Fuel 317 (2022) 123449

Contents lists available at ScienceDirect

Fuel
journal homepage: www.elsevier.com/locate/fuel

A critical review on the two-stage biohythane production and its viability as

a renewable fuel
K.B. Sasidhar a, b, P. Senthil Kumar a, b, *, Leilei Xiao c, d
a
Department of Chemical Engineering, Sri Sivasubramaniya Nadar College of Engineering, Kalavakkam, Chennai 603110, India
b
Centre of Excellence in Water Research (CEWAR), Sri Sivasubramaniya Nadar College of Engineering, Kalavakkam, Chennai 603110, India
c
Key Laboratory of Coastal Biology and Biological Resources Utilization, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, PR
China
d
CAS Key Laboratory of Coastal Environmental Processes and Ecological Remediation, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai
264003, PR China

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

Keywords: With over 130 countries signing the net-zero coalition treaty to reduce the global warming, the interest to replace
Anaerobic digestion fossil-fuels is constantly increasing. Also, there exists an increased concern to reduce the pollution load from
Biohydrogen waste biomass. Though the socio-economic benefits of biomethane production from these wastes have been an
Biohythane
enticing study of the recent past, a sequential biohydrogen and biomethane production is said to offer more in
Biomethane
terms of operational stability, energy yield, and cleaner by-products. This review thus tries to provide insights to
Dark-fermentation
Two-stage production have a better understanding on the process of biohydrogen production and subsequent biomethane production
and the challenges associated with upscaling. With over 40% reduction in carbon monoxide and carbon dioxide
emissions, the viability of Hythane as a clean and renewable fuel are also discussed.

1. Introduction acknowledged as one of the eco-friendly ways of disposal as it offers

The United Nations expect the population to increase by 2.1 billion in
the urban centres by 2030 [1]. With an increase in population, the en­
ergy requirement is said to increase by almost 50% in the next 20 years
[2]. These high energy demands are currently met with fossil fuels
which impart a negative effect on the earth. Thus, to alleviate the
pollution load, and fossil fuel depletion, renewable energy resources are
looked upon for sustainable development. Modern research in renew­
able fuel focus mainly on the energy from biomass, as this method not
only provides energy, but also reduces the waste load. Researchers have
proposed the production of valuable fuels such as biodiesel, bioethanol,
biohydrogen and biomethane from the waste biomass. Biodiesel pro­
duction from waste biomass such as Moringa Olifera Oil [3], Chicken fat
[4], and other edible oils [5] have been established in recent times.
Though biodiesel is a viable alternative to conventional diesel, the
extraction part requires a fatty or oil substrate, and hence cannot be
adopted for all organic wastes. Thereby, biohydrogen and sequential
biomethane production is more suitable in treatment of solid wastes. In
comparison with the conventional treatment of solid wastes by incin­
eration, compositing, or landfilling, anaerobic digestion (AD) has been
revenue via energy in addition to a stable treatment [6–7]. AD of
wastewater is also considered to be one of the widely sought-after
technologies since it produces bio-energy and other useful bio-
products [8–9]. Angelidaki et al., describe that AD is used for: (1) acti­
vated sludge and secondary clarifier sludge from wastewater treatment
plant, (2) industrial wastewater from agro, biomass and food-based in­
dustries, (3) wastes generated from agriculture and livestock, and (4)
organic fractions of municipal solid waste (OFMSW) [10]. The diverse
microbiological population in the waste can be utilized to degrade the
organic fraction of both solid and liquid wastes into biogas, electricity,
fuel, and other energy rich by-products [11–13]. Researchers in recent
times have begun to extend the reclamation of more useful products
from AD than biogas which could potentially have more value. In
retrospect, more importance is given to the reclamation of organic
carbon through carboxylic acids, volatile fatty acids (VFA), and
hydrogen [14]. Hydrogen is considered to be a clean fuel as combusting
hydrogen results in the formation of only water vapor. Since it is clean
and sustainable with zero-carbon emissions and has a high energy
density of 122–142 kJ/g, it is described as the fuel of the future society
[15–17]. AD for the co-production of biohydrogen and biomethane is

* Corresponding author.
E-mail address: senthilkumarp@ssn.edu.in (P.S. Kumar).

