase (MCR) enzyme is an important
factor for biomethane production. MCR is a hexamer,
comprising of 2 alpha, 2 beta, and 2 gamma sub-units and 2
2Hþ þ Fd (red) / H2 þ Fd (ox) (2) identical nickel porphinoid active sites. The stepwise reduc-
tion of coenzyme-bound and activated C1 intermediates helps
In facultative anaerobic bacteria like Enterobacteria sp and
in the energy metabolism in Archaea. Metabolite methyl-
E. coli, pyruvate oxidation is catalysed by pyruvate formate
coenzyme M (CH3eSCoM) binds with the electron donor co-
lyase (PFL). This leads to the formation of acetyl- CoA and
enzyme B (HSeCoB) to form methane and heterodisulfide
formate (Eq. (3)) [28]. Thereafter, the formate is cleaved and
CoMeSeSeCoB with the help of methyl-coenzyme M reduc-
CO2 and H2 are produced. The overall reaction is catalysed by
tase as catalyst under anaerobic environment. The overall
the formate hydrogen lyase (FHL) enzyme (Eq. (4)) [29].
process requires major factors like coenzyme F430 and nickel-
porphinoid prosthetic group [35,36]. A ferredoxin-like alpha/
Pyruvate þ CoA / acetyl-CoA þ Formate (3) beta-sandwich fold with a duplicated beta-alpha-beta topol-
ogy is attached to the N-terminal region of the alpha subunit.
HCOOH / COL2 þ H2 (4) Coenzyme M binding initiates the changes in molecular
mechanism ensuring the entry of methyl-coenzyme M in the
During the conversion of glucose, 4 mol of H2 are generated
substrate channel before coenzyme B.
if acetate is obtained as the product of pyruvate oxidation.
In hydrogenotrophic methanogenesis, CO2 and H2 are
However, only 2 mol of H2 are generated when butyrate is
generated in acidogenic DF mechanism as energy sources in
produced as the end-product (Eqs. (5) and (6)). Thus, the yield
metabolic pathways of methanogens. The partial hydrogen
of H2 in DF mainly depends upon the fate of pyruvate
pressure in methanogenic environment is between 1 and 10
oxidation.
pa, with the associated free energy change from CO2 and H2
between 20 and 40 kJ mol1, respectively [37,38]. At least
C6H12O6 þ 2H2O / 2CH3COOH þ 2CO2 þ 4H2 (5)
50 kJ mol1 of free energy is required for in vivo ATP synthesis
from ADP and inorganic phosphates. Therefore, less than
C6H12O6 þ 2H2O / CH3CH2 CH2COOH þ 2CO2 þ 2H2 (6) 1 mol ATP per mol of CH4 is produced during growth under
regular physiological conditions [39].
During the production of H2 using facultative and obliga-
tive anaerobes, hydrogenase enzyme plays a major role.
Facultative anaerobes produce four types of nickel-iron [NiFe]
Two-stage AD for biohythane production
hydrogenases: namely, hydrogenase-1 (Hyd-1), hydrogenase-
2 (Hyd-2), hydrogenase-3 (Hyd-3), and hydrogenase-4 (Hyd-
DF is one of the most promising technologies for biohydrogen
4), respectively [7]. Hyd-1 and Hyd-2 are encoded by hya and
production [40]. After producing H2 via DF, the spent media
hyb operons, while Hyd-3 is encoded by hyc operon. Hyd-4
consists of volatile fatty acids (VFAs) derived from residues of
does not play any role in hydrogen production [30,31]. At pH
acetate, propionate, butyrate, etc, among others. These VFAs
6.2e6.5, Hyd-3 catalyses the degradation of formate to H2. fdhF
are used for the conversion of methane by methanogens [41].
gene encodes formate dehydrogenase-H (FDH-H),
Thus, the integration of biohydrogen with biomethane under
hycBCDEFGH encodes formate hydrogen lyase (FHL) system,
the eponym of biohythane will help in improving the recovery
hyp operon codes for 5 genes used in the maturation of hy-
of gaseous fuels. The overall production mechanism requires
drogenase, and fhlA gene encodes the formate regulon [7]. The
the adjustment of pH between 7.0 and 7.8 before the meth-
fhlA gene is a transcriptional activator protein group activated
anogenesis step [17]. The growth of hydrogenotrophic
by the expression of FHL system. Formate activates FDH-H
methanogens is influenced by the dissolved H2 in the media.
and other components of the FHL system by binding to the
Biohydrogen and biomethane integration into biohythane
upstream activator sites [32].
could be worth for industrial commercialization [15].
 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 6 ( 2 0 2 1 ) 1 1 2 0 1 e1 1 2 1 6
Biohythane is comparatively cleaner, more eco-friendly and
energy-efficient, and better combustible than the already
available fuels. The two-stage anaerobic fermentation for
biohythane follows various strategies and conditions [42].
