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 4 ( 2 0 1 9 ) 1 7 3 6 3 e1 7 3 8 0

Available online at www.sciencedirect.com

ScienceDirect

journal homepage: www.elsevier.com/locate/he

Biohythane production in two-stage anaerobic

digestion system

Meenu Hans a,b,c,1, Sachin Kumar a,*

a
Biochemical Conversion Division, Sardar Swaran Singh National Institute of Bio-Energy, Kapurthala, 144601, India
b
~ o Carlos, University of Sa
Institute of Physics of Sa ~o Paulo, Sa
~ o Carlos, Sa
~o Paulo, 13560-970, Brazil
c
Department of Microbiology, Guru Nanak Dev University, Amritsar, Punjab, 143005, India

article info abstract

Article history: Hydrogen (H2) and methane (CH4) are the potential alternative energy carriers with
Received 21 August 2018 autonomous extensive and viable importance. These fuels could complement the advan-
Received in revised form tages, and discard the disadvantages of each other, if produced simultaneously. Consid-
30 September 2018 ering their complementary properties, co-production of a mixture of H2 and CH4 in the
Accepted 3 October 2018 form of biohythane in two-stage anaerobic digestion (AD) process is gaining more interest
Available online 30 October 2018 than their individual production. Biohythane is a better transportation fuel than com-
pressed natural gas (CNG) in terms of high range of flammability, reduced ignition tem-
Keywords: perature as well as time, without nitrous oxide (NOx) emissions, improved engine
Anaerobic digestion performance without specific modification, etc. Other than production of biohythane,
Hydrogen performing two-stage AD is advantageous over one-stage AD due to short HRT, high energy
Methane recovery, high COD removal, higher H2 and CH4 yields, and reduced carbon dioxide (CO2) in
Biohythane biogas. For improved biohythane production, various aspects of two-stage AD need to be
Biowastes emphasized. Keeping the facts in mind, the process of two-stage AD along with microbial
diversity in comparison to one-stage AD has been discussed in the previous sections of this
review. For large scale commercial production, and utilization of biohythane in automobile
sector, its execution needs evaluation of process parameters, and problems associated
with two-stage AD. Hence, the later part of this review describes the production process of
biohythane, concerned microbial diversity, operational process parameters, major chal-
lenges and their solutions, applications, and economic evaluation for enhanced production
of biohythane.
© 2018 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.

thermo-chemical and biochemical which are usually used in

Introduction
Biofuels are emerging as relatively cleaner and significant
renewable alternate energy carriers to the continuously
diminishing fossil fuels. There are various processes including
biofuel production. However, biochemical processes have ad-
vantages over thermo-chemical processes i.e. low cost, less
energy intensive and environment friendly. A variety of
lignocellulosic biomass (LCB) such as agricultural residues,

* Corresponding author. Biochemical Conversion Division, Sardar Swaran Singh National Institute of Bio-Energy, Jalandhar-Kapurthala
Road, Wadala Kalan, Kapurthala, 144601, Punjab, India
E-mail addresses: sachin.biotech@gmail.com, sachin.kumar20@gov.in (S. Kumar).
1
~ o Carlos, University of Sa
Present address: Institute of Physics of Sa ~ o Paulo, Sa
~ o Carlos, Sa
~ o Paulo, 13560-970, Brazil.
https://doi.org/10.1016/j.ijhydene.2018.10.022
0360-3199/© 2018 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
17364 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 4 ( 2 0 1 9 ) 1 7 3 6 3 e1 7 3 8 0
forestry residues, industrial and municipal wastes, etc. are
being utilized for the biofuels production [1]. Hydrogen (H2) and
methane (CH4) are significantly desirable biofuels due to their
variety of applications. Although, hydrogen is considered as
the most efficient among all the biofuels and widely used in
chemical industries due to high calorific value of 143 kJ/g,
electricity production via fuel cells, and clean due to formation
of only water as a combustion product [2]. Further, combustion
of both CH4 and H2 is not associated with release of nitrous
oxide (NOx), and sulphur dioxide (SOx) unlike other fuels [3].
Biogas, which is typically consists of CH4 as the major
component, is used for heating, cooking and electricity gen-
eration, and the digestate is further utilized as a biofertilizer
[4]. Additionally, CH4 is also used as compressed natural gas
(Bio-CNG), which is an efficient and clean transportation fuel
than gasoline or diesel. However, application of CH4 as bio-
CNG is limited by its narrow range of flammability, slow
burning speed and high ignition temperature [5]. Interestingly,
low flammability range of CH4 in bio-CNG can be extended by
adding a small quantity of H2 due to its about 2½-folds higher
mass specific heating value (119,930 kJ/kg) than CH4 (50,020 kJ/
kg), and the burning time of engine can also be reduced due to
about 7-fold higher burning speed of H2 flame (265e325 cm/s)
over CH4 [5,6]. In addition, the presence of CO2 and hydrogen
sulphide (H2S) in biogas limits its use. For H2 production, dark
fermentation (DF) is considered as the most efficient process.
However, theoretical yields of H2 are never achieved due to
problems such as high cost of operation, formation of inhibi-
tory metabolites (VFAs), and co-existence of H2 producers
with methanogens. Only 20% of chemical oxygen demand
(COD) was removed with low energy recovery during the
process of individual H2 production, thus, alone H2 is not
sufficient in reducing the organic content of biowastes [7,8].
The major part of energy of organic matter is remained as
volatile fatty acids (VFAs) at end of the process. Thus, the VFAs
need to be utilized further for maximum energy recovery.
Anaerobic digestion (AD) is best way to utilize these VFAs for
CH4 production in the form of biogas as it already leads to H2
production during first three steps using VFAs due to co-
existence of methanogens with H2 producing bacteria in na-
ture. However, conventional AD process (one-stage) is ar-
ranged in the way which results in only CH4 as large part of
biogas with only traces of H2.
By designing AD as two-stage process for co-production of
H2 along with CH4 simultaneously rather than their individ-
ual production, the heat efficiency of bio-CNG could be
improved, makes the engine easy to ignite, reduces the en-
ergy requirements, and could also improve the H2 production
by combating the problems associated with DF alone. Both H2
and CH4 could complement their beneficial effects, and
compensate each other's disadvantages and difficulties,
therefore, their co-production in the form of “hythane” is
gaining attention as valuable fuel for vehicles [9]. Hythane® is
a trademark introduced by Hydrogen Component Inc. (HCI) in
their patented study of using a mixture of H2 and CH4 as a fuel
for internal combustion engines, and reported that burning of
this H2 rich CNG could reduce the release of NOx into the
atmosphere with comparable energy efficiency to CNG [10].
Using this blend of gases is beneficial due to relatively inex-
pensive, non-requirement of specific storage system and
changes in CNG engines as well as infrastructures. Hythane is
significantly advantageous over CNG in terms of high flam-
mability range due to presence of H2 as it has a flame speed of
7-fold than that of CH4 [11]. Excellent reducing power of H2
stimulates the combustion of CH4, and increases the burning
even at low temperatures [2]. Moreover, it is eco-friendly
because it causes reduced GHG emissions into atmosphere
as H2 replaces, and reduces the CO2 in a mixture of gaseous
products. Nowadays, term “hythane” is being replaced by
“biohythane” as organic wastes (biowastes) are used as sub-
strate for its production [12]. Buitron et al. [13] reported
73e75% organic removal from tequila vinasses using pre-
heated sludge as the inoculum in sequencing batch reactor
(SBR) and upflow anaerobic sludge blanket (UASB) for pro-
duction of H2 and CH4, respectively. In another study, Yang
et al. [14] reported CH4 production using effluent of the H2
forming phase. Formally, biohythane can be defined as a
mixture of 10e15% H2, 50e55% CH4 and 30e40% CO2 and
traces of H2S, ammonia (NH3), etc. produced via two-stage AD
of biowastes [15]. The biohythane was first utilized as a
transportation fuel in 1995 in a project named as Montreal
Hythane Bus Project, and achieved 45% reduction in NOx
emission compared to the conventional CNG fuelled buses,
followed by Sweden and China in Beijing Hythane Bus Project
[10]. Currently, various car manufacturing companies like
Toyota, Volvo and Fiat are developing cars based on bio-
hythane as fuel [16,17]. Some of the countries like United
States of America (USA), and India are being commercializing
biohythane as a transportation fuel [18]. However, from
scaling up point of view, two-stage AD plants correspond to
only <1% of full scale AD plants at present [2]. Thus, scaling
up of biohythane production via two-stage AD along with its
utilization needs to be implemented to expand its applica-
tions in automobile sector along with other industrial appli-
cations. The widely accepted and easiest method of
biohythane production is two-stage AD, which involves the
production of H2 in the first, and CH4 in the second stage [19].
This review is focused on the important aspects of two-stage
AD such as the process mechanism, microbial diversity
involved, operational factors, key challenges and solutions,
applications, and economic assessment of biohythane
production.
Anaerobic digestion
Anaerobic digestion (AD) is an anaerobic breakdown of
organic matter by a variety of microorganisms biochemically
to produce energy in the form of ATP. Generally, AD consists
of four steps; hydrolysis, acidogenesis, acetogenesis and
methanogenesis [20]. In hydrolysis, complex organic matters
that are carbohydrates, proteins, and lipids undergo hydro-
lysis to simpler monomers that are sugars, amino acids and
fatty acids, respectively (Eqs. (1)e(3)). In acidogenesis, the
acidogens convert the products of hydrolysis into VFAs, H2,
and CO2 along with H2S, ethanol and NH3 in traces (Eqs.
(4)e(7)). In acetogenesis, acetogenic bacteria convert VFAs into
acetic acid, more H2, CO2, and other products in traces (Eqs
(8)e(10)). Finally, in methanogenesis, methanogens convert
acetic acid, H2, CO2 into CH4 and CO2 (Eqs. (11)e(13)) [21e23].
 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 4 ( 2 0 1 9 ) 1 7 3 6 3 e1 7 3 8 0
Hydrolysis
ðC6 H10 O4 Þn þ 2H2 O / C 6 H12 O6 þ O2
Cellulose Glucose
ð  RCHðNH2 ÞCOO  Þn þ ðn  1ÞH2 O / nRCHðNH2 ÞCOOH
Protein Amino acids



H2 COOCðCH2 Þn CH3 / n H2 COOCðCH2 Þn CH3
n (3)
Fat Fatty acids
Acidogenesis
C6 H12 O6 /2CH3 CH2 OH þ 2CO2
C6 H12 O6 þ 2H2 /2CH3 CH2 COOH þ 2H2 O
C6 H12 O6 /CH3 CH2 CH2 COOH þ 2CO2 þ 2H2
C6 H12 O6 /3CH3 COOH
Acetogenesis
CH3 CH2 COO þ 3H2 O4CH3 COO þ Hþ þ HCO3 þ 3H2
C6 H12 O6 þ 2H2 O42CH3 COOH þ 2CO2 þ 4H2
CH3 CH2 OH þ 2H2 O4CH3 COO þ 2H2 þ Hþ
Methanogenesis
CH3 CH2 OH/CH4 þ 2CO2
CO2 þ 4H2 /CH4 þ 2H2 O
2CH3 CH2 OH þ CO2 /CH4 þ 2CH3 COOH
In fact, first three steps of AD: hydrolysis, acidogenesis
and acetogenesis, where H2 is formed, are similar to DF.
These steps (or DF) involves degradation of complex mate-
rials preferably carbohydrates to simple sugars such as
glucose, which is metabolized to pyruvate via glycolysis. In
glycolytic pathway, generated ATP are utilized by microbes
to get energy, while pyruvate is converted to acetate and
butyrate, and H2 is produced as a result of neutralization of
released electrons in the process by protons (Hþ) with the
help of hydrogenase enzyme (H2ase) (Fig. 1). H2 is produced
via different routes followed by obligate and facultative
anaerobes. Obligate anaerobes (Clostridia) first, oxidizes py-
ruvate to acetyl coenzyme A (CoA) with the help of
pyruvate-ferredoxin oxidoreductase (PFOR) and reduction of
ferredoxin (Fd), which is further oxidized with the formation
of H2 by H2ase (Eqs. (14) and (15)) [24]. On the other hand,
facultative anaerobes (Enterobacter) oxidize pyruvate to
acetyl-CoA and formate by pyruvate formate lyase (PFL), and
the formate is further cleaved to CO2 and H2 by formate
hydrogen lyase (FHL) (Eqs. (16) and (17)) [25]. For one mol of
glucose, 4 mol of H2 are produced if acetate is the sole end
product of pyruvate oxidation whereas, 2 mol of H2 are
produced if butyrate is the sole end product (Eqs. (6) and (9))
[26,27].
17365
C3 H3 O3 þ CoA þ 2FdðoxÞ/acetyl  CoA þ 2FdðredÞ þ CO2
(1) (14)
2Hþ þ FdðredÞ/H2 þ FdðoxÞ (15)
(2) C3 H3 O3 þ CoA/acetyl  CoA þ CHOO (16)
HCOOH/CO2 þ H2 (17)
In last step of AD i.e. methanogenesis, the principle
enzyme system involved is methyl-coenzyme M reductases
(MCR). This enzyme catalyzes two important transformations:
(4)
CH4 formation and anaerobic oxidation of CH4. It uses methyl
thioether methyl-coenzyme M (CH3eSeCH2CH2eSOe 3 or
(5)
MeeSeCoM) and thiol coenzyme B (CoB-SH) as substrates, and
converts them reversibly to CH4 and corresponding hetero-
(6) disulfide (CoBeSeSeCoM) [28]. However, the catalytic mech-
anism is still unknown. On the basis of desired final products,
(7) AD could be divided into two types: one-stage AD leading to
CH4 as predominant and desirable product along with other
products in traces, and two-stage AD leading to both CH4 and
(8) H2 simultaneously as desirable products, discussed in further
sections. Difference in one-stage and two-stage AD is
(9) described in Fig. 2.
(10) One-stage anaerobic digestion
In one-stage AD, all four steps of the process that are hydro-
(11) lysis, acidogenesis, acetogenesis and methanogenesis are
performed in a single reactor/digester. Thus, one-stage pro-
(12) cess leads to 60e65% of CH4 as major product including H2,
CO2, H2S, etc. in traces. One-stage AD is an ancient, naturally
(13) occurring and efficient process, however, it is associated with
one inescapable drawback that is acidification of reactor due
to formation of volatile fatty acids (VFAs) as inhibitory me-
tabolites during acidogenesis and acetogenesis. Higher con-
centration of VFAs beyond a certain limit decreases the pH
value, and cause toxicity to H2 producers, and results in
reduced CH4 yield [29]. H2 producers could not tolerate
excessive accumulation of VFAs in the reactor and process
stops at acidogenesis and acetogenesis phases, and causes the
process to halt. Dominating VFAs are acetic acid, butyric acid
and propionic acid [30]. Tolerance limit of acetic acid, butyric
acid and propionic acid are reported to be 2400, 1800 mg L1
and 900 mg L1, respectively [31]. Moreover, accumulation of
H2 in excess also causes complete failure of methanogenesis
[32]. Along with CH4 as the major component, CO2 constitutes
a large contribution of 30e40% in biogas in one-stage AD
which is major cause of GHG emissions if it is released into
atmosphere in an uncontrolled manner.
Two-stage anaerobic digestion (AD)
In case of two-stage AD, first three steps/phases; hydrolysis,
acidogenesis and acetogenesis are carried out in one vessel/
bioreactor as first stage, and last and fourth step/phase i.e.
methanogenesis is carried out in a separate vessel/reactor as
second stage. In first reactor (first-stage), hydrolytic bacteria
17366 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 4 ( 2 0 1 9 ) 1 7 3 6 3 e1 7 3 8 0