https://doi.org/10.1016/j.fuel.2022.123449
Received 8 September 2021; Received in revised form 19 January 2022; Accepted 24 January 2022
Available online 3 February 2022
0016-2361/© 2022 Elsevier Ltd. All rights reserved.
K.B. Sasidhar et al. Fuel 317 (2022) 123449

Fig. 1. Two-stage Biohydrogen and Biomethane (Biohythane) Production from Organic Solid Wastes.

currently a growing topic and quite extensive research on the topic have
been documented [13,17–29,241,242]. A graphical representation of
sequential hydrogen and methane production from organic solid wastes
through a two-stage anaerobic digestion process can be seen in Fig. 1.
An inclusion of hydrogen (10–25% by volume [17] or 5–7% by en­
ergy [30]) to methane, is often suggested in the blend of Hythane.
Though methane is a cleaner fuel when compared to the conventional
fuels, it has a very poor range of lean flammability [31], leading to a
poor combustion efficiency [17]. Combining even trace amounts of
hydrogen to methane leads to a significant increase in the lean flam­
mability range of the fuel [30], thus improving the efficiency. Hythane
has been tested as an alternate fuel since 1980 s, with its applicability in
Spark-Ignition (SI) engines [31–34]. Although the feasibility of
hydrogen production has been proven in lab-scale, an industrial-scale
system has not been built. Main reason behind this can be attributed
to the production of hydrogen. Current technologies in hydrogen pro­
duction focus on the physicochemical extraction from decarbonization
of methane [35]. The process involves technologies such as partial
oxidation of methane, autothermal reforming, coal gasification, biomass
gasification, photocatalytic water splitting, nuclear-fission powered
electrolysis, etc., [35–36], which are not sustainable due to their fossil
driven energy [17]. Alternatively, biological hydrogen production

Fig. 2. Bio-Process Involved in the Anaerobic Degradation of Organic Polymers to Biohydrogen and Biomethane. Adopted from Gujer and Zehnder, (1983) [44].