Two-stage anaerobic fermentation involves the optimized
functioning of acidogens and methanogens in nutrition,
physiology, growth, and environment sensitivity. In the first
stage of fermentation, acidogens produce hydrogen, CO2,
VFAs, alcohols, and lactic acids using the substrate. The pH
required for the first stage is in the range of 5.0e6.0, requiring
around 1e3 days for the entire process [43]. During the second
stage of fermentation, methanogens convert the remaining
lactic acids, alcohols, and VFAs into CH4 and CO2 within 10e15
days at pH in the range of 7.0e8.0 [44]. Therefore, the eco-
friendly nature, flexibility in H2/CH4 ratio, high degradation
efficacy, high recovery, less fermentation time and potential
usage as transportation fuel are the major advantages of
biohythane [45]. Generally, there are four processes involved
in one-stage AD, namely, hydrolysis, acidogenesis, aceto-
genesis, and methanogenesis, in a single reactor/fermenter. In
hydrolysis, the complex macromolecules are broken down
into smaller molecules (Eqs. (7)e(9)).
Hydrolysis
(C6H10O4) n þ 2H2O / C6H12O6
Cellulose Glucose
(-RCH (NH2) COO-) n þ (n-1) H2O / nRCH (NH2) COOH
Protein Amino Acids
(H2COOC (CH2) nCH3) n / n(H2COOC (CH2) nCH3)
Fig. 3 e Overall process scheme of a two-stage anaerobic digestion process for biohythane production.
11205
Fat fatty acids
In acidogenesis, VFAs and hydrogen are released. In the
third stage, the products of acidogenesis act as substrates to
produce acetic acid under acetogenesis. Finally, methane and
carbon dioxide are produced during methanogenesis [17]. CH4
(60e65%) is produced as the major product along with other
compounds like CO2, H2, H2S, etc. Higher concentration of
VFAs produced during acidogenesis and acetogenesis causes a
reduction in the pH and increased toxicity of H2, which lead to
lower yield of CH4 [7]. H2 producers cannot tolerate excessive
accumulation of VFAs in the reactor, and the process stops at
the acidogenesis and acetogenesis phases. The dominating
VFAs are usually acetic acid, butyric acid and propionic acid.
The tolerance limit of acetic acid, butyric acid and propionic
acid are reported to be 2400, 1800 mg L1 and 900 mg L1,
respectively. Moreover, the accumulation of H2 in excess also
causes a complete failure of the methanogenesis step. Along
with CH4 as the major component, CO2 constitutes a large
component of 30e40% in biogas in one-stage AD which is a
major cause of GHG emissions, if it is released into atmo-
sphere in an uncontrolled manner [46].
Conventionally, a two stage AD is performed in two
different vessels involving several steps (Fig. 3). Hydrolysis,
acidogenesis, and acetogenesis occurs in the first stage, while
(7) methanogenesis is carried out in the second stage. During the
first stage of anaerobic digestion, the hydrolysation of com-
plex organic polymers are converted to monomers by the
hydrolytic bacteria. The acidogens produce H2, CO2, and
(8)
organic acids by converting the organic monomers. While in
the second stage of the bioreactor, the effluents are utilized by
methanogens to form CH4 and CO2 [18,47]. Further, inhibitory
molecules like VFAs produced during the first stage is also
(9)
converted by the methanogens for improving the yield of CH4.
This process of transferring metabolites from the first to the
second stage improves the H2, CH4 yield and energy content of
11206 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 6 ( 2 0 2 1 ) 1 1 2 0 1 e1 1 2 1 6
the overall process [48]. In a report by Kvesitadze et al. [49],
two-stage AD of municipal solid waste showed ~ 23% and 26%
increase in H2 and CH4, respectively, as compared to the one-
stage digestion process. Willquist et al. [50] produced bio-
hythane by a combination of DF and AD by pre-treating wheat
straw in the presence of Caldicellulosiruptor saccharolyticus
leading to the production of 46% of H2, 43% of CH4, and 0.4% of
CO2. In a study performed by Massanet-Nicolau et al. [51], two-
stage AD increased the CH4 yield from 261 L/kg-volatile solids
(VS) in single stage to 359 L/kg-VS within 20 days. Micolucci
et al. [13], reported that 310 days of a long-term pilot scale trial
using food waste as substrate produced 7% of H2, 58% of CH4
and 35% of CO2 using a two phase thermophilic AD process.
Multistage AD of glucose with coffee seed coated bovine
manure as the substrate in one-stage and three-stage AD
produced high rates of H2 and CH4. Three-stage AD using
lignocellulosic materials as substrate produced 2.4 times
higher energy than the former substrate/feedstock [52]. Food
waste substrate was used to produce biohythane via single
stage anaerobic fermentation at mesophilic temperature. In
that study, the production of 714 mL/L-d of H2 and 254 mL/L-
d of CH4 was reported within 10e12 days. Overall, 8.6% of H2
and 48% of methane was produced [53]. In a recent study by Si
et al. [45], post-hydrothermal liquefaction wastewater was
used to produce 29 mL g1 COD of H2 and 254 mL g1 COD of
CH4 via two-stage fermentation. Thus, all the previous reports
suggest that adapting a two-stage AD is more beneficial than a
one-stage process in terms of higher energy efficiency, high
recovery of energy, short HRT, production of multiple prod-
ucts, better COD reduction, and the process is eco-friendly in
nature. Two-stage AD is more reliable when considering the
operation aspects: good pH regulation, high resistance to-
wards shocks during organic loads, efficient conversion of
organic substrate to energy, and low chances of process fail-
ure due to the accumulation of VFAs [54]. Therefore, a two-
stage AD is the most efficient and suggested fermentation
technique for the production of biohythane.