Fig. 1 e Fermentation for hydrogen production (PFOR: Pyruvate-Ferredoxin Oxidoreductase; PFL: Pyruvate Formate Lyase;
FHL: Formate Hydrogenlyase).

Fig. 2 e Single-stage and two-stage anaerobic digestion (AD) processes.

 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 4 ( 2 0 1 9 ) 1 7 3 6 3 e1 7 3 8 0
hydrolyze the complex organic polymers to monomers, acid-
ogens convert monomeric organic molecules into H2, CO2 and
organic acids, and further acetogens convert all the organic
acids into acetic acid, H2, and CO2 (Eqs. (1)e(10)). In second
reactor (second stage), methanogens utilize effluents and
products of first-stage (organic acids including acetic acid, H2,
and CO2) to form CH4 and CO2 [33].
In case of two-stage AD process, inhibitory metabolites
(VFAs) of first-stage are supplied to methanogens to improve
CH4 yield in the second-stage. It not only results in the
improved production and collection of H2 in first-stage, but
also improves the CH4 yield and energy content. Kvesitadze
et al. [34] reported 23% and 26% increased production of H2
and CH4, respectively, in the two-stage process as compared
to the one-stage process from municipal solid wastes. Malave
et al. [35] conducted one-stage and two-stage AD with glucose,
and one-stage and three-stage AD with coffee seed coatings
using bovine manure as inoculum. The two-stage AD effi-
ciency using glucose was 48% higher than the one-stage AD,
while the efficiency using lignocellulosic material was 2.4
times higher in three-stage AD than the one-stage AD. The
CH4 yields of one-stage and two-stage were 50 L/kg-VS and
150 L/kg-VS, respectively. This study confirms that the two-
stage AD process results in more output of energy from
organic wastes than the one-stage AD. Massanet-Nicolau et al.
[36] reported the comparison of one-stage and two-stage
digestion with real time gas production data from wheat
feed pellets using effluent of a local sewage treatment plant as
inoculum. CH4 yield increased from 261 L/kg-VS in single-
stage to 359 L/kg-VS in two-stage AD process in 20 days. In a
study, biohythane was produced by combining DF (H2 pro-
duction) and AD (CH4 production) using pretreated wheat
straw with Caldicellulosiruptor saccharolyticus for H2 production
and an undefined consortium for CH4 production and reported
46e57% H2, 43e54% CH4 and 0.4% CO2 [37].
One-stage AD takes about 28e30 days for completion of
whole process to CH4 as the end product. Whereas, hydraulic
retention time (HRT) of acidogenic step is only 1e3 days to
produce acids along with H2. Thus, in separate reactor in two-
stage AD, H2 could be collected immediately after production,
and effluents are supplied to methanogenic reactor where it
takes about 10e15 days for CH4 production, thus process
generally takes lesser time for production of both of the
products [38]. Adapting two-stage AD is advantageous over
one-stage AD in terms of short HRT, high energy efficiency
and recovery, co-production of two-valuable fuels, high COD
reduction, environmental concerns, etc. This system gives
reliable operation due to better pH self-adjusting capacity, a
higher resistance to organic loading shocks, higher conversion
rate of organic substrate, and prevents eventual failure by
VFAs accumulation [39].
Microbial diversity
First-stage anaerobic digestion
In both one-stage and two-stage AD, bacteria carry out the
first three steps. In nature, hydrogen producers co-exist with
methanogens as they are mainly responsible for substrate
17367
formation for methanogens i.e. organic acids. Various micro-
organisms involve in H2 production, which are strict anaer-
obes, facultative anaerobes and some aerobes [40]. Clostridia
are the industrially most important bacteria engaged in H2
production [41]. These are Gram-positive, rod-shaped, spore-
forming obligate anaerobes with low G þ C content, and
have lower doubling time and stress tolerance. Clostridia can
utilize pentose as well as hexose sugars present in LCB effi-
ciently [42]. Enterobacter sp. another important H2 producers,
are Gram-negative, rod-shaped, facultative anaerobes with
higher growth rates as compared to obligate anaerobes.
Enterobacter sp. also utilize LCB efficiently, however, they are
resistant to lower traces of dissolved oxygen. Escherichia coli
are Gram-negative, rod-shaped, with low G þ C content and
produce H2 primarily from formate. E. coli is the key organism
for genetic manipulation for the improvement of H2 produc-
tion. In recent times, selective knockout of frdC (furmarate
reductase), ldhA (lactate dehydrogenase), fdnG (formate de-
hydrogenase), ppc (phosphoenolpyruvate carboxylase), narG
(nitrate reductase), focA (formate transporter), hyaB (the large
subunit of hydrogenase 1), aceE (pyruvate dehydrogenase),
mgsA (methylglyoxal synthase), and hycA (a regulator of the
transcriptional regulator FhlA) in E. coli resulted into improved
H2 using glycerol as substrate [43]. Bacillus sp. is a Gram-
positive, spore forming facultative bacterium, and tolerates
unfavorable conditions.
Second-stage anaerobic digestion
The last step i.e. methanogenesis is carried out by archea.
Archea are strict anaerobes that consume H2 and CO2 to form
CH4. Two types of methanogens are involved in the process
depending upon utilizing H2 and CO2 and acetic acid to form
CH4 as the end product. The H2 consuming methanogens are
called hydrogenotrophs, which consume H2 and CO2 to form
CH4 [7], whereas, acetic acid utilizing methanogens are called
as acetoclastic methanogens e.g. Methanoculleus sp. [44]. In
two-stage AD, hydrogenotrophs (H2 consumers) are undesir-
able as H2 is required as end-product whereas, there is need of
H2 producers and acetoclastic methanogens in the process.
Thus, hydrogenotrophs get deactivated in the two-stage AD
process. Their deactivation is discussed further in Section
Inoculum. Under the kingdom archaea, the methanogens
were classified under Euryarchaeota, which also comprise of
extremehalophiles e.g. Thermoplasma. These are usually
Gram-positive, rod-shaped methanogens that use CO2 as en-
ergy source to reduce methanol to CH4. Methanosphaera sp.,
however, is an exception to the above group as they are cocci
in shape and use H2 as energy source. They mainly comprise
of two families, Methanobacteriaceae, and Methanothermaceae.
The Methanobacteriaceae family is highly diverse and includes
several genera such as Methanobacterium, Methanothermobacter
gen. nov., Methanobrevibacter and Methanosphaera [26]. Various
studies have been reported the difference in microbial com-
munity for both the stages in biohythane production process
[7,44,45]. H2 producers dominate in acidogenic reactor i.e.
when H2 forms as a by-product, while, the microbial domi-
nance shifts to methanogens in methanogenic reactor. Zahedi
et al. [46] investigated the microbial community in two-stage
thermophilic AD from co-digestion of organic fraction of
17368 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 4 ( 2 0 1 9 ) 1 7 3 6 3 e1 7 3 8 0
municipal solid waste (OFMSW) and waste active sludge
(WAS). They found bacteria as the dominating phylum in
acidogenic and methanogenic stages. Microorganisms in
acidogenic stage that are non-firmicutes and non-clostridia,
were lower in amount than methanogens. Clostridium sp.
were 76% of total firmicutes in acidogenesis, but they got
reduced in second step due to substrate deficiency. Other fir-
micutes increased due to increase in pH and decrease in H2
partial pressure, and Methanobacteriales constitute the major
group of archaea. In a study of thermophilic and mesophilic
biohythane production, acidogenic and methanogenic re-
actors were observed to be dominated by Thermoanaer-
obacterium thermosaccharolyticum and acetoclastic
Methanoculleus sp., respectively [47].
Zahedi et al. [33] reported while working on municipal solid
waste that hydrolytic-acidogenic bacteria constitute 44e77%
of total population, and archaea, acetogens and sulphate-
reducing bacteria were insignificant in the first-stage, while
dominated in the second-stage. The relative population of
acidogens, family Clostridiaceae got reduced and acetogens
(Syntrophaceae, Syntrophomonadaceae and Desulfovibrionaceae)
and acetate-oxidizing bacteria (Spirochaetes) increased in bio-
hythane system as compared to single-stage AD system [48].
The archaea remained stable consisting of acetoclastic
methanogens of family Methanosaetaceae. The dominating H2
producers were Thermoanaerobacterium sp. and Clostrdium sp.,
while dominating methanogens were Methanosarcina mazei
and Methanothermobacter defluvii in another study [49]. The
microbial diversity and dynamics in the two-stage AD is still
limited.
Factors affecting process
Both the stages in two-stage AD are controlled by specific
microorganisms with different optimal process conditions.
Mesophilic
Temperature
Industrial/Municipal
Agricultural
Type of Biohydrogen
biowastes
Kitchen/Food
Aquatic
Biohydrogen
= 5.5
Fig. 3 e Process parameters affecting the biohythane production in two-stage AD.
Therefore, a sensitive balance between process parameters
such as substrate/feedstock type, temperature, pH, pretreat-
ment, reducing equivalents, etc. is required for biohythane
production. Some of the factors affect differently for their
individual production, while some factors affect collectively.
Thus, it is essential to control these factors accordingly to
increase the yield of biohythane. Some important process
factors are described in Fig. 3.
Type of biowastes
Previously, H2 production was mainly focused on simple and
pure substrates such as starch, glucose or sucrose as feed-
stock. However, use of pure sugar is very expensive. Instead of
using simple and pure substrates, naturally occurring com-
plex sources of carbohydrates and other biomolecules like
proteins and fats are proved to be more effective in bio-
hythane production due to cheap and availability in abun-
dance. The most suitable complex sources are biowastes as
they are largely generated and cause management problems
in today's life. Thus, biowastes are the most promising
feedstock for biohythane production from structural, envi-
ronmental and economical point of view. The availability of
these types of lignocellulosic biowastes to utilize them as
feedstock for biochemical conversion to bio-fuels depends
upon various factors such as geographical, climatic and
anthropogenic, agricultural and forestry, etc. [50].
Almost all types of biowastes can be used for biohythane
production. Table 1 shows biohythane production from
different biowastes as feedstock along with seed/inoculum for
acidogenic and methanogenic reactors, HRT and temperature
conditions. Composition of LCB significantly influences H2 as
well as CH4 production [62,63]. Most of the substrates have
complex composition as formed of organic molecules such as
carbohydrates, proteins and lipids. As compared to protein
and lipid, feedstocks with high carbohydrate content exhibit
Thermophilic
Feedstock
Pretreatment
Inoculum
Biohythane
Biomethane
Reducing
equivalents NAD+/NADH
pH
Biomethane
= 6.55
Table 1 e Biohythane (H2þCH4) production from different biowastes.
Feedstock Seed (Hydrogen) Seed (Methane) Temperature HRTa Hydrogen yield Methane yield Reference
( C) (L/Kg-VSb (L/Kg-VSb
Palm oil mill effluent (POME) Thermoanaerobacterium-rich Sludge from POME treating 55 H2-2 days; CH4-10 days 73 342 [15]
sludge from POME treating thermophilic methane reactor
thermophilic acidogenic