2
K.B. Sasidhar et al.
techniques can be looked upon to abate the use of fossil fuels. Bio­
hydrogen production from the dark fermentation of organic waste is
considered to be one of the simplest techniques [17,27]. The biological
system can be manipulated by altering the operating conditions to
obtain the necessary H2/CH4 (ratio) required for the Hythane blend
[17]. However, this method also has its own cons which has prevented
the research circuit into building an industrial-scale biohythane plant.
Biohydrogen production via dark fermentation, a precursor to the
anaerobic digestion, is a challenge of its own. This includes availability
of proper substrates for fermentation, process inhibitors, operating
conditions, hydrogen storage and Hythane blending [26–27]. This study
firstly summarises the works and applications carried out in the field of
two-stage sequential hydrogen and methane production, in terms of
microbial synthesis, substrate usage and process inhibitors. This litera­
ture will also address the challenges in the production (and co-
production), and storage of hydrogen, methane and Hythane, by
running through the effect of operating conditions, and provide possible
remediations in terms of pre-treatment, process optimization, co-
digestion, and bioreactor configuration. Finally, as a novel approach,
this study will assess the potential of Hythane as a clean fuel based on a
cost-benefit analysis and environmental impact with respect to other
fuels.
2. Hythane production via dark fermentation and anaerobic
digestion of organic wastes
2.1. Overview of anaerobic digestion process
Anaerobic Digestion is a series of biochemical reduction reactions
where a variety of micro-organisms degrade the complex organic poly­
mers into methane, carbon dioxide, ammonium, hydrogen sulphide and
water in the absence of oxygen [36–38]. The degradation happens in
four different stages namely hydrolysis, acidogenesis, acetogenesis and
methanogenesis [36–38,251] as shown in Fig. 2. Anaerobic microor­
ganisms function together in syntropic associations to breakdown the
complex substrate to biogas [36–38]. During the hydrolysis stage,
complex organic polymers are converted to simple monomers yielding
fermented sugars, carbohydrates, and amino acids [36,39]. During
acidogenesis, the acidogens ferment the sugars, carbohydrates, amino
acids, and lipids into intermediate volatile fatty acids (VFA) such as
pyruvic acid, lactate, ethanol, etc., through various bio-chemical path­
ways [40]. Acidogenic pathways are classified trifold; the butyric
fermentation yields acetic and butyric acid along with hydrogen and
carbon dioxide, propionic fermentation produces propionic, valeric, and
acetic acid with little carbon dioxide and hydrogen. The ethanol
fermentation involves production of ethanol and acetic acid with
meagre amounts of carbon dioxide and hydrogen [41]. The acetogenesis
stage is a crucial step in hydrogen production as the VFAs from the
previous process are reduced acetate, carbon dioxide, and hydrogen gas
[26,39,42]. During this process, the protons act as electron acceptors
and are hence reduced to hydrogen gas [43]. In the final step of digestion
(methanogenesis), acetate and hydrogen are converted to methane and
carbon dioxide by the methanogenic archaea [13,17,36–39,243,244].
The aceteclastic methanogens convert acetate to methane and carbon
dioxide and the hydrogenotrophs convert hydrogen and carbon dioxide
to methane [17,26,39,245,246]. Butyric and acetic acid are the main
precursors to methane production with acetic acid contributing to
65–95% of methane production [45]. The system has to be modified in
order to supress the activity of hydrogenotrophic methanogens and
homo-acetogens to ensure maximum biohydrogen and subsequent bio­
hythane yield.
2.2. Two-Stage digestion vs single-stage dark fermentation or anaerobic
digestion
A two-stage or two-phase anaerobic system is more advantageous
3
Fuel 317 (2022) 123449
than a single-stage system producing methane. Energy recovery,
methane yield, and lactate degradation have been found to be thermo­
dynamically superior in a two-stage anaerobic system when compared
to a single-phase system [19,42,46–51]. Hythane co-production in a
two-phase system increases the energy recovery by 100% when
compared to single-phase hydrogen production and by 30% when
compared to single-phase methane production [17]. In addition to the
energy recovery, co-production also significantly reduces the fermen­
tation time and ergo, the retention time [23,42,52–54]. This can be
related to the shorter retention during hydrogenesis when compared to
the methanogenesis, which subsequently permits larger organic loading
rates and stable operations in two-phase systems when compared to
single-phase systems [55–57]. The two-phase fermentation can be
effectively used for the degradation of complex organic solids with high
lignin contents such as rice husk, corn-straw, etc., and the subsequent
VFA produced from the first stage can be converted to methane in the
second stage [20,47,58]. Finally, the production of hydrogen and
methane in two separate stages provide adequate control over the mi­
crobial community with different functions, thus, making the operation
more stable than single-phase systems [59–61]. This further gives a
better control over the hydrogen to methane (H2/CH4) ratio as the mi­
crobial consortium can be controlled separately for better hydrolysate
stabilization and energy recovery [62].
2.3. Production of VFAs from organic wastes
VFA production is a precursor to the hydrogen and methane pro­
duction (Fig. 2). Fermentation of various organic carbon sources have
been explored by various researchers in the recent past for the produc­
tion of the VFAs [63–65]. For maximized production of VFAs, pure
sugars have been tested which yield minimal by-products [65–66].
Higher yield of VFAs come with a higher price as purchasing pure sugars
for raw materials is costly. However, other intermediate by-products can
be beneficial, which does not require fermentation of pure sugars
[14,67]. These includes intermediate products such as ethyl alcohol,
isopropyl alcohol, succinate, lactic acid, etc. [42,68–70]. Acetic acid can
be produced by Thermoanaerobacter, Acetomicrobium, Acetobacter, Glu­
conobacter, and Clostridium through fermentation of distinct kinds of
sugars [71,72]. As mentioned earlier, pure sugars are costly raw mate­
rials, hence, alternative carbon sources have to be explored. Studies
using Clostridium lintocellum show acetic acid yield from cellulosic and
lignocellulosic substrates can be as high as 30.98 g.l-1 and 17 g.l-1
respectively [73]. Propionic acid is a high demand VFA with an esti­
mated 470 kiloton requirement by 2020 [74]. Production of propionic
acid mostly involves the bacteria of genus Propionibacterium which in­
cludes P. jensenii, P. shermanii, P. acidipropionici, P. theonii, and
P. freudenreichii [75]. Propionic acid from glycerol (0. 71 g.g− 1) was
observed to be higher than glucose (0.35 g.g− 1) which is a costlier raw
material [76]. Pure culture of P. acidopropionici yielded 30 g.l-1 of pro­
pionic acid from low-cost cane molasses [77]. The fermentative organ­
isms which degrade the organic carbon can be natural or genetically
modified to carry out the required degradation. For instance, genetically
modified P. acidopropionici increased by 25% after modification [78]
and P. jensenii yielded 26.95 g.l-1 and 34.93 g.l-1 of propionic acid after
genetic modification [79]. Butyric acid is also considered one of the high
commodities VFAs with a demand of about 105 kilotons by 2020 [74].
Clostridium thermobutyricum, Clostridium Barkeri, Butyribacterium sp.,
Butyrivibrio fibrisolvens, Sarcina, Fusobacterium nucleatum, and Eubacte­
rium limosum are some of the microbial strains observed to produce
butyric acid through fermentation pathway [74]. Fermentation of
glucose and xylose with Clostridium tyrobutyricum yielded 34.2 g.l-1 of
Butyric Acid [80], and a maximum of 86.9 g.l-1 of Butyric Acid was
observed in a fibrous bed reactor with fed-batch fermentation strategy
[81]. Although beneficial, pure cultures have a lot of restrictions in day-
to-day applications. These cultures require sterile operating conditions
and extremely pure substrates as raw materials [13]. In order to reduce
K.B. Sasidhar et al. Fuel 317 (2022) 123449