Microbes involved in biohythane production
To generate biohythane through the two-stage fermentation
process, it is necessary to identify the potential microbes
which are capable of producing both H2 and CH4 in the desired
ratio [55]. Acetogens and methanogens involved in bio-
hythane production are enriched separately owing to their
differences in physiology, growth kinetics, nutritional re-
quirements, and sensitivity towards environmental stimuli
[56]. Hence, these microorganisms act synergistically to
decompose the feedstock in different reaction conditions and
aid in the production of H2 and CH4 [57]. Microorganisms
involved in biohythane production (both acetogens and
methanogens) along with their metabolic routes have been
enlisted in Table 2.
Dark fermentative H2 production
During the first stage of fermentation in a biohythane reactor,
various bacterial strains are involved in H2 production from
different feedstocks through DF reaction. Among the various
microbes involved in H2 production, strict facultative anaer-
obic bacteria (chemoautotrophs) that belong to the mesophilic
and thermophilic domains were found to be efficient H2 pro-
ducers. Under mesophilic conditions, obligate anaerobic Clos-
tridium sp., are well known for high H2 production from various
renewable feedstocks [58]. They are capable of producing H2
with a theoretical yield of 2.1e2.2 mol H2/mol sugar consumed
[59]. The facultative anaerobes Enterobacter sp. were capable of
producing H2 at the yield of 1.0 mol H2/mol sugar under mes-
ophilic conditions [60]. Under anaerobic conditions, E. coli could
convert formate into H2 and CO2 using FHL enzyme and pro-
duce 0.6e1.3 mol H2/mol sugar [61]. Also, the sporulating
facultative mesophilic bacterium Bacillus sp produces H2 with a
yield of 0.5 mol H2/mol glucose, resulting in lactic acid as a
major metabolic end product [62]. Further, DF in the thermo-
philic range (55e60  C) displays much more favourable kinetics
and stoichiometry for H2 production, which also aid in
reducing the risk associated with partial hydrogen pressure
and contaminants associated with inoculum or the feedstocks.
Based on the optimal growth temperature, the thermophilic
bacteria involved in dark fermentative H2 production were
classified as moderate thermophiles (45 Ce55  C), true ther-
mophiles (55 Ce75  C), and extremophiles (above 75  C). Ther-
moanaerobacterium sp. and certain thermophilic Clostridium sp.
were found to be involved in DF under thermophilic conditions.
Thermoanaerobacterium sp. are moderate thermophiles which
produce H2 through butyrate and acetate fermentation [63],
whereas the obligatory anaerobe C. thermocellum utilizes
various hexoses to produce H2 with a yield of 1.2e1.6 mol/H2
mol hexose [64]. DF, under extreme thermophilic conditions,
was performed by Caldicellulosiruptor sp and Thermotoga sp.,
which were able to ferment various hexoses obtained from
lignocellulosic waste, showing a yield of 3.3 mol H2/mol hexose
and 3.3 mol H2/mol hexose, respectively [65,66].
Anaerobic methane digester
During the second stage of biohythane production, methane
production is achieved by various types of bacteria and
Archaea in anaerobic digesters. Several classes of Archaea
classified based on metabolic pathways, e.g. CO2-reducing,
methylotrophs, and acetoclastic, tend to generate CH4
through anaerobic fermentation of bicarbonate, lactic acid,
alcohols, VFAs, and CO2. Under mesophilic conditions, Meth-
anoculleus sp, Methanospirillum sp, and Methanococcus sp play
an important role in CH4 production from CO2, H2 and acetic
acid [57,67,68]. Methanothermobacter sp and Methanosarcina sp
were found to be actively engaged in CH4 formation in ther-
mophilic conditions, in which these hydrogenotrophic
methanogens utilize acetic acid or methyl containing com-
pounds and H2 to reduce CO2 to CH4 [69,70]. Methanothermus
sp. and Methanothermococcus sp grow under extreme thermo-
philic conditions (85  C) and assimilate CO2, H2, or formate as
the energy source for CH4 production [71,72].
Substrates used in biohythane production
The cost-effective production of biohythane (H2 and CH4) re-
lies on the availability of cheap and renewable substrates
Table 2 e Predominant microbes and its metabolic routes involved in biohythane reactors.
Microorganism Anaerobic Reactor’s temperature condition Metabolic feature Reference

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 6 ( 2 0 2 1 ) 1 1 2 0 1 e1 1 2 1 6
characteristics
Acidogenesis (hydrogen producers)

Clostridium sp. Obligately anaerobic Mesophilic condition Able to ferment a wide range of sugars from various renewable substrates to produce [27,59]
bacterium H2 with a yield of 2.1e2.2 mol H2/mol sugar.