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 4 ( 2 0 1 9 ) 1 7 3 6 3 e1 7 3 8 0
reactor
Food waste Digested sludge from Digested sludge from H2-55 ± 2; H2-3.5 days; CH4-1.5 days 115.2 ± 5.3 334.7 ± 18.6 [51]
mesophilic ADc plant treating mesophilic ADc plant treating CH4-37 ± 2
buffalo manure, dairy buffalo manure, dairy
wastewater wastewater
Corn silage and cattail Granular sludge from apple Granular sludge from apple 37 60 days 59.4 ± 4.1 328.8 ± 16.8 [52]
juice waste water treatment juice waste water treatment
plant plant
Food waste Sludge of cassava waste water Sludge of cassava waste water 37 H2-12 h; CH4-24 h 292.7 391.6 [53]
treatment plant treatment plant
Wheat bran Slurry from first stage of a Slurry from second stage of a 37 ± 1 H2-4 days; CH4-40 days 18.9 243.5 [54]
biogas plant biogas plant
Wheat feed pellets Effluent of a sewage treatment Effluent of a sewage treatment 35 20 days 7 359 [36]
plant plant
Food waste Mixture of minced organic Mixture of minced organic 55 H2-3.3 days; CH4-12.6 days 66.7 720 [55]
waste and tap water waste and tap water
Carbon-rich macro-algae Anaerobic digestate from an Laboratory anaerobic digester 37 H2-4 days; CH4-26 days 97.0 224.3 [56]
(Laminaria digitata), Irish farm digester effluent
nitrogen-rich micro-algae
(Chlorella pyrenoidosa and
Nannochloropsis oceanica)
Sargassum sp. Caldicellulosiruptor Anaerobic granular sludge from H2-70; CH4-37 H2-10 days; CH4-42 days 91.3 ± 3.3 541 ± 10 [57]
saccharolyticus a brewery industry
Food waste Anaerobic digester sludge Anaerobic digester sludge 35 25 days 310 210 [58]
Ethanol stillage, cake Anaerobic digested manure Anaerobic digested manure 35 H2-3 days; CH4-12 days 48 344 [7]
and glycerol waste
Wheat straw Granule sludge Granule sludge þ digested >60 4 days 89 307 [38]
hydrolysate manure
Grass silage Cow manure sludge Cow manure sludge H2-35; CH4-55 H2-6 h; CH4-6 days 6.46 467 [59]
Sweet sorghum Indigenous microflora Anaerobic digester sludge 35 12 h 10.4 107 [60]
biomass
Biowaste from e Seed sludge of biowaste 55 32 days 51 780 [61]
treatment plant treatment plant
a
HRT-Hydraulic retention time.
b
VS-Volatile solid.
c
AD-Anaerobic Digestion.

17369
17370 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 4 ( 2 0 1 9 ) 1 7 3 6 3 e1 7 3 8 0
higher H2 potential, while lipids exhibit higher CH4 potential
[64,65]. Thus, starch and fat are preferred substrates for bio-
hythane production followed by proteins. However, it has also
been reported that higher concentration of starch leads to less
production of H2, while lower concentration leads to more H2
production due to incomplete hydrolysis of starch and for-
mation of more VFAs at higher concentration of starch [66,67].
It is clear from Table 1 that food waste (FW) yielded good
amount of H2 and CH4 [53,55]. Kitchen/food waste is consid-
ered as the promising feedstock for H2 production due to
higher amount of carbohydrate content [10]. It may also be
somehow linked to dominance of H2 producers over H2 con-
sumers, however, the exact reason of having vast potential of
H2 production in kitchen/food waste is still unknown.
Enhancement in H2 yield was found in a study when FW was
added in pretreatment before fermentation which might help
in eliminating H2 consumers [68]. H2 production from FW
without inoculum or additional processes was studied, and it
was found that H2 was produced at high yield (1.79 mol/mol
glucose added) when fermented at 50e60  C [69]. The lactate is
a major VFA observed in the kitchen waste digestion. The
lactate degradation is somewhere believed to be connected to
H2 production but the process is unknown till now. However,
lower hydrogen production rate was observed in kitchen
waste in comparison to corn starch due to lack of degradable
carbohydrate, and inhibitors like ammonia, oil (fat) and grease
[70]. Moreover, it is believed that cooked material has more
starch like easily degradable carbohydrates. Several studies
have been conducted from municipal, industrial and agricul-
tural waste due to presence of large amount of complex
organic composition [71e73]. A study of biohythane produc-
tion from co-digestion of palm oil mill effluent (POME) with
decanter cake (DC) and empty fruit bunches (EFB) was con-
ducted and the H2 yield was observed almost similar as
16.26 L/Kg-VS and 16.52 L/Kg-VS in co-digestion of POME with
DC and EFB, respectively, whereas, the maximum CH4 yield of
391.62 L/Kg-VS was reported from co-digestion of POME with
DC [74].
Pulp and paper sludge (PPS) contains lignin, carbohydrates
and 2e8% cellulose and thus, used rarely for biohythane
production due to more lignin content (20e58%) than carbo-
hydrates (0e23%), proteins (22e52%) and lipids (2e10%).
However, anaerobic co-digestion of PPS and FW can effec-
tively produce higher H2/CH4 yields due to complementary pH
and C/N ratio. Aquatic and marine weeds have attracted
worldwide attention due to fast growth rate, causing serious
problems in navigation and irrigation. However, they appear
to be a valuable resource for energy production through AD.
Water hyacinth (Eichhornia crassipes) is fast growing, perennial
aquatic weed with broad, thick, glossy leaves with long,
spongy and bulbous stalks, non-toxic in nature and available
in many parts of the world. It has great potential for biogas
production and many studies have been reported for biogas
production [75,76]. Chuang et al. [77] reported H2 production
and evaluated H2 and CH4 production parameters from water
hyacinth using pig slurry as inoculum for H2 production. H2
and CH4 yields were reported as 31.3 GJ/ha/y and 853.9 GJ/ha/y,
respectively. Algae are the third-generation biofuel and have
potential in AD. Recently, algae are grown (Anabaena, Chlorella,
Scendesmus) to produce biogas under autotrophic conditions.
Aquatic algae have also been used for the same purpose.
Higher productivity potential of algae is due to low lignin
content, thus reduces the extensive energy requirements for
degradation [78,79].
Temperature
Temperature has a very profound effect on the progress of
two-stage AD for H2 as well as CH4 in separate reactors for
biohythane production [80]. Temperature influences the
nutritional requirements, metabolic end product formation,
and characteristics of microbial cells in the conversion pro-
cesses. Biochemical conversion pathways are greatly influ-
enced by temperature, because microorganisms as well as
enzymes have an optimum temperature (threshold) range at
which they shows maximum productivity. Any deviation
from this optimum range results in denaturation/inactivation
of the enzymes and cells thus, leads to process inhibition.
Generally, a 1  C rise in temperature is associated with double
increase in enzymatic activity till the approach of optimum
range [26]. Methanogens are generally considered as more
sensitive to temperature fluctuations than others microor-
ganisms in the process, and can be suppressed by even as
small as 2e3  C variation in temperature which could lead to
accumulation of VFAs in the reactor, which eventually causes
pH drop [81].
Growth of methanogens with temperature could be
observed from rate of organic matter or volatile solid (VS)
degradation, COD removal and CH4 production in methano-
genesis stage. For instance, Trisakti et al. [82] studied the ef-
fect of temperature on methanogenesis in two-stage AD, they
observed considerable increase in rate of VS degradation and
COD reduction with rise in temperature. VS decomposition,
COD removal and biogas yield were reported to be 67.44%,
79.16% and 37.03 L/kg-VS, respectively under thermophilic
conditions whereas, mesophilic conditions resulted in VS
decomposition, COD removal and biogas yield as 79.82%,
51.56% and 31.77 L/kg-VS. Depending upon the microorgan-
isms to be used, the process is conducted at respective tem-
perature range. Generally, H2 producers as well as
methanogens have different temperature ranges; viz. psy-
crophilic (0e20  C), mesophilic (20e45  C) and thermophilic
(42e75  C) 2015 [22,26]. The rate of hydrolysis of cellulose in
thermophilic conditions is higher than in mesophilic condi-
tion, and requires smaller volume of reactor [83]. Optimum
temperature ranges for mesophilic and thermophilic metha-
nogens are 30e35  C and 50e65  C, respectively [84]. A variety
of studies have been reported at mesophilic as well as ther-
mophilic conditions.
However, thermophilic conditions are considered as more
efficient than mesophilic conditions in terms of improved
kinetics, reduced risk of contamination, and short HRT [85].
Thermodynamically, extreme thermophilic bacteria could
follow an efficient acetic acid pathway to obtain maximum
theoretical H2 yield of 4 mol/mol of glucose [86]. The extreme
thermophiles for instance, Thermotoga maritima, C. saccha-
rolyticus, and Caldicellulosiruptor bescii have high potential of
hydrolysis, and to utilize reducing equivalents (NADþ/NADH)
(discussed later in Section Reducing equivalent content) dur-
ing glycolysis in first-stage of process [87]. C. saccharolyticus
 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 4 ( 2 0 1 9 ) 1 7 3 6 3 e1 7 3 8 0
and T. maritime are found to release thermostable cellulases
and xylanases to breakdown complex LCB, thus could be used
for co-digestion of C5 and C6 sugars [88,89]. Moreover, C. bescii
could degrade LCB without need of any pretreatment [90].
Proportion of H2 in biohythane via two-stage AD could be
enhanced by using co-culture of extreme thermophiles [91].
Abreu et al. [92] evaluated the effect of co-cultures of C. sac-
charolyticus and C. bescii from garden waste, and reported
enhanced H2 yield of 98.3 ± 6.9 L/kg-VS along with CH4 yield of
322 L/kg of total COD. Resulting biohythane was enriched with
15% H2 with 22.2 MJ/kg-VS of energy. In addition, activity of
main enzyme responsible for H2 production i.e. hydrogenase
is strongly influenced by temperature, whose optimal value
for maximum activity was observed to be 55  C [93]. Further-
more, thermophilic conditions are significantly beneficial for
two-stage AD because the undesirable H2 consumers are
sensitive to high temperature, and get deactivated at high
temperature whereas, most of the H2 producers are spore
forming in stress conditions like at high temperatures thus,
these desirable H2 producing microorganisms can survive at
thermophilic conditions [94]. But this is not always the case, so
care of microbial community should be taken during while
choosing the temperature conditions of the process Kim et al.
[95] and Lin et al. [96] were the first to investigate the ther-
mophilic digesters in both the stages of two-stage AD. Ventura
et al. [97] investigated two-stage AD of FW by controlling the
temperature profiles of acidogenic and methanogenic reactors
by adapting three systems that were mesophilic-mesophilic;
mesophilic-thermophilic, and thermophilic-mesophilic.
They reported the maximum COD removal of 86.6% with
methane yield, and production rate of 440 L/Kg-VS, and 1.23 L/
L-day, respectively in mesophilic-thermophilic reactor. They
also investigated the effect of the temperature alternation to
two-stage AD on the microbial structure of the system using
16s ribosomal DNA gene amplification and terminal-
restriction fragment length polymorphism (TRFLP) analysis.
The distinct microbial community of the acidogenic and
methanogenic reactors was observed. System consisting of
mesophilic-thermophilic was considered as the best for the
operation of two-stage AD in this study. In an another study of
using thermophilic acidogenic and mesophilic methanogenic
digester conditions from POME using pretreated and un-
treated anaerobic sludge from a POME biogas plant as inoc-
ulum, the highest H2 and CH4 yields were reported as 200 L/Kg-
COD and 292 L/Kg-COD, respectively, with 51% CH4, 14% H2
and 35% CO2 contents and total energy yield of 15.34 MJ kg/
COD [47]. In this study, the hydrogen yield and rate of growth
of H2 producing bacteria were observed to be increased with
thermophilic temperature conditions. Adapting thermophilic
acidogenic and mesophilic methanogenic digester system
was a promising approach for the combined energy recovery
and removal of COD. In another study on evaluating bio-
hydrogen potential (BHP) and biomethane potential (BMP) of
skim latex serum (SLS) using mixed inoculum (AD
sludge þ Thermoanaerobacterium 112YL pure culture) and
sludge from two tanks of POME based AD system as thermo-
philic inoculum for H2 and CH4 production, respectively, the
reported H2 and CH4 yields were found to be 41.3 L/Kg-VS and
321.05 L/Kg-VS, respectively [49]. Thermophilic conditions and
inoculum used in this study effectively removed organic
17371
content in SLS in terms of economic feasibility. Thermophilic
conditions are also advantageous as they increase the lipid
degradation due to the enhanced solubility of lipid at higher
temperatures [98]. The thermophilic conditions also increase
the pH of the acidogenic reactor, which affect the microbial
activity in the process [97].
pH
In a biochemical conversion process, pH is an important and
sensitive parameter, which affects the microbial growth and
determines stability of the process via controlling the effi-
ciency of enzymatic machinery [26]. Unlike temperature, pH
in two-stage AD required to be different in both the reactors
for the respective microbial growth, and their associated en-
zymes. The continuous accumulation of VFAs during H2 pro-
duction in first reactor disrupts the microbial membrane, and
in due course leads to interruption of pH balance, finally stops
H2 production at pH 3.8e4.2 [99]. On the other hand, pH is
maintained at 7.29e7.55 in methanogenic reactor due to the
presence of acetate and bicarbonate ions in effluent owing to
action of acetogens and archaea [100]. Further, low pH leads to
shift from acidogenesis to solventogenesis (formation of sol-
vents other than desired product and H2). Low pH is toxic for
methanogens, but beneficial for H2 producers as optimum pH
value for hydrogenase activity is 5.5, and also to keep
methanogens away, first reactor needs to be maintained at
this pH or at least in the range of 5e6, whereas optimal pH
range for methanogenesis i.e. for second reactor is 6.5e7.5
[94]. Generally, chemicals are used as buffering agent to con-
trol the pH during the process. However, to avoid use of
expensive chemicals, and to make the process reasonable
from scaling up point of view, nowadays, an advanced recir-
culation system is adapted for pH maintenance in two-stage
AD in which the digested sludge or slurry from second
digester (methanogenic) is recirculated to the first digester
(acidogenic). The digested effluent acts as buffering agent
(including residual ammonium, and bicarbonate) for first
reactor, and helps to balance the pH through intake of nutri-
ents and feedstock intensity [61,101].
However, excessive recirculation may be toxic for the
process due to accumulation of ammonia reducing the H2 and
CH4 yields [55,102]. Keeping this issue in view, Micolucci et al.
[103] developed a semi-automatic control system based on
ammonia concentration. They used variable recirculation
flow to control the process, which prevented ammonia inhi-
bition in first reactor and VFA accumulation in second reactor.
Recirculation flow can be regulated by keeping VFAs/alkalinity
ratio below 0.3, to prevent the process inhibition [104]. Got-
tardo et al. [105] by adapting recirculation system achieved a
significant biohythane production with H2 and CH4 yields of
170 L/kg-VS and 750 L/kg-VS, respectively, with composition
of 40% H2 and 67% CH4. Thus, recirculation is an efficient and
cost effective method for pH maintenance in two-stage AD
without requirement of any external supply of chemicals.
Pretreatment
Pretreatment is a significant step in AD for getting higher
product yields. Main substrate of digestion i.e. cellulose is
17372 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 4 ( 2 0 1 9 ) 1 7 3 6 3 e1 7 3 8 0
packed within hemicelluloses and lignin. Lignin removal and
unpacking of hemicelluloses is essential to make cellulose
accessible for digestion. In biohythane studies, both feedstock
and inoculum used for acidogenic reactor are usually
pretreated.
Feedstock
It is essential to remove complex and rigid structure of lignin
and hemicelluloses for easy digestion of cellulose. There are
different methods of pretreatment including chemical, phys-
ical, physico-chemical and biological [106]. The pretreatment
of biomass efficiently enhances the production of biofuels
[107]. In most of the studies, LCB is pretreated by physico-
chemical means using acid, alkali, stream explosion, micro-
wave, ionizing radiation, hot water, or combination [108].
Alkaline pretreatment removes the lignin by breaking the
linkage between lignin and hemicelluloses, thus, causes
unpacking of cellulose for hydrolytic enzymes and makes the