Enterobacter sp. Facultative Mesophilic condition Resistant to trace amount of dissolved oxygen and able to produce 1.0 mol H2/mol [60]
anaerobic bacterium sugar.
E. coli Facultative Mesophilic condition Able to ferment glucose and produce H2 with the yield of 0.6e1.3 mol H2/mol glucose [61]
anaerobic bacterium through formate pathway.
Bacillus sp. Facultative Mesophilic condition Able to ferment glucose and produce H2 with the yield of 0.5 mol H2/mol glucose with [62]
anaerobic bacterium lactic acid as main metabolite.
Thermoanaerobacterium sp. Obligately anaerobic Thermophilic condition Able to produce H2 from various sugars through butyrate and acetate fermentation. [63]
bacterium
Thermophilic Obligatory anaerobic Thermophilic condition Able to ferment cellulose from lignocellulosic biomass and capable of producing H2 [64]
Clostridium bacterium with the yield ranges from 1.2 to 1.6 mol H2/mol hexose.
(C. thermocellum)
Caldicellulosiruptor sp. Obligatory anaerobic Extreme thermophilic Able to ferment a wide range of lignocellulosic substrate for H2 production with the [66]
bacterium condition yield of 3.3 mol H2/mol hexose; major metabolites are acetic acid and lactic acid.
Thermotoga sp. Obligatory anaerobic Extreme thermophilic Able to generate H2 at extreme temperature (>90  C) with the yield of 3.5 mol H2/mol [65]
bacterium condition hexose.
Methanogenesis (methane producers)

Desulfobacterium sp. Anaerobic e sulfate Mesophilic condition Involved in initial CH4 production stage which was able to utilize lactic acid and [24]
reducing bacterium pyruvate to the formation of acetate, H2 and CO2 as the major end product.
Methanoculleus sp. Obligate anaerobic Mesophilic condition Plays an important role in second stage production of CH4 from CO2 and H2. [68]
archaea
Methanospirillum sp. Obligate anaerobic Mesophilic condition Able to ferment acetic acid for CH4 production. [57]
archaea
Methanococcus sp. Obligate anaerobic Mesophilic condition Utilize CO2 and H2 or formate as the energy source for CH4 production. [67]
archaea
Methanothermobacter sp. Obligate anaerobic Thermophilic condition Hydrogenotrophic methanogen involved in the consumption of H2 at CH4 production [69]
archaea stage.
Methanosarcina sp. obligate and Thermophilic condition Hydrogenotrophic or acetoclastic able to reduce CO2 or utilize acetic acid and methyl- [70]
facultative anaerobic group containing compounds to produce CH4.
archaea
Methanothermus sp. Obligate anaerobic Extreme thermophilic condition Able to integrate CO2 and H2 for CH4 production at extreme temperature (83e85  C). [72]
archaea
Methanothermococcus sp. Obligate anaerobic Extreme thermophilic condition Hydrogenotrophic methanogen consumes CO2 and H2 or formate for CH4 production. [71]

11207
archaea
11208 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 6 ( 2 0 2 1 ) 1 1 2 0 1 e1 1 2 1 6
Fig. 4 e Schematic representation of various waste substrates involved in biohythane production.
which can be used with minimal pretreatment process,
thereby effectively improving the overall economy of the
process (Fig. 4). Most of the previous studies for H2 production
were based on the exploitation of simple sugars (glucose, su-
crose, starch) for the production of H2. However, the utiliza-
tion of simple sugars is often expensive which makes the
whole process economically unreliable. To address this issue,
the replacement of simple sugars with naturally occurring
inexpensive renewable organic substrates, i.e. those pos-
sessing high content of fermentable complex sugars, has been
shown to be a promising alternative. From an economic
standpoint, the utilization of renewable organic substrates
(complex carbohydrates, lipids, fat and proteins) generated
during day-to-day-life activities can be considered to make
the process economically viable [73]. Organic biomass/sub-
strates, e.g. agri-based processing wastes, oil processing
wastes, brewery processing wastes, food substrates and algal
substrates have been successfully utilized for biohythane
production (Table 3).
Agriculture based substrates
In comparison with lipids, fats and proteins, lignocellulosic
biomass comprising of complex fermentable sugars was
found to be efficient in H2 production, whereas the former
substrates are involved in CH4 production [74]. Lignocellulosic
biomass and its derivatives (e.g. cassava residues, wheat bran,
corn silage and cattail, sweet sorghum and cow manure sug-
arcane syrup) generated from agri-based sector assure unin-
terrupted supply of cheap renewable substrates for
biohythane production. In addition to their cheap availability,
these substrates also have a large volume of nitrogen and
carbohydrates which ensure the appropriate C/N ratio for H2
and CH4 production [75]. Chavadej et al. [76] reports that the
pretreatment of cassava residues produced 15 mL H2 g1
CODremoved and 259 mL CH4 g1CODremoved at the operating
temperature 55  C, where the increased starch content was
likely involved in increased biohythane production. When
sugarcane syrup obtained from sugar processing waste was
utilized at the concentration of 25 g COD L1 in an integrated
continuous stirred tank reactor (CSTR) and upflow anaerobic
sludge blanket (UASB) reactor, H2 and CH4 were produced at
17.5 L1d1 and 2.25 L1d1, respectively [77]. Wheat bran was
utilized to produce biohythane in a mesophilic reactor with a
yield of 8.9 and 243.5 L kg1 VS of H2 and CH4, respectively [78].
Bioaugmentation strategy was implemented for the utiliza-
tion of corn silage and cattail for biohythane production in
UASB reactor in which, the bioaugmentation strain, an
anaerobic fungus, Piromyces rhizinflata YM600 was acclima-
tized for the desired utilization of corn silage which resulted in
its involvement in the specific production of H2 and CH4 in
comparison to CH4 production alone [79]. Sorghum crop,
which is available all through the year, can be efficiently uti-
lized for biohythane production, where it was found to spe-
cifically enhance CH4 production up to 78 L kg1 [80].
Oil processing waste substrates
Palm oil mill effluent (POME) wastes are rich in organic solid
fractions as well as micronutrients (total nitrogen:
0.83e0.92 g L1 and total phosphorus: 0.097e0.125 g L1). Its
low pH at higher temperatures favours its potential to be
utilized for biohythane production [81]. Suksong et al. [82]
produced biohythane via co-digestion strategy by utilizing
POME waste along with the empty palm fruit bunches by
two-stage solid anaerobic digesters. The resultant effluent
from the H2 reactor was converted as feedstock for CH4
production which resulted in the production of
16.26 mL g1 VS (H2) and 240.62 mL g1 VS (CH4), respec-
tively. The two-stage anaerobic digester resulted in an
enhanced variolization of lignocellulosic biomass of co-
substrate [Cellulose: 57e59%, hemicellulose:35e40% &
lignin 16e27%]. Further, the feasibility of using palm oil in-
dustry solid waste residue co-digested with POME waste was
also investigated for biohythane production. The resultant
digestion of oil industry solid waste along with POME effi-
ciently improved the production of biohythane in a signifi-
cant and cost-effective manner [83]. Recently,
Table 3 e Production of biohythane (H2 and CH4) from various organic substrates.
Type of Substrate Reactor Seed Operating Yield Reference
wastes mode conditions
Hydrogen Methane
Agri-based Cassava residue Continuous two- Anaerobic sludge pH 5.5; Temp. 55  C 15 mL H2 259 mL CH4 [76]
processing stage UASB reactor sample taken from g1 CODremoved g1 CODremoved
waste the UASB
substrates
Sugarcane syrup Integrated CSTR Clostridium butyricum pH 4.5e6.1 (H2) and 17.5 L1d1 2.25 L1d1 [77]
with UASB reactor TISTR1032 (H2); Anaerobic 7.0e8.0 (CH4); Temp

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 6 ( 2 0 2 1 ) 1 1 2 0 1 e1 1 2 1 6
granular sludge (CH4) 37 ± 1  C
Wheat bran e Sludge collected from two- 37 ± 1  C 8.9a 243.5a [78]
stage mesophilic
commercial biogas plant
Corn silage UASB reactor Anaerobic fungus P. 37  C 59.4 ± 4.1a 328.8 ± 16.8a [79]
and cattail rhizinflata YM600 and
anaerobic
sludge from
agro-industrial
wastes
treatment plant
Mixture of CSTR systems e pH 5.5 (H2) and 8.0 2.14 L1 LRd 0.90 L1 LRd [113]
ensiled sorghum (CH4); Temp. 37  C
and cow manure
Sweet sorghum CSTR systems Anaerobic sludge pH 5.3 (H2) and 7.5 10.4 L kg1 78 L kg1 [80]
(CH4); Temp. 35  C
Oil processing POME (palm oil Two stage reactor e Thermoanaerobacterium-rich 55  C 73 mL g1 COD 342 mL g1 COD [63]
waste substrates mill effluent) ASBR with UASB Sludge
Palm oil mill e Anaerobic sludge from palm pH 5.5 (H2) and 7.0 16.26 mLb 240.62 mLb [82]
effluent (POME) oil mill wastewater (CH4); Temp. 60  C
treatment (H2); Inoculum (Thermophilic)
from thermophilic biogas
plant (CH4)
Palm oil mill Two-stage anaerobic Anaerobic sludge from Two-stage, 21.9 mLb 364.3 mLb [83]
effluent (POME) fermenter POME biogas plant Thermophilic (55  C).