organic matter more accessible [109].
About 2% (v/v) sulphuric acid (H2SO4) at 121  C is re-
ported to be the best conditions for the hydrolysis of sug-
arcane bagasse (SCB) for H2 production, whereas, the
production ceases with increasing H2SO4 concentrations to
more than 2% due to continuous rise of acetic acid and
furfural resulting in the process inhibition [110]. Some re-
searchers used Pt/TiO2 aerogel for photo degradation
[111,112]. In a study, 0.8% reduction in lignin was investi-
gated using 0.5% (w/v) TiO2 in 6.4% (w/v) H2O2 and 23% (w/v)
acetic acid at 130  C for 2e3 h [113]. Jafari and Zilouei [30]
performed combined pretreatment of SCB with nano-TiO2,
H2SO4 and ultraviolet (UV) irradiation to use it as feedstock
for biohythane production, and achieved H2 and biogas
yields of 101.5 L/kg-VS and 591 L/kg-VS, respectively, under
mesophilic conditions. They observed that nano-TiO2 pre-
treatment along with H2SO4 and UV irradiation improved H2
and CH4 production up to 127% and 74%, respectively, as
compared to the raw bagasse.
Physico-chemical pretreatment provides efficient struc-
tural changes in LCB and increases the rate of cellulose hy-
drolysis for enhanced biohythane production, however,
requirement of special instrumentation, higher energy, and
associated inhibitors make it less suitable from both
economical as well as environmental point of view [114].
Biological pretreatment using microorganisms or their me-
tabolites (usually enzymes), is a promising and eco-friendly
approach for lignocellulosic biofuels production due to non-
requirement of chemicals and without release of toxic prod-
ucts [115]. For instance, white-rot fungi (WRF) are most
effective naturally found lignin-degrading microorganisms
having a vast system of efficient ligninolytic enzymes; lac-
cases, manganese peroxidases (MnP), lignin peroxidases (LiP),
and accessory enzymes [116,117]. WRF are widely utilized for
biomass pretreatment for fermentation studies under natural
physiological conditions.
Kumari and Das [114] studied pretreatment of sugarcane
top with WRF Pleurotus pulmonarium MTCC 1805 with 30% solid
loading at 28  C for 21 days for biohythane production, and
reported 60.4% (w/w) lignin removal with 87.40 L/kg-VS and
180.86 L/kg-VS of H2 and CH4 yields, respectively along with
37.7% of overall gaseous energy recovery. Bioaugmentation is
a technique of enrichment of specific microorganisms, which
is used in AD for the improvement of lignocelluloses di-
gestibility, biogas production and recovery from ammonia and
VFA formation. However, in a study of bioaugmentation of
corn silage and cattail using the anaerobic fungus, Piromyces
rhizinflata YM600, there was no improved CH4 yield of either
corn silage or cattail, but improved H2 and CH4 production
rates were observed. VFA degradation for both the corn silage
and cattail was improved, which is useful to avoid VFA
accumulation [52].
However, biological pretreatment is the slower and less
efficient method than thermo-chemical methods. Hydro-
thermal pretreatment is significantly efficient method of
biomass pretreatment which involves heating the biomass at
higher temperatures keeping in water as medium. In a recent
study, Jehlee et al. [19] applied hydrothermal pretreatment to
Chlorella sp. with 16% solid loading at 95  C for 24 h, and re-
ported H2 and CH4 yields of 190.0 L/kg-VS and 319.8 L/kg-VS,
respectively with 47% biodegradation efficiency as compared
to H2 and CH4 yields from untreated biomass as 124.9 L/kg-VS
and 230.1 L/kg-VS, respectively with biodegradation efficiency
of 34%. Hydrothermal pretreatment was proved to have great
potential for H2 and CH4 production in this study of two-stage
AD.
Thermal pretreatment was also reported to be significant
by Bong et al. [118], it was demonstrated that heating the
feedstock at high temperature (>120  C) for about 30 min is
efficient for cellulose solubilisation. Ultrasound irradiation
is another type of pretreatment, which involves passing of
ultrasonic waves into the biomass kept in aqueous solution
or slurry [119,120]. Passage of ultrasonic waves creates the
expansion and compression regions through pressure dif-
ference in the biomass creating large surface area resulting
into efficient lignin depolymerization during pretreatment
[121]. Martinez-Jimenez et al. [122] recorded 60e80% in-
crease in CH4 content in biohythane by sonication of sug-
arcane straw and vinasse in two-stage AD using 180 W of
ultrasonic power at 37 kHz for 30 min. In the study, soni-
cation changed the composition of biomass to variable
extent leading to destruction of lignin, thus, increased bio-
hythane production.
Inoculum
In biohythane production studies, treatment of inoculum is an
important step to be used in acidogenic reactor (Stage I) due to
the presence of both H2 producers and consumers. H2 con-
sumers are highly sensitive to extreme pH, temperature and
feedstock concentration, whereas, many of the H2 producers
such as Clostridium and Bacillus species form endospores under
unfavorable environmental conditions like high temperature,
desiccation and chemical toxicity, which regerminate on re-
turn of favorable conditions [16]. Thus, H2 consumers could be
removed from the reaction mixture by pretreating the inoc-
ulum to enhance the H2 yield. Wet heat treatment by boiling
for 15 min of AD sludge, and dry heat treatment (baking at
104  C for 2 h) of compost and soil have been used in some
studies [123,124]. Variety of inoculum pretreatments have
been applied in the reported studies for instance, chemical
(acidic or alkaline), load-shock treatment (LST) and heat-
shock treatment (HST) [26]. Load-shock treatment involves
 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 4 ( 2 0 1 9 ) 1 7 3 6 3 e1 7 3 8 0
the frequently changing higher loadings of nutrients with
inoculum at slightly acidic pH (5.0e6.0), whereas, HST is
heating the inoculum at higher temperatures, and then cool-
ing down at ambient temperatures to deactivate the heat
sensitive H2 consumers [26,49].
It is reported in studies that LST of inoculum gives more
efficient results in terms of H2 yield than that of HST due to
undesirable destruction of the non-spore forming H2 pro-
ducing [94]. The similar fact was observed in a study, where
efficient pretreatment method was investigated on meso-
philic and thermophilic inoculums out of LST and HST using
high glucose loading of 50 g/L and heating in an autoclave at
100  C for 30 min, respectively for H2 production. It was
observed that LST pretreated thermophilic inoculum gave
the highest H2 yield of 205.5 L/kg-VS in 3 days, and HST
resulted in poor yield which might be due to destruction of
non-spore forming H2 producers [49]. Thermophilic inoc-
ulum was also observed to be more acclimatized to growth
than mesophilic inoculum in this study. Although, heat
treatment at 100  C generally inactivates the H2 consumers,
but it was observed in a study that there was not much dif-
ference in H2 yields using the untreated sludge and sludge
treated at 100  C. However, it was higher than the sludge
treated at 50 and 80  C [125]. This little influence of heat
treatment on H2 production might be due to complexity of
corn stalk that was generated due to wet steam-explosion,
which, caused degradation of produced inhibitors in the
process, and eventually favour H2 production [126]. To avoid
persistence of H2 consumers in case of effluent recirculation
from methanogenic reactor to acidogenic reactor, it was
suggested that larger fraction of the return sludge may be
heated, and less frequent heating of sludge could be prac-
ticed to avoid the prevention of germination of all the H2
producer's spores since it takes longer time of 60e90 min to
germinate the spores [127].
Reducing equivalent content
During the process of H2 production, reducing equivalents
(NADþ/NADH) formed are disposed of through proton (Hþ)
reduction and electron carriers, which led to H2formation
[128]. Thus, yield of H2 is directly proportional to content of
reducing equivalents. However, these reducing equivalents
are utilized in the formation of metabolites such as alcohols
(ethanol, butanediol, etc.). Therefore, to enhance the H2 yields,
reducing equivalents need to be recovered or increased by
blocking the metabolite formation pathway. Two studies have
been reported on improvement of H2 yield by increasing the
reducing equivalents through mutagenesis technique in
thermophilic H2 producers by blocking the metabolites (alco-
hols) production [129,130]. Ito et al. [129] reported 15%
improved H2 yield by developing alcohol production pathway
defective mutant Enterobacter aerogens by exposing to N-
methyl-N-nitro-N-nitrosoguanidine (NTG) as mutagen. Roy
et al. [130] developed alcohol production pathway defective
mutant Thermoanaerobacterium thermosaccharolyticum IIT BT-
ST1 by exposing to ethyl methane sufonate and reported
13.5% improved H2 yield. Both these studies succeeded in
decreasing alcohol (ethanol) production, increasing reducing
equivalents and thus, improved overall H2 yields.
17373
Benefits of biohythane and its applications
Biohythane production is more advantageous than individual
production of H2 and CH4 for instance, it would be cost
effective to produce two clean and valuable biofuels in a single
process [92]. Unlike one-stage AD, biohythane production via
two-stage AD offers reduction of process time due to lower
HRT of DF (acidogenic reactor), and working volume of the
reactors [48]. Its production has environmental benefits in
terms of reduced CO2 content, and NOx emissions. To utilize it
as bio-CNG, H2 addition aids in improving the performance of
engines by reduction in the CH4 number, and better burning
due to its lower ignition energy (0.02 MJ) than that of CH4
(0.29 MJ) [10]. Due to more stable combustion, biohythane
could expand the narrow burning limit [131]. There is
increased net energy by recovering 100% hydrogen and 30%
methane and reduces process time [132]. Thus, biohythane
improves energy recovery from organic wastes to make the
process commercially feasible. There is about 35e93% COD
reduction while treating wastes and CO2 emission reduction
from 15.2 to 23.7 tons and thus, it has more potential of waste
treatment and eco-friendly [37,133]. A net energy yield of
7400e7700 kJ/kg-TS and 13,363 kJ/kg-COD has been reported
by Willquis et al. [37] and Khongkliang et al. [66]. It is reported
that addition of 10% of H2 in biogas enhanced combustion
with a consequent improvement in thermal efficiency and
power output [134]. High diffusivity and the low density of H2
allow biohythane jet to extend more efficiently in the com-
bustion. The combustion decreases the entrainment of the
ambient fluid and raises the viscosity of the flow, leading to an
increase of the longitudinal velocity along the biohythane jet,
and a reduction of the turbulence in comparison with non-
reacting configuration [135]. Biohythane is a promising alter-
native fuel to fossil fuels and as vehicle fuel, exhibits advan-
tages over CNG in terms of extended flammability range,
reduced GHG emissions, and shortened quenching distance
[16]. Its use as a vehicle fuel for operation of a four-stroke
spark ignition gas engine has also been reported [136]. Addi-
tion of 5e20% of H2 to biogas increases engine efficiency as
compared to CH4 combustion and reduces NOX emission [55].
In addition, biohythane could be utilized as better fuel for
methanol production than pure CH4 or biogas alone due to
addition of H2 [137,138].
Key challenges and solutions
Despite of wide advantages and applications of combined
production of H2 and CH4 as biohythane in one process, there
are some problems associated with the process of two-stage
AD due to which desirable yield is not achieved. Some of the
major challenges with possible solutions are highlighted
further in the section.
Challenges of metabolic pathway
As discussed in Section Anaerobic digestion and Section
Reducing equivalent content metabolic pathways of H2 pro-
duction are associated with VFAs and some alcohols such as
17374 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 4 ( 2 0 1 9 ) 1 7 3 6 3 e1 7 3 8 0
ethanol and butanediol, etc. It is observed that the predomi-
nant metabolite of glucose metabolism in the acidogenic
reactor is lactic acid. Low yield and production rate of H2 are
associated with dominance of lactic acid bacteria as compared
to Clostridia dominated cultures as lactate consumes electrons
in the process thus, interferes with the neutralization process
conducted by hydrogenase [139]. Thus, blocking the lactate
production pathway would theoretically improve H2 yield.
Metabolic engineering and genetic modification strategies are
required to develop lactate production pathway deficient
strains to improve H2 production. Further, the effect of VFAs
on methanogenesis in two-stage AD needs to be evaluated for
possible solutions of VFA related problems. Recently, butyric
acid and propionic acids at 8 g/L shown to have inhibitory
effect, thus, preventing the high concentration of butyric and
propionic acids in the effluent could enhance methanogenesis
in two-stage AD [140].
Technical challenges for scaling up
Energy evaluation
Biohythane is known to have higher theoretical energy re-
covery on the basis of chemical energy of the feedstock.
However, in comparison to the one-stage AD, two-stage AD
requires two reactors which consume additional energy for
heating up two reactors, energy losses taking place through
shifting of effluents between stages, and release of products
[47,141]. Energy balance on large-scale two-stage AD needs to
be fully examined.
Reactor integration
Although biohythane is a promising renewable alternative in
automobile sector, however, most of the reported studies are
still at the lab scale. Implementation of biohythane produc-
tion on large scale, and desirable yields of both H2 as well as
CH4 could only be achieved through efficient integration of
reactors in two-stage process. Working of the methanogenic
reactor completely depends upon the working of acidogenic
reactor or DF stage as it provides the substrate for metha-
nogens; VFAs including other effluents. Growth rate of
hydrogenogens in acidogenic reactor is higher than metha-
nogens because the change in Gibbs free energy of H2 pro-
duction is much larger than that of methanogenesis [94]. HRT
of DF in first reactor is lower than the methanogenesis in
second reactor, and it is relatively more prone to higher bac-
terial growth rates [142,143]. On the other hand, second
reactor performance is often poor in terms of instability and
reduced CH4 yields due to slow growth rate and washout of
methanogens [144,145]. Generally, continuous DF processes
are carried out in continuous stirred tank reactor (CSTR) sys-
tem due to simple design, and selection of desirable H2 pro-
ducers by washing out methanogens [18,146]. For one-stage
AD process for sole CH4 production, anaerobic fixed bed
reactor (AFBR) is suitable option for maintaining the high
concentration of slow growing archaea by holding the active
cells on inert packing support along with high loading
competence, and higher treatment efficiency [147,148]. Thus,
integration of CSTR and AFBR could give the best results for
biohythane production via two-stage AD. However, there is
lack of stability for continuous performance of CSTR for longer
times [55]. Yeshanew et al. [51] evaluated a successful inte-
gration of both stages using CSTR and AFBR for acidification
and methanation, respectively with higher biohythane pro-
duction. The AFBR showed a stable, excellent performance,
and effective effluent recirculation to the CSTR leading to the
balanced pH in reactor for efficient H2 production. Other
technical challenges also need to be considered before com-
mercial scale applications such as the stability of the reactors,
reactor configuration, and energy efficiency.
Operational elucidation of acidogenic stage
As discussed in previous section, the performance of acido-
genic stage is critical for the overall performance of two-stage
AD therefore, it is important to focus on the problems asso-
ciated with progress of this stage such as VFA accumulation,
other metabolites, persistence of methanogens in mixed
consortia particularly in case of recirculation of effluent from
methanogenic reactor to acidogenic, etc. for efficient working
of the acidogenic reactor thus, overall process. Increasing the
organic loading rate (OLR) is considered to affect the perfor-
mance of acidogenic reactor positively [149]. However, higher
loading could lead to load-shocks due to excessive VFA
accumulation which disturbs the pH of the system, thus, in-
hibits the process. An advanced approach of pre-aeration
strategy could be adapted to increase the hydrolysis rate of
complex substrate molecules in reactor by hydrolases
secreted by aerobic microorganisms, and results in more en-
ergy recovery [150]. Aeration not only assists in hydrolysis rate
of process but also suppresses the methanogens in acidogenic
reactor. In fact, pre-aeration of feedstock is reported to affect
positively on the VFA production in relation to efficient H2
production [151]. Continuous air supply reduces the VFA
accumulation, which prevents the frequent pH drop during
the process, and improves process efficiency [152]. Pre-
aeration is also a cost effective and simple approach
compared to other pretreatment techniques to regulate the DF
as well as overall AD process.
Economic assessment
Economic evaluation of two-stage AD is one of the most
important step that provides useful data including profit-
ability and feasibility for commercialization of the process.
The various steps of economic evaluation includes total cap-
ital investment (TCI), annual production cost (APC) and
annual revenue (Fig. 4). First of all, TCI is estimated, which
consists of fixed capital cost (FCC), and working capital cost
(WCC). Apart from the capital cost, other hazardous factors
like pollution, irradiations, etc., should also be considered.
APC is calculated after selection of the equipment with
optimal operating conditions. Annual revenue of biohythane
production plant deals with feedstock and its treatment, and
the products of process that are H2 and CH4. After the esti-
mation of TCI, APC, and annual revenue, the profitability
performance indicators such as the net present value (NPV),
internal rate of return (IRR), and payback period (PBP) are
further determined. NPV is the cash flow along duration of the
concerned project, the project is significant for more than zero
value. IRR is an interest rate for zero value of NPV, and it is
 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 4 ( 2 0 1 9 ) 1 7 3 6 3 e1 7 3 8 0
Fig. 4 e Economic assessment of biohythane production
(TCI: Total Capital Investment; APC: Annual Production
Cost; FCC: Fixed Capital Cost; WCC: Working Capital Cost;
NPV: Net Present Value; IRR: Internal Rate of Return; PBP:
Payback Period).
desirable to have higher IRR of the projects. PBP is the time
period to return the money invested in the start of the project
[153].
For economic analysis of biohythane production, various
factors like type of process, feedstock, scale of the process (lab
scale/pilot scale/large scale or batch/continuous), play
important role. Cost of H2 production is directly related to type
of process, and feedstock. DF due to low energy requirements
(ambient temperature and pressure) provides promising pro-
cess for clean H2 fuel than other conventional chemical pro-
cesses [154]. But due to lower yields, it is beneficial to produce
biohythane in two-stage AD process rather than by individual
DF to reduce the cost, and to get the valuable fuels with higher
yields. As far as feedstock is concerned, food waste is prom-
ising substrate for H2 production as it contains rich amount of
carbohydrates, which are direct substrates for hydrogen pro-
ducers. Thus, substrates with higher carbohydrate content are
cost effective, and more productive. Further, it is reported that
hydrogen yield is directly proportional to the operating costs
while rate is directly proportional to the reactor costs or the
installation costs. Lee and Chung [155] demonstrated eco-
nomic feasibility of biohythane production from FW and corn
flour in two-stage AD and they found FW was more econom-
ical than corn flour as production cost of $ 0.19/m3 using FW
was quite low. In another study, economic feasibility of H2
production from FW was evaluated, which was estimated on
the basis of overall costs such as cost of installation, oper-
ating, labor, equipment, etc. About 42,858 m3 of H2 was found
to be produced in a year from 1095 tons of FW, which costs
$115,717 [156]. Chu et al. [157] studied biohythane production
from FW and reported 1,862,258 kcal/ton energy generated,
which was 7.48% from H2, and 92.52% from CH4. Therefore, the
17375
two-stage AD is the perfect combination that can maximize
the energy recovery from FW and minimizes the production
cost of process. The production scale is another factor that
directly influences the expense and revenue. H2 production
cost from molasses was estimated in a study, it was observed
that the 40 m3 plant would gave positive results with 0.63% IRR
and PBP of 9.7 years however, it was suggested in this study
that H2 production would be more economically feasible if
operated at larger scale [158]. In another comparative study,
batch and larger reactors with 0.3 and 3 L of working volumes
gave the H2 yield of 1.71 mol/mol glucose and 1.57 mol/mol
glucose, respectively, thus, batch scale shown somewhat
higher yield than that of larger scale but difference was not
enough to drawing up any conclusion [159]. Thus, scaling
studies also play important role before economic estimations
for scale up of biohythane production.
Storage costs of fuels are also an important part in eco-
nomic evaluation. H2 can be stored in three ways: compressed
gas in high-pressure tanks; liquid in tanks at 253  C; solid by
either absorbing or reacting with metals or chemical com-
pounds or storing in an alternative chemical form. Com-
pressed gas requires high pressures (5000e10,000 psi), liquid
H2 requires to be stored at 253  C, and materials-based
storage in solids like metal hydrides, high surface area sor-
bents, chemical hydrogen storage, etc. H2 as a cryogenic liquid
requires higher energy than compression of gas to 3600e5000
psi. Thus, storage in compressed form is economically more
feasible than liquid form particularly for transportation pur-
poses. Storage of CH4 is costly factor, thus if possible, part of
produced CH4 should be used directly, without any storage.
However, wherever it is not possible to use it directly, storage
should be practiced so that it may greatly facilitate the reli-
able, efficient, cheap and clean integrated energy systems.
CH4 storage requires pipeline gas, electricity and heat, which
may be achieved by applying gas grid, compressed tank, liq-
uefied storage, bottling, adsorbed storage, physical and
chemical conversion, and/or CH4 generation infrastructure.
Transportation of pressurized CH4 in gas grids requires lesser
energy than by high voltage cables, thus gas grid storage is
more economically favorable and efficient method especially
in countries with higher production of CH4. Moreover, grid
injection is more feasible at larger scales as capital costs are
less prominent at higher scale. Another way is compressed
storage called as compressed biomethane (CBM), which is
defined as the compressed CH4 stored in large pressurized gas
containers (cylindrical, pipe tanks or spherical), filled by pro-
ducers directly near the production site for easy trans-
portation [160]. The cost of the compressor for compressing
the gas is the main cost in a CBM. However, CBM transport is
suitable for short distances. Further, lower pressures require
less efforts for compression, energy consumption, lower
costs, cheap and easy to use, thus are economically more
feasible than at storing at higher pressures.
In countries with limited gas grid development, CH4 is
stored in the compressed form in pressurized containers.
Furthermore, CH4 bottling is suitable for distribution purposes
to few consumers. Bottled CH4 is more economical for use as a
vehicle fuel as it requires simple dispensing/filling stations.
Moreover, bottled CH4 can be economical for cooking in re-
gions with limited feedstocks for cooking purposes [161].
17376 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 4 ( 2 0 1 9 ) 1 7 3 6 3 e1 7 3 8 0
Thus, by considering the production process, scale of process,
feedstocks and storage of both fuels in one process i.e. two-
stage AD, costs of fuels via separate processes that are DF
and one-stage AD could be minimized, and infrastructure can
be more optimally used, eventually minimizing capital costs
required for storage implementation.
Conclusion
Two-stage anaerobic digestion (AD) could be a viable process
for the combined production of two valuable biofuels; H2 and
CH4, collectively called as biohythane. Biohythane could be
proved as better alternative of CNG due to addition of H2
which improves the low inflammability range of CH4, reduces
the burning time of engine without NOx emissions into at-
mosphere. Thus, biohythane is economical as well as eco-
friendly energy carrier. Currently, only <1% full scale plants
are based on two-stage AD, thus, for practical implementa-
tions of biohythane as transportation fuel on commercial
scale, aspects of two-stage AD are required to be evaluated.
Although an efficient process than one-stage AD, two-stage
AD process is associated with many challenges, which needs
to be discussed and resolved for successful implementation of
biohythane. The key challenges are related to metabolic
pathway and technical problems of scaling up of process.
Technical challenges are further related to energy elucidation,
reactor integration and performance of acidogenic reactor.
Further, economic assessment of two-stage AD is also
necessary before establishment of full scale plants.
Acknowledgement
One of the authors (Meenu Hans) is very thankful to Sardar
Swaran Singh National Institute of Bio-Energy, Kapurthala for
providing Research Fellowship and Guru Nanak Dev Univer-
sity, Amritsar for providing Ph.D. registration (Reg. No.
2007.KJ/A.519). Authors gratefully acknowledge the Ministry
of New and Renewable Energy, India for providing financial
supports to carry out the research activities.
references
[1] Kumar S, Dheeran P, Singh SP, Mishra IM, Adhikari DK.
Bioprocessing of bagasse hydrolysate for ethanol and xylitol
production using thermotolerant yeast. Bioproc Biosyst Eng
2015;38:39e47.
[2] Roy S, Das D. Biohythane production from organic wastes:
present state of art. Environ Sci Pollut Res 2016;23:9361e410.
[3] Gaffney JS, Marley NA. The impacts of combustion
emissions on air quality and climate-from coal to biofuels
and beyond. Atmos Environ 2009;43:23e36.
[4] Patil VS, Deshmukh HV. Anaerobic digestion of vegetable
waste for biogas generation: a Review. Int Res J Environ Sci
2015;4(6):80e3.
[5] Bauer CG, Forest TW. Effect of hydrogen addition on the
performance of methane fueled vehicles. Part I: effect on
S.I. engine performance. Int J Hydrogen Energy
2001;26:55e70.
[6] Liu X, Wang W, Shi Y, Zheng L, Gao X, Qiao W, et al. Pilot-
scale anaerobic co-digestion of municipal biomass waste
and waste activated sludge in China: effect of organic
loading rate. Waste Manag 2012;32:2056e60.
[7] Luo G, Xie L, Zhou Q, Angelidaki I. Enhancement of
bioenergy production from organic wastes by two-stage
anaerobic hydrogen and methane production process.
Bioresour Technol 2011;102:8700e6.
[8] Xie B, Cheng J, Zhou J, Song W, Chen K. Cogeneration of
hydrogen and methane from glucose to improve energy
conversion efficiency. Int J Hydrogen Energy 2008;33:5006e11.
[9] Sen B, Aravind J, Kanmani P, Lay CH. State of the art and
future concept of food waste fermentation to bioenergy.
Renew Sustain Energy Rev 2016;53:547e57. https://doi.org/
10.1016/j.rser.2015.08.065.
[10] Bolzonella D, Battista F, Cavinato C, Gottardo M, Micolucci F,
Lyberatosd G, et al. Recent developments in biohythane
production from household food wastes: a review.
Bioresour Technol 2018;257:311e9.
[11] Moreno F, Munoz M, Arroyo J, Magen O, Monne  C, Suelves I.
Efficiency and emissions in a vehicular spark ignition
engine fuelled with hydrogen and methane blends. Int J
Hydrogen Energy 2012;37:11495e503.
[12] Mishra P, Balachandar G, Das D. Improvement in
biohythane production using organic solid waste and
distillery effluent. Waste Manag 2017;66:70e8.
[13] Buitron G, Kumar G, Martinez-Arce A, Moreno G. Hydrogen
and methane production via a two-stage processes (H2-SBR
þ CH4-UASB) using tequila vinasses. Int J Hydrogen Energy
2014;39(33):19249e55.
[14] Yang Z, Xu X, Guo R, Fan X, Zhao X. Accelerated
methanogenesis from effluents of hydrogen-producing
stage in anaerobic digestion by mixed cultures enriched
with acetate and nano-sized magnetite particles. Bioresour
Technol 2015;190:132e9.
[15] Seengenyoung J, Mamimin C, Prasertsan P, O-Thong S.
Pilot-scale of biohythane production from palm oil mill
effluent by two-stage thermophilic anaerobic Fermentation.
Int J Hydrogen Energy 2018:1e9. https://doi.org/10.1016/
j.ijhydene.2018.08.021.