Brewery processing Agave bagasse Two stage reactors: Anaerobic granular sludge pH 5.5 (H2) and 7.5 105 mL g1 225 mL g1 [114]
waste substrates CSTR with UASB from Tequila vinasses (CH4); Temp. 35  C
reactor reactor (H2) and 23e25  C
(CH4)
Vinasse Serum bottles Granule sludge obtained pH 5.5 (H2) and 7.2 14.8 mLb 274 mLb [87]
from brewery plant (CH4); Temp. 37  C
Tequila vinasse Two-stage reactors: Anaerobic sludge from a pH 5.5 (H2) and 6.8 to 57.4 mL Lh 257.9 mL g1 COD [86]
(glucose adapted) SBR with UASB brewery wastewater 7.5 (CH4); Temp.
reactors treatment plant 35  C
Ethanol stillage CSTR system Thermophilic anaerobic 35  C 48a 344a [85]
digested manure from
biogas plant

11209
(continued on next page)
 11210
Table 3 e (continued )
Type of Substrate Reactor Seed Operating Yield Reference
wastes mode conditions
Hydrogen Methane
Food waste substrates Tomato pomace (TP) HSAD Dry beef manure and Temp 30  C (H2) and 73.17 mL g1 dry GP 201 mL g1 dry TP; 132 mL g1dry GP [89]
and grape pomace mature green waste 55  C (CH4)
waste (GP) compost
Fruit and vegetable CSTR and AFBR Digested sludge from 55 ± 2  C (H2) and 115.2 ± 5.3a 334.7 ± 18.6a [90]
processing wastes mesophilic ADc plant 37 ± 2  C (CH4)
treating buffalo manure,

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 6 ( 2 0 2 1 ) 1 1 2 0 1 e1 1 2 1 6
dairy wastewater
Food waste CSTR system Sludge of cassava waste 37  C 292.7a 391.6a [91]
water treatment plant up
flow anaerobic sludge
blanket (UASB) reactor
Wheat feed pellets Two-stage Anaerobic digester effluent 35  C 7a 359a [51]
fermentation from a local sewage
system treatment works
Mixture of minced CSTR system e 55  C 66.7a 720a [115]
organic waste
Sugar cane Molasses Integrated Anaerobic digester pH 5.5 (H2) and 7.0; 2.8 LR1 d1 1.48 LR1d1 [92]
two stage reactors sludge (CH4); Temp. 35  C
Macro-algae Laminaria digitata, Serum bottles Anaerobic digestate from an 37  C 97.0a 224.3a [95]
substrates Chlorella pyrenoidosa Irish farm digester (H2) and
and Nannochloropsis anaerobic digester effluents
oceanica mainly based on seaweeds
(CH4)
Sargassum sp. Serum bottles Caldicellulosiruptor 70  C (H2) and 37  C 91.3 ± 3.3a 541 ± 10a [94]
saccharolyticus DSM 8903 (CH4)
(H2) and anaerobic granular
sludge from a brewery
industry (CH4)

UASB - Upflow anaerobic sludge blanket; CSTR - Continuous stirred tank reactor; ASBR - Anaerobic sequencing batch reactor; SBR - Sequencing batch reactor; HSAD - High solid anaerobic digestors;
AFBR - anaerobic fixed bed reactor; AD - Anaerobic digestor; VS - Volatile solids.
a
L Kg1 VS.
b
g1 VS substrate.
 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 6 ( 2 0 2 1 ) 1 1 2 0 1 e1 1 2 1 6
Seengenyoung et al. [63], developed a two-stage thermo-
philic reactor for biohythane production from POME waste
substrate. The ratio of produced biohythane of 0.13:0.18 (H2/
CH4) makes an ideal fuel for vehicles, thereby making POME
waste as an ideal candidate for cleaner biofuel production.
Brewery processing waste substrates
Brewer’s spent grain, ethanol stillage, and vinasse are among
the wide range of brewery waste substrates which are ob-
tained during brewery processing and production [84]. The
stillage obtained from an ethanol plant was employed as
substrate in a two-stage aerobic thermophilic reactor which
resulted in the production of 48 and 344 L kg1 VS of H2 and
CH4, respectively. Microbial community analysis revealed that
Thermoanaerobacterium thermosaccharolyticum and Meth-
anosarcina acetivorans played major roles in the H2 and CH4

n et al. [86] analyzed the feasibility of uti-
reactors [85]. Buitro
lizing tequila vinasses in a two-stage sequencing batch reactor
(SBR) and a UASB reactor, resulting in the production of
57.4 mL Lh H2 and 257.9 mL g1 COD CH4. The methane
composition dominated at 68% in biohythane production with
an influent concentration of 1636 mg COD L1 which resulted
in 73e75% of organic matter removal. Fu et al. [87] also eval-
uated the two-stage AD for biohythane production which
significantly improved the efficiency of substrate utilization
and energy recovery from vinasse and resulted in 14.8 mL g1
VSsubstrate H2 and 274 mL g1 VSsubstrate CH4.
Food waste substrates
Food wastes are ideal candidates that can serve as feedstocks
for anaerobes due to the availability and abundance of energy
associated with this waste substance, which also provide
added benefits for managing solid organic waste disposal [88].
Achmon et al. [89] demonstrated the efficacy of using tomato
pomace (TP) and grape pomace (GP) waste feedstock for
enhanced biohythane production. Microbial community
analysis results showed that Methanoculleus, Methanosarcina
(Archaeal genera) and Halanaerobiaceae (Bacteriales order) as
the dominant species involved in the production of H2 and
CH4 with a yield of 201 mL g1 dry TP and 132 mL g1dry GP,
respectively. Yeshanew et al. [90] carried out continuous
production of biohythane using food wastes by employing
integrating CSTR and an anaerobic fluidized bed reactor
(AFBR) which resulted in the generation of
115.2 ± 5.3 L kg1VSadded H2 along with 334.7 ± 18.6 kg1
VSadded CH4. The recirculation of AFBR effluent to CSTR was
effective in maintaining the pH of the hydrogen reactor which
significantly improved the economy of the process. Pisut-
paisal et al. [91] also investigated the total energy recovery
from food waste in which hydrogen and methane production
were accomplished sequentially. The results revealed that the
hydrogen and methane yields were 292.7 and 391.6-mL g1 VS,
respectively, with the total energy recovery from two-stage
process of 6.5  102 kW-h. The two-stage AD of sugarcane
molasses, a food industry by-product at mesophilic conditions
produced H2 and CH4 with the yield of 2.8 LR1d1 and
1.48 LR1d1, respectively. Clostridium butyricum, Meth-
anobacterium beijingense, and Methanothrix soehngenii were
11211
found to be the dominant microflora involved in biohythane
production [92].
Macro-algae substrates
Aquatic macroalgae are ubiquitous organisms which possess
a high content of polysaccharides and are devoid of or possess
low content of lignin making them an ideal candidate for the
production of biofuels [93]. Hence, researchers have investi-
gated the possibilities of utilizing macroalgae as feedstock for
biohythane production. Preliminarily, Costa et al. [94] inves-
tigated the potential of using macroalgae Sargassum sp. for
biohythane production using Caldicellulosiruptor saccharolyticus
DSM 8903 (H2) and the addition of anaerobic granular sludge
(CH4) as the inoculum resulted in a yield of 91.3 ± 3.3 and
541 ± 10 L kg1 VSadded for H2 and CH4, respectively. Similarly,
Ding et al. [95] also investigated the utilization of both carbon-
rich macro algae and nitrogen-rich micro-algae (Chlorella pyr-
enoidosa and Nannochloropsis oceanica) in order to maintain a C/
N ratio of 20 for a two-stage co-fermentation process for bio-
hythane production. The co-fermentation technique eventu-
ally resulted in the production of 97.0 and 224.3-mL g1VS of
H2 and CH4, respectively, and enhanced the energy conversion
efficiency from 6.6% to 70.9% with mono-fermentation.
Technical challenges and possible solutions for
enhanced biohythane production from organic
waste biomass
Two-stage anaerobic digestion-based biohythane production
is emerging as a potential technology for the sustainable
generation of biofuel from organic biomass. However, there
are several shortcomings, which need to be addressed to
attain the desirable biohythane yield at larger scales. Some of
the major challenges with plausible solutions are summarized
below:
Pretreatment
Biohythane production by two-stage anaerobic fermentation
primarily relies on the carbohydrate content of the feedstock.
Organic waste biomass comprises diverse feedstock (agri-
based, food-based and microalgal substrates) rich in complex
fermentable sugars which can be efficiently exploited for
biohythane production. However, these substrates are present
as complex polymers which hinder effective bio-conversion
by microorganisms for biofuel production [58]. Hence, it is
essential to pre-treat these biomasses to improve their
biodegradability, which would eventually enhance both rate
and yield of biohythane [96]. Numerous pretreatment
methods based on physical, chemical and biological steps,
have been proposed to achieve sustainable gaseous biofuel
production from organic biomass. However, these methods
are associated with their own merits and demerits and no
single method is optimal to treat all types of biomass/feed-
stock. Hence, more in-depth research is needed to understand
the biomass composition as well as its suitable/optimal pre-
treatment conditions. In particular, a separate pretreatment
stage aids in enhancing the efficiency of hydrolysis and also
11212 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 6 ( 2 0 2 1 ) 1 1 2 0 1 e1 1 2 1 6
prevents the formation of inhibitors during acetogenesis and
methanogenesis [97]. Hence, the exploitation of a combined
pretreatment step, where two or more techniques are com-
bined to treat various feedstocks will be be more efficient than
a single pretreatment method.
Microbial consortium management
The preliminary challenge in achieving enhanced biohythane
production from organic feedstock is developing a consistent
design and rational control of the microbial consortium
involved in the two stages of anaerobic fermentation [98]. In
biohythane production, two groups of microorganisms take
part in both acetogenesis and methanogenesis as a synergistic
consortium [99]. However, the native microbial consortium
involved in DF/AD is limited due to its instability in treating
complex organic feedstock for long-term controlled opera-
tions and inefficiency in simultaneously generating bio-
hydrogen/biomethane from the treated feedstocks [23]. The
indigenous consortium is inefficient in hydrolyzing biomass
and it primarily relies on the pretreatment, which increases
the process economy in large-scale installations. Besides, in
DF, the native biohydrogen producers present in a consortium
are inferior in producing high rate and yield of H2 than the
pure culture [19]. Hence, a fundamental understanding of the
microbial community involved and its mechanism/pathway
for the hydrolysis of feedstock with simultaneous biohythane
production via two-stage anaerobic fermentation is required.