[16] Liu Z, Zhang C, Lu Y, Wu X, Wang L, Wang L, et al. States
and challenges for high-value bio-hythane production from
waste biomass by dark fermentation technology. Bioresour
Technol 2013;135:292e303.
[17] Genovese A, Ortenzi F. Enriched methane for city public
transport buses. In: De Falco Marcello, Basile, Angelo,
editors. Enriched Methane: the first step toward the
hydrogen economy. Switzerland: Springer International
Publishing; 2016. p. 195e213.
[18] Cavinato C, Bolzonella D, Pavan P, Cecchi F. Two-phase
anaerobic digestion of food wastes for hydrogen and
methane production. In: De Falco Marcello, Basile, Angelo,
editors. Enriched Enriched Methane: the first step toward
the hydrogen economy. Switzerland: Springer International
Publishing; 2016. p. 75e90.
[19] Jehlee A, Khongkliang P, Suksong W, Rodjaroen S,
Waewsak J, Reungsang A, et al. Biohythane production from
Chlorella sp. biomass by two-stage thermophilic solid-state
anaerobic digestion. Int J Hydrogen Energy
2017;42:27792e800.
[20] Mathew AK, Bhui I, Banerjee SN, Goswami R, Shome A,
Chakraborty AK, et al. Biogas production from locally
available aquatic weeds of Santiniketan through anaerobic
digestion. Clean Technol Environ Policy 2014. https://
doi.org/10.1007/s10098-014-0877-6.
[21] Bajpai P. In: anaerobic technology in pulp and paper
industry. Springer Briefs in Applied Sciences and
Technology; 2017.
 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 4 ( 2 0 1 9 ) 1 7 3 6 3 e1 7 3 8 0
[22] Zupanc ic
 GD, Grilc V. Anaerobic treatment and biogas
production from organic waste. In: Kumar S, editor.
Management of organic waste. Croatia: Tech; 2012. p. 1e28.
[23] Biarnes M. Biomass to biogas-anaerobic digestion. E
Instruments International; 2013. http://www.e-inst.com/
biomass-to-biogas.
[24] Hay JXW, Wu TY, Juan JC. Biohydrogen production
through photo fermentation or dark fermentation using
waste as a substrate: overview, economics, and future
prospects of hydrogen usage. Biofuel Bioprod Bior
2013;7:334e52.
[25] Kumar G, Bakonyi P, Sivagurunathan P, Kim SH,
Nemesto  thy NB, Be
lafi-Bako
 K, et al. Enhanced biohydrogen
production from beverage industrial wastewater using
external nitrogen sources and bioaugmentation with
facultative anaerobic strains. J Biosci Bioeng
2015;120:155e60.
[26] Roy S, Das D. Biohythane production from organic wastes:
present state of art. Environ Sci Pollut Res 2015:5469e74.
[27] Nath K, Das D. Improvement of fermentative hydrogen
production: various approaches. Appl Microbiol Biotechnol
2004;65:520e9.
[28] Friedrich MW. Methyl-coenzyme M reductase genes: unique
functional markers for methanogenic and anaerobic
methane-oxidizing archaea. Methods Enzymol
2005;397:428e42.
[29] Franke-Whittle IH, Walter A, Ebner C, Insam H.
Investigation into the effect of high concentrations of
volatile fatty acids in anaerobic digestion on methanogenic
communities. Waste Manag 2014;34(11):431.
[30] Jafari O, Zilouei H. Enhanced bio-hydrogen and subsequent
bio-methane production from sugarcane bagasse using
nano-titanium dioxide pretreatment. Bioresour Technol
2016;214:670e8.
[31] Wang Y, Zhang Y, Wang J, Meng L. Effects of volatile fatty
acid concentrations on methane yield and methanogenic
bacteria. Biomass Bioenergy 2009;33:848e53.
[32] Panyadee S, Petiraksakul A, Phalakornkule C. Biogas
production from co-digestion of Phyllanthus emblica residues
and food waste. Energy Sustain Dev 2013;17:515e20.
[33] Zahedi S, Sales D, Romero LI, Solera R. Optimisation of the
two-phase dry-thermophilic anaerobic digestion process of
sulphate-containing municipal solid waste: population
dynamics. Bioresour Technol 2013;148:443e52.
[34] Kvesitadze G, Sadunishvili T, Dudauri T, Zakariashvili N,
Partskhaladze G, Ugrekhelidze V, et al. Two-stage anaerobic
process for bio-hydrogen and bio-methane combined
production from biodegradable solid wastes. Energy
2012;37(1):94e102.
[35] Malave ACL, Bernardi M, Fino D, Ruggeri B. Multistage
anaerobic digestion (MAD) as a tool to increase energy
production via H2þCH4. Int J Hydrogen Energy
2015;40:5050e61.
[36] Massanet-Nicolau J, Dinsdale R, Guwy A, Shipley G. Use of
real time gas production data for more accurate comparison
of continuous single-stage and two-stage fermentation.
Bioresour Technol 2013;129:561e7.
[37] Willquist K, Nkemka VN, Svensson H, Pawar S,
Ljunggren M, Karlsson H, et al. Design of a novel biohythane
process with high H2 and CH4 production rates. Int J
Hydrogen Energy 2012;37:17749e62.
[38] Kongjan P, O-Thong S, Angelidaki I. Performance and
microbial community analysis of two-stage process with
extreme thermophilic hydrogen and thermophilic methane
production from hydrolysate in UASB reactors. Bioresour
Technol 2011;102:4028e35.
[39] Ariunbaatar J, Perta ESD, Panico A, Frunzo L, Esposito G,
Lens PNL, et al. Effect of ammoniacal nitrogen on one-stage
17377
and two-stage anaerobic digestion of food waste. Waste
Manag 2015;38:388e98.
[40] Ghimire A, Frunzo L, Pirozzi F, Trably E, Escudie R, Lens PNL,
et al. A review on dark fermentative bio-hydrogen
production from organic biomass: process parameters and
use of by-products. Appl Energy 2015;144:73e95.
[41] Lee MJ, Song JH, Hwang SJ. Enhanced bio-energy recovery in
a two-stage hydrogen/methane fermentation process.
Water Sci Technol 2009;59:21e37.
[42] Wei H, Fu Y, Magnusson L, Baker JO, Maness PC, Xu Q, et al.
Comparison of transcriptional profiles of Clostridium
thermocellum grown on cellobiose and pretreated yellow
poplar using RNA-Seq. Front Microbiol 2014;5:142e7.
[43] Tran KT, Maeda T, Wood TK. Metabolic engineering of
Escherichia coli to enhance hydrogen production from
glycerol. Appl Microbiol Biotechnol 2014;98:4757e70.
[44] Merlino G, Rizzi A, Schievano A, Tenca A, Scaglia B, Oberti R,
et al. Microbial community structure and dynamics in two-
stage vs. single-stage thermophilic anaerobic digestion of
mixed swine slurry and market bio-waste. Water Res
2013;47:1983e95.
[45] Shin SG, Han G, Lim J, Lee C, Hwang S. A comprehensive
microbial insight into two-stage anaerobic digestion of
food waste-recycling wastewater. Water Res
2010;44:4838e49.
[46] Zahedi S, Solera R, Micolucci F, Cavinato C, Bolzonella D.
Changes in microbial community during hydrogen and
methane production in two-stage thermophilic anaerobic
co-digestion process from bio-waste. Waste Manag
2016;49:40e6.
[47] Mamimin C, Singkhala A, Kongjan P, Suraraksa B,
Prasertsan P, Imai T, et al. Two-stage thermophilic
fermentation and mesophilic methanogen process for bio-
hythane production from palm oil mill effluent. Int J
Hydrogen Energy 2015;40(19):6319e28.
[48] Si B, Liu Z, Zhang Y, Li J, Shen R, Zhu Z, et al. Towards bio-
hythane production from biomass: influence of operational
stage on anaerobic fermentation and microbial community.
Int J Hydrogen Energy 2016;41(7):4429e38.
[49] Jariyaboon R, O-Thong S, Kongjan P. Bio-hydrogen and bio-
methane potentials of skim latex serum in batch
thermophilic two-stage anaerobic digestion. Bioresour
Technol 2015;198:98e206.
[50] Kumar S, Dheeran P, Singh SP, Mishra IM, Adhikari DK.
Kinetic studies of two-stage sulphuric acid hydrolysis of
sugarcane bagasse. Renew Energy 2015;83:850e8.
[51] Yeshanew MM, Frunzo Lb, Pirozzi F, Lens PNL, Esposito G.
Production of biohythane from food waste via an integrated
system of continuously stirred tank and anaerobic fixed bed
reactors. Bioresour Technol 2016;220:312e22.
[52] Nkemka VN, Gilroyed B, Yanke J, Gruninger R, Vedres D,
McAllister T, et al. Bioaugmentation with an anaerobic
fungus in a two-stage process for bio-hydrogen and biogas
production using corn silage and cattail. Bioresour Technol
2015;185:79e88.
[53] Pisutpaisal N, Nathao C, Sirisukpoka U. Biological hydrogen
and methane production from food waste in two-stage
CSTR. Energy Procedia 2014;50:719e22.
[54] Corneli E, Dragoni F, Adessi A, Philippis RD, Bonari E,
Ragaglini G. Energy conversion of biomass crops and
agroindustrial residues by combined bio-hydrogen/bio-
methane system and anaerobic digestion. Bioresour
Technol 2016;211:509e18.
[55] Cavinato C, Giuliano A, Bolzonella D, Pavan P, Cecchi F. Bio-
hythane production from food waste by dark fermentation
coupled with anaerobic digestion process: a long-term pilot
scale experience. Int J Hydrogen Energy
2012;37(15):11549e55.
17378 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 4 ( 2 0 1 9 ) 1 7 3 6 3 e1 7 3 8 0
[56] Ding L, Cheng J, Xia A, Jacob A, Voelklein M, Murphy JD. Co-
generation of bio-hydrogen and bio-methane through two-
stage batch co-fermentation of macro- and micro-algal
biomass. Bioresour Technol 2016;218:224e31.
[57] Costa JC, Oliveira JV, Pereira MA, Alves MM, Abreu AA. Bio-
hythane production from marine macroalgae Sargassum sp.
coupling dark fermentation and anaerobic digestion.
Bioresour Technol 2015;190:251e6.
[58] Han SK, Shin HS. Performance of an innovative two-stage
process converting food waste to hydrogen and methane. J
Air Waste Manag Assoc 2004;54:242e9.
[59] Pakarinen OM, Tahti HP, Rintala JA. One-stage H2 and CH4
and two-stage H2þCH4 production from grass silage and
from solid and liquid fractions of NaOH pre-treated grass
silage. Biomass Bioenergy 2009;33:1419e27.
[60] Antonopoulou G, Gavala HN, Skiadas IV, Angelopoulos K,
Lyberatos G. Bio-fuels generation from sweet sorghum:
fermentative hydrogen production and anaerobic digestion
of the remaining biomass. Bioresour Technol 2008;99:110e9.
[61] Cavinato C, Bolzonella D, Fatone F, Giuliano A, Pavan P.
Two-phase thermophilic anaerobic digestion process for
bio-hythane production treating bio-waste: preliminary
results. Water Sci Technol 2011;64:715e21.
[62] Alibardi L, Cossu R. Composition variability of the organic
fraction of municipal solid waste and effects on hydrogen
and methane production potentials. Waste Manag
2015;36:147e55.
[63] Li WW, Yu HQ. From wastewater to bioenergy and
biochemicals via two-stage bioconversion processes: a
future paradigm. Biotechnol Adv 2011;29:972e82.
[64] Alibardi L, Cossu R. Effects of carbohydrate, protein and
lipid content of organic waste on hydrogen production and
fermentation products. Waste Manag 2016;47:69e77.
[65] De Gioannis, Muntoni G, Polettini AA, Pomi R. A review of dark
fermentation hydrogen production from biodegradable
municipal waste fractions. Waste Manag 2013;33:1345e61.
[66] Khongkliang P, Kongjan P, O-Thong S. Hydrogen and
methane production from starch processing wastewater by
thermophilic two-stage anaerobic digestion. Energy
Procedia 2015;79:827e32.
[67] Hasyim R, Imai T, O-Thong S, Sulistyowati L. Bio-hydrogen
production from sago starch in wastewater using an
enriched thermophilic mixed culture from hot spring. Int J
Hydrogen Energy 2011;36:14162e71.
[68] Kim DH, Kim SH, Shin HS. Hydrogen fermentation of food
waste without inoculum addition. Enzym Microb Technol
2009;45(3):181e7.
[69] Kim M, Lee D, Kim D. Continuous hydrogen production
from tofu processing waste using anaerobic mixed
microflora under thermophilic conditions. Int J Hydrogen
Energy 2011;36:8712e8.
[70] Li SL, Kuo SC, Lin JS, Lee ZK, Wang YH, Cheng SS. Process
performance evaluation of intermittent-continuous stirred
tank reactor for anaerobic hydrogen fermentation with
kitchen waste. Int J Hydrogen Energy 2008;33:1522e31.
[71] Dong L, Zhenhong Y, Yongming S, Longlong M. Anaerobic
fermentative co-production of hydrogen and methane from
an organic fraction of municipal solid waste. Energy
Sources Part A 2011;33:575e85.
[72] Cota-Navarro CB, Carrillo-Reyes J, Davila-Vazquez G,
Alatriste-Mondragon F, Razo-Flores E. Continuous hydrogen
and metahe production in a two-stage cheese whey
fermentation system. Water Sci Technol 2011;64:7.
[73] Venetsaneas N, Antonopoulou G, Stamatelatou K,
Kornaros M, Lyberatos G. Using cheese whey for hydrogen
and methane generation in a two-stage continuous process
with alternative pH controlling approaches. Bioresour
Technol 2009;100:3713e7.
[74] Suksong W, Kongjan P, Thong S. Bio-hythane production
from co-digestion of palm oil mill effluent with solid
residues by two-stage solid state anaerobic digestion
process. Energy Procedia 2015;79:943e9.
[75] Patil JH, Raj MA, Muralidhara PL, Desai SM, Mahadeva
Raju GK. Kinetics of anaerobic digestion of water hyacinth
using poultry litter as inoculum. Int J Environ Sustain Dev
2012;3(2):94e8.
[76] Sarkar S, Banerjee S. Studies on biomethanation of water
hyacinth (Eichhornia crassipes) using biocatalyst. Int J Energy
Environ 2013;4(3):449e58.
[77] Chuang YS, Lay CH, Sen B, Chen CC, Gopalakrishnan K,
Wud JH, et al. Bio-hydrogen and Bio-methane from water
hyacinth (Eichhornia crassipes) fermentation: effects of
substrate concentration and incubation temperature. Int J
Hydrogen Energy 2011;36:14195e203.
[78] Sialve B, Bernet N, Bernard O. Anaerobic digestion of
microalgae as a necessary stage to make micro algal
biodiesel sustainable. Biotechnol Adv 2009;27:409e16.
[79] Jones CS, Mayfield SP. Algae bio-fuels: versatility for the
future of bioenergy. Curr Opin Biotechnol 2012;23:346e51.
[80] Roy S, Vishnuvardhan M, Das D. Improvement of hydrogen
production by newly isolated Thermoanaerobacterium
thermosaccharolyticum IIT BT-ST1. Int J Hydrogen Energy
2014;39:7541e52.
[81] Schunurer A, Jarvis A. Microbiological handbook for biogas
plants (Malmo € ) Sweden. Sweedish waste management; 2009.
[82] Trisakti B, Irvan M, Taslim Turmuzi M. Effect of
temperature on methanogenesis stage of two-stage
anaerobic digestion of palm oil mill effluent (POME) into
biogas. Mater Sci Eng 2017;206, 012027. https://doi.org/
10.1088/1757-899X/206/1/012027.
[83] Gaby JC, Zamanzadeh M, Horn SJ. The effect of temperature
and retention time on methane production and microbial
community composition in staged anaerobic digesters fed
with food waste. Biotechnol Biofuels 2017;10:302. https://
doi.org/10.1186/s13068-017-0989-4.
[84] Ge X, Yang L, Sheets JP, Yu Z, Li Y. Biological conversion of
methane to liquid fuels: status and opportunities.
Biotechnol Adv 2014;32:1460e75.
[85] Abreu AA, Karakashev D, Angelidaki I, Sousa DZ, Alves MM.
Biohydrogen production from arabinose and glucose using
extreme thermophilic anaerobic mixed cultures. Biotechnol
Biofuels 2012:5e6.
[86] Willquist K, Zeidan AA, van Niel EW. Physiological
characteristics of the extreme thermophile
Caldicellulosiruptor saccharolyticus: an efficient hydrogen cell
factory. Microb Cell Factories 2010;22(9):89. https://doi.org/
10.1186/1475-2859-9-89.
[87] Verhaart MRA, Bielen AAM, van der Oost J, Stams AJM,
Kengen SWM. Hydrogen production by hyperthermophilic
and extremely thermophilic bacteria and archaea:
mechanisms for reductant disposal. Environ Technol
2010;31(8e9):993e1003.
[88] de Vrije T, Bakker RR, Budde MA, Lai MH, Mars AE,