It can be achieved by sensibly designing a well-defined or
engineered microbial consortium (e.g. the supplementation of
bioaugmentation strain/acclimatization), where it directly
involves both feedstock hydrolysis and biohythane produc-
tion simultaneously through a consolidated bioprocessing
strategy. In bioaugmentation, exogenous supplementation of
microbial consortia or a strain enriched from the nature, with
the appropriate enzymatic arsenal, into the microbial com-
munity improves the efficiency of substrate hydrolysis and
fermentation [58,100]. The introduction of bioaugmentation
strategy in biohythane production offers several advantages:
(i) the process is simplified, (ii) no pretreatment of feedstocks,
(iii) ability to recover the system that suffers from high OLR
and (iv) prevents the over accumulation of VFA in AD [101].
However, based on its metabolic characteristics, exogenous
microorganisms fail to integrate into the indigenous popula-
tion, which also affects its contribution to the process of bio-
hythane production [102]. Hence, further research should be
focused on understanding the metabolic capabilities of
different microbial population involved and their interactions
for enhanced biohythane production.
Genetic modification of microbes
Investigations on microbial populations and their interactions
involved in biohythane generation will provide deep insights
about the feasibility of altering the microbes for enhanced
production. Microorganisms involved in the production of H2
is affected by competitive by-products, which consume the
reducing equivalents, i.e. NADþ/NADH and Hþ and they have a
major impact on the yield and rate of H2 production [103].
Lactic acid is a predominant competitive metabolic by-
product, which consumes the electron required for the
enzyme hydrogenase and interferes with the neutralization
process [104]. Further, the production of butyric and propionic
acids has an inhibitory effect on the methanogenesis process
[105]. Hence, metabolic engineering studies associated with
the targeted blocking of pathways associated with the pro-
duction of lactic acid and alcohols will eventually increase the
reducing equivalents, thus, improving the overall H2 and CH4
production.
Energy evaluation
Based on the chemical energy of the feedstock used in two-
stage anaerobic fermentation, biohythane has a higher theo-
retical energy recovery [23]. However, compared to the single-
stage AD for biogas production, biohythane requires a two-
stage reactor for the production of H2 and CH4, which re-
quires additional energy for heating up reactors, shifting of
effluents between the reactors, followed by the release of
products [25,106]. These technical challenges, along with the
operating complexity, reactor configuration and high invest-
ing cost limit the application of two-stage digesters for bio-
hythane production [107]. These problems can be addressed
using a single-stage process in which both H2 and CH4 can be
produced simultaneously [53]. Recently, a novel single-stage
dark fermentation system using entrapped hydrogenic and
methanogenic bacteria was proposed for biohythane pro-
duction [8]. However, the mechanism behind the phase sep-
aration of H2 and CH4 needs to be elucidated in order to
implement these bioreactors at the full-scale.
Process integration
The cost-efficient production of biohythane primarily de-
pends on the individual production yields of H2 and CH4 from
renewable organic feedstocks. However, this is often associ-
ated with intense energy consumption for heating, digestate
recirculation and separation, which eventually increases the
cost of the process [107]. Hence, an efficient integration of
two-stage process into a single-stage continuous operation
can significantly improve the overall economy of biohythane
production. The most influential process parameters that play
an important role in the integration of the processes are mi-
crobial population, accumulation of organic compounds,
reactor design, HRT and OLR [108]. From a microbial
perspective, the design and integration of the reactors aid in
improved mixing, cautiously maintaining the metabolic
characteristics of the microbes, which significantly improves
the conversion and synergetic use of substrate that enhance
biohythane production [16]. HRT and digestate recirculation
help in maintaining the desired microbial population in the
reactors. Also, the hydrogen producers present in the aceto-
genic stage play a key role in the production of VFAs and other
effluents, which typically act as a substrate for CH4 production
in the methanogenic phase [17]. However, feeding the acido-
genic digestate toward the methanogenic reactor in a
continuous reactor is more difficult during continuous reactor
operation. Hence, an advanced quality control algorithm,
online monitoring of key parameters and an integrated pro-
cess control system should be considered for the efficient
 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 6 ( 2 0 2 1 ) 1 1 2 0 1 e1 1 2 1 6
production of biohythane [109]. Furthermore, the recirculation
of digestate from the methanogenic stage to the cetogenic
reactor also poses a harmful effect over the hydrogen pro-
ducing microbial consortium. However, it can be circum-
vented by the introduction of innovative solutions like an
increase in OLR [85], temperature-based process [110], a
membrane filter mediated digestate recirculation [111] and
enhancement of hydrogen producers by bio-augmentation.
Conclusions
This review provides a comprehensive description of the
recent advancements in biohythane production from various
organic substrates. The presence of hydrogen in biohythane
significantly improves its combustion yield with reduced CO2
and NOx emission that results in a cleaner and superior
transport biofuel with reduced carbon footprint compared to
biogas. This review also summarizes the production of bio-
hythane from waste organic biomass via two-stage anaerobic
fermentation, which is found to be a promising technology for
renewable biofuel production with higher energy recovery
and waste biomass valorization. Further, the various strate-
gies that help to overcome the technical challenges in scaling
up biohythane production and the possibilities of commer-
cialization of different technologies have been discussed.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
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