Claassen PA. Efficient hydrogen production from the
lignocellulosic energy crop Miscanthus by the extreme
thermophilic bacteria Caldicellulosiruptor saccharolyticus and
Thermotoga neapolitana. Biotechnol Biofuels 2009;2(1):12.
https://doi.org/10.1186/1754-6834-2-12.
[89] Frock AD, Kelly RM. Extreme thermophiles: moving beyond
single-enzyme biocatalysis. Curr Opin Chem Eng
2012;1(4):363e72. https://doi.org/10.1016/j.coche.2012.07.003.
[90] Basen M, Rhaesa AM, Kataeva I, Prybol CJ, Scott IM, Poole FL,
et al. Degradation of high loads of crystalline cellulose and
of unpretreated plant biomass by the thermophilic
bacterium Caldicellulosiruptor bescii. Bioresour Technol
2014;152:384e92.
 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 4 ( 2 0 1 9 ) 1 7 3 6 3 e1 7 3 8 0
[91] Pachapur VK, Sarma SJ, Brar SK, Le Bihan Y, Soccol CR,
Buelna G, et al. Co-culture strategies for increased
biohydrogen production. Int J Energy Res 2015;39:1479e504.
[92] Abreu AA, Tavares F, Alves MM, Pereira MA. Boosting dark
fermentation with co-cultures of extreme thermophiles for
biohythane production from garden waste. Bioresour
Technol 2016;219:132e8.
[93] Reith JH, Wijffels RH, Barten H. Bio-methane and
biohydrogen status and perspective of biological methane
and hydrogen production. Rotterdam: Dutch Bio-Hydrogen
Foundation; 2003. p. 118e25.
[94] Liu Z, Si B, Li J, He J, Zhang C, Lu Y, et al. Bioprocess
engineering for biohythane production from low-grade
waste biomass: technical challenges towards scale up. Curr
Opin Biotechnol 2018;50:25e31.
[95] Kim S, Bae J, Choi O, Ju D, Lee J, Sung H, et al. A pilot scale
two-stage anaerobic digester treating food waste leachate
(FWL): performance and microbial structure analysis using
pyrosequencing. Process Biochem 2013;49(2):301e8.
[96] Lin YQ, Wu SB, Wang DH. Hydrogen-methane production
from pulp & paper sludge and food waste by mesophilic and
thermophilic anaerobic co-digestion. Int J Hydrogen Energy
2013;38(35):15055e62.
[97] Ventura JRS, Lee J, Jahng D. A comparative study on the
alternating mesophilic and thermophilic two-stage anaerobic
digestion of food waste. J Environ Sci 2014;26:1274e83.
[98] Chipasa KB, Medrzycka K. Behavior of lipids in biological
wastewater treatment processes. J Ind Microbiol Biotechnol
2006;33:635e45.
[99] Khanal SK, Chen WH, Sung LLS. Biological hydrogen
production: effects of pH and intermediate products. Int J
Hydrogen Energy 2004;29:1123e31.
[100] Moraes BS, Triolo JM, Lecona VP, Zaiat M, Sommer SG.
Biogas production within the bioethanol production chain:
use of co-substrates for anaerobic digestion of sugar beet
vinasse. Bioresour Technol 2015;190:227e34.
[101] Lee DY, Ebie Y, Xu KQ, Li YY, Inamori Y. Continuous H2 and
CH4 production from high-solid food waste in the two-stage
thermophilic fermentation process with the recirculation of
digester sludge. Bioresour Technol 2010;101:42e7.
[102] Wu C, Huang Q, Yu M, Ren Y, Wanga Q, Sakai K. Effects of
digestate recirculation on a two-stage anaerobic digestion
system, particularly focusing on metabolite correlation
analysis. Bioresour Technol 2018;251:40e8.
[103] Micolucci F, Gottardo M, Bolzonella D, Pavan P. Automatic
process control for stable bio-hythane production in two-
phase thermophilic anaerobic digestion of food waste. Int J
Hydrogen Energy 2014;39(31):17563e72.
[104] Battista F. Resolutions for the anaerobic digestion of agro-
food wastes: the case of olive oil production's refuses. Lap
Lambert Academic Publishing; 2015.
[105] GottardoM, Micolucci F, Bolzonella D, Uellendahl H,
Pavan P. Pilot scale fermentation coupled with anaerobic
digestion of food waste-Effect of dynamic digestate
recirculation. Renew Energy 2017;114:455e63.
[106] Ramadoss G, Muthukumar K. Influence of dual salt on the
pretreatment of sugarcane bagasse with hydrogen peroxide
for bioethanol production. Chem Eng J 2015;260:178e87.
[107] Behera S, Arora R, Nandhagopal N, Kumar S. Importance of
chemical pretreatment for bioconversion of lignocellulosic
biomass. Renew Sustain Energy Rev 2014;36:91e106.
[108] Saratale GD, Oh SE. Lignocellulosics to ethanol: the future of
the chemical and energy industry. Afr J Biotechnol
2012;11:1002e13.
[109] Mahdy A, Mendez L, Ballesteros M, Gonzalez-Fernandez C.
Autohydrolysis and alkaline pretreatment effect on
Chlorellavulgaris and Scenedesmus sp. methane production.
Energy 2014;78:48e52.
17379
[110] Rai PK, Singh SP, Asthana RK, Singh S. Bio-hydrogen
production from sugarcane bagasse by integrating dark-
and photo-fermentation. Bioresour Technol
2014;152:140e6.
[111] Zheng XJ, Wei LF, Zhang ZH, Jiang QJ, Wei YJ, Xie B, et al.
Research on photocatalytic hydrogen production from
acetic acid solution by Pt/TiO2 nanoparticles under UV
irradiation. Int J Hydrogen Energy 2009;34(22):9033e41.
[112] Puskelova J, Baia L, Vulpoi A, Baia M, Antoniadou M,
Dracopoulos V, et al. Photocatalytic hydrogen production
using TiO2-Pt aerogels. Chem Eng J 2014;242:69e101.
[113] Kuznetsov BN, Danilov VG, Kuznetsova SA,
Yatsenkova OV, Aleksandrova NB. Optimization of fire
wood delignification by acetic acid in the presence of
hydrogen peroxide and a TiO2 catalyst. Theor Found
Chem Eng 2009;43(4):499e503.
[114] Kumari S, Das D. Biologically pretreated sugarcane top as a
potential raw material for the enhancement of gaseous
energy recovery by two stage biohythane process. Bioresour
Technol 2016;218:1090e7.
[115] Sindhu R, Binod P, Pandey A. Biological pretreatment of
lignocellulosic biomass-An overview. Bioresour Technol
2015;199:76e82.
[116] Rameshaiah GN, Reddy MLJ. Applications of ligninolytic
enzymes from a white-rot fungus Trametes versicolor.
Univers J Environ Res Technol 2015;5(1):1e7.
[117] Shirkavand E, Baroutian S, Gapes DJ, Young BR.
Combination of fungal and physicochemical processes for
lignocellulosic biomass pretreatment- A review. Renew
Sustain Energy Rev 2016;54:217e34.
[118] Bong CPC, Lim LY, Lee CT, Kleme s JJ, Ho CS, Ho WS. The
characterization and treatment of food waste for
improvement of biogas production during anaerobic
digestion- a review. J Clean Prod 2018;172:1545e58.
rez M. Upgrading the temperature-
[119] Riau V, De la Rubia MA, Pe
phased anaerobic digestion of waste activated sludge by
ultrasonic pretreatment. Chem Eng J 2015;259:672e81.
[120] Ofori-Boateng C, Lee KT. Ultrasonic-assisted simultaneous
saccharification and fermentation of pretreated oil palm
fronds for sustainable bioethanol production. Fuel
2014;119:285e91.
[121] Bussemaker MJ, Zhang D. Effect of ultrasound on
lignocellulosic biomass as a pretreatment for biorefinery
and biofuel applications. Ind Eng Chem Res
2013;52:3563e80. https://doi.org/10.1021/ie3022785.
[122] Martinez-Jimeneza FD, Pintoa MPM, Mudhoob A, de Alencar
Nevesa T, Rostagnoc MA, Forster-Carneiroa T. Influence of
ultrasound irradiation pre-treatment in biohythane
generation from the thermophilic anaerobic co-digestion of
sugar production residues. J Environ Chem Eng 2017;5:3749e58.
[123] Lay JJ. Modeling and optimization of anaerobic digested
sludge converting starch to hydrogen. Biotechnol Bioeng
2000;68:269e78.
[124] Ginkel SV, Sung S, Lay JJ. Bio-hydrogen production as a
function of pH and substrate concentration. Environ Sci
Technol 2001;35:4726e30.
[125] Liu Z, Li Q, Zhang C, Wang L, Han B, Li B, et al. Effects of
operating parameters on hydrogen production from raw
wet steam-exploded cornstalk and two-stage fermentation
potential for bio-hythane production. Biochem Eng J
2014;15:234e8.
[126] Li D, Chen H. Biological hydrogen production from steam-
exploded straw by simultaneous saccharification and
fermentation. Int J Hydrogen Energy 2007;32:1742e8.
[127] Sung S, Raskin L, Duangmanee T, Padmasiri S, Simmons JJ.
Hydrogen production by anaerobic microbial communities
exposed to repeated heat treatments. Proceedings of the
2002 US DOE hydrogen program review nrel/cp-610-32405.
17380 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 4 ( 2 0 1 9 ) 1 7 3 6 3 e1 7 3 8 0
[128] Li S, Lai C, Cai Y, Yang X, Yang S, Zhu M, et al. High
efficiency hydrogen production from glucose/xylose by the
ldh-deleted Thermoanaerobacterium strain. Bioresour
Technol 2010;101:8718e24.
[129] Ito T, Nakashimada Y, Kakizono T, Nishio N. High-yield
production of hydrogen by Enterobacter aerogenes mutants
with decreased alpha-acetolactate synthase activity. J
Biosci Bioeng 2004;97:227e32.
[130] Roy S, Banerjee D, Dutta M, Das D. Metabolically
redirected bio-hydrogen pathway integrated with
biomethanation for improved gaseous energy recovery.
Fuel 2015;158:471e8.
[131] De Simio L, Gambino M, Iannaccone S. Using natural gas/
hydrogen mixture as a fuel in a 6-cylider stoichiometric
spark ignition engine. In: De Falco Marcello, Basile, Angelo,
editors. Enriched methane: the first step toward the
hydrogen economy. Switzerland: Springer International
Publishing; 2016. p. 175e94.
[132] Perera KRJ, Ketheesan B, Arudchelvam Y,
Nirmalakhandan N. Fermentative bio-hydrogen production
II: net energy gain from organic wastes. Int J Hydrogen
Energy 2012;37:167e78.
[133] Intanoo P, Rangsanvigit P, Malakul P, Chavadej S.
Optimization of separate hydrogen and methane
production from cassava wastewater using two-stage
upflow anaerobic sludge blanket reactor (UASB) system
under thermophilic operation. Bioresour Technol
2014;173:256e65.
[134] Porpatham E, Ramesh A, Nagalingam B. Effect of hydrogen
addition on the performance of a biogas fuelled spark
ignition engine. Int J Hydrogen Energy 2007;32:2057e65.
[135] Hraiech I, Sautet JC, Mergheni MA, Ticha HB, Touati H,
Mhimid A. Effects of hydrogen addition and carbon-dioxide
dilution on the velocity field in non reacting and reacting
flows. Int J Hydrogen Energy 2014;39(34):19818e31.
[136] Chuayboon S, Prasertsan S, Theppaya T, Maliwan K,
Prasertsan P. Effects of CH4, H2 and CO2 mixtures on SI gas
engine. Energy Procedia 2014;52:659e65.
[137] Patel SKS, Mardina P, Kim D, Kim SY, Kalia VC, Kim IW,
et al. Improvement in methanol production by regulating
the composition of synthetic gas mixture and raw biogas.
Bioresour Technol 2016;218:202e8.
[138] Patel SKS, Singh RK, Kumar A, Jeong J-H, Jeong SH, Kalia VC.
Biological methanol production by immobilized Methylocella
tundraeusing simulated biohythane as a feed. Bioresour
Technol 2017;241:922e7.
[139] Cooney M, Maynard N, Cannizzaro C, Benemann J. Two-
phase anaerobic digestion for production of hydrogen-
methane mixtures. Bioresour Technol 2007;98:2641e51.
[140] Mamimin C, Prasertsan P, Kongjan P, O-Thong S. Effects of
volatile fatty acids in biohydrogen effluent on biohythane
production from palm oil mill effluent under thermophilic
condition. Electron J Biotechnol 2017;29:78e85.
[141] 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;41:22619e25.
[142] Dahiya S, Sarkar O, Swamy YV, Venkata Mohan S.
Acidogenic fermentation of food waste for volatile fatty acid
production along with cogeneration of biohydrogen.
Bioresour Technol 2015;182:103e13.
[143] Ma J, Frear C, Wang Z-W, Yu L, Zhao Q, Li X, et al. A simple
methodology for rate-limiting step determination for
anaerobic digestion of complex substrates and effect of
microbial community ratio. Bioresour Technol
2013;134:391e5.
[144] Ziganshin AM, Schmidt T, Lv Z, Liebetrau J, Richnow HH,
Kleinsteuber S, et al. Reduction of the hydraulic retention
time at constant high organic loading rate to reach the
microbial limits of anaerobic digestion in various reactor
systems. Bioresour Technol 2016;217:62e71.
[145] Khemkhao M, Techkarnjanaruk S, Phalakornkule C. Effect
of chitosan on reactor performance and population of
specific methanogens in a modified CSTR treating raw
POME. Biomass Bioenergy 2016;86:11e20.
[146] Carrillo-Reyes J, Trably E, Bernet N, Latrille E, Razo-Flores E.
High robustness of a simplified microbial consortium
producing hydrogen in long term operation of a biofilm
fermentative reactor. Int J Hydrogen Energy
2016;41(4):2367e76.
[147] Karadag D, Koroglu OE, Ozkaya B, Cakmakci M. A review on
anaerobic biofilm reactors for the treatment of dairy
industry wastewater. Process Biochem 2015;50:262e71.
[148] Van Lier JB, Van der Zee FP, Frijters CTMJ, Ersahin ME.
Celebrating 40 years anaerobic sludge bed reactors for
industrial wastewater treatment. Rev Environ Sci
Biotechnol 2015:1e20.
[149] Goud RK, Venkata Mohan S. Regulating biohydrogen
production from wastewater by applying organic load-
shock: change in the microbial community structure and
bio-electrochemical behavior over long-term operation. Int J
Hydrogen Energy 2012;37:17763e77.
[150] Pedizzi C, Regueiro L, Rodriguez-Verde I, Lema JM,
Carballa M. Effect of oxygen on the microbial activities of
thermophilic anaerobic biomass. Bioresour Technol
2016;211:765e8.
[151] Sarkar O, Mohan SV. Pre-aeration of food waste to augment
acidogenic process at higher organic load: valorizing
biohydrogen, volatile fatty acids and biohythane. Bioresour
Technol 2017;242:68e76.
[152] Cossu R, Morello L, Raga R, Cerminara G. Biogas production
enhancement using semi-aerobic pre-aeration in a hybrid
bioreactor landfill. Waste Manag 2016;55:83e92.
[153] Jarunglumlert T, Prommuak C, Putmai N, Pavasant P.
Scaling-up bio-hydrogen production from food waste:
feasibilities and challenges. Int J Hydrogen Energy
2018;43:634e48.
[154] Dincer I, Acar C. Review and evaluation of hydrogen
production methods for better sustainability. Int J Hydrogen
Energy 2015;40(34):11094e111.
[155] Lee YW, Chung J. Bioproduction of hydrogen from food
waste by pilot-scale combined hydrogen/methane
fermentation. Int J Hydrogen Energy 2010;35(21):11746e55.
[156] Han W, Fang J, Liu Z, Tang J. Techno-economic evaluation of a
combined bioprocess for fermentative hydrogen production
from food waste. Bioresour Technol 2016;202:107e12.
[157] Chung F, Li YY, Xu KQ, Ebie Y, Inamori Y, Kong HN. A pH
and temperature-phased two-stage process for hydrogen
and methane production from food waste. Int J Hydrogen
Energy 2008;33:4739e46.
[158] Han W, Liu Z, Fang J, Huang J, Zhao H, Li Y.
Technoeconomic analysis of dark fermentative hydrogen
production from molasses in a continuous mixed
immobilized sludge reactor. J Clean Prod 2016;127:567e72.
[159] Jang S, Kim DH, Yun YM, Lee MK, Moon C, Kang WS, et al.
Hydrogen fermentation of food waste by alkali-shock
pretreatment: microbial community analysis and limitation of
continuous operation. Bioresour Technol 2015;186:215e22.
[160] Budzianowski WM, Brodacka M. Biomethane storage:
evaluation of technologies, end uses, business models, and
sustainability. Energy Convers Manag 2017;141:
254e73.
[161] Suwansri S, Moran JC, Aggarangsi P, Tippayawong N,
Bunkham A, Rerkkriangkrai P. A biomethane solution for
domestic cooking in Thailand. Energy Sust Dev
2014;23:68e77.