such high costs, researchers are focussing on mixed culture degradation Table 1
of widely available lignocellulosic biomass as substrates [47,82–85]. List of Pure Culture Mesophilic, Thermophilic and Hyperthermophilic Organ­
Suitable pre-treatment has to be employed to reduce the lignin content isms and Corresponding Hydrogen Yield Recorded. (The Data Covers only the
in the biomass for the microorganisms to function effectively. This in­ Highest Yield Reported).
volves physicochemical or enzymatic pre-treatment of lignin rich wastes Organisms Operating Hydrogen yield References
which again increases the cost of treatment [85–86]. Thus, an optimized Conditions
mix culture does not depend one any particular cultural strain and can Clostridium butyricum Mesophilic 31.95 (ml H2 .g- [101]
be operated in non-sterile conditions with little contamination [87–89]. 1
COD)
Mesophilic 2.2 (mol H2.mol- [102]
1
glucose)
2.4. Formation of hydrogen through dark fermentation Mesophilic 2.4 (mol H2.mol- [104]
1
glucose)
Mesophilic 27.71 (ml H2 .g- [105]
Hydrogen production is quite similar and goes hand-in-hand with the 1
VSadded)
VFA production. The marked similarity between the two process is that Mesophilic 2.29 (mol H2.mol- [106]
1
both are intermediates of methanogenesis and act as a pre-cursor for glucose)
Mesophilic 1.4 (mol H2.mol- [108]
biogas production. Thus, in order to obtain either VFA or Hydrogen or 1
glucose)
both, methanogenesis has to be limited [89–91]. Thereby, hydrogen can Clostridium Mesophilic 1.8 (mol H2.mol- [106]
be considered as the by-product of VFA production [13,26,250]. acetobutylicum 1
glucose)
Hydrogen is produced by facultative and strict anaerobes under anaer­ Mesophilic 1.97 (mol H2.mol- [108]
1
obic conditions [92]. The theoretical or stoichiometric production of glucose)
Clostridium beijerinckii Mesophilic 2.81 (mol H2.mol- [106]
hydrogen is governed by the conversion of glucose to acetate as seen in 1
glucose)
Equation (1) [93]. Mesophilic 2 (mol H2.mol- [108]
1
glucose)
C6 H12 O6 + 4H2 O→2CH3 COO −
+ 2HCO−3 +
+ 4H + 4H2 (1) Clostridium Mesophilic 1.47 (mol H2.mol- [106]
1
tyrobutyricum glucose)
Thus, it can be inferred that for 1 mol of glucose consumed, 4 mol of
Mesophilic 223 (mlH2.g- [107]
H2 will be yielded. Subsequently, hydrogen conversion from glucose can 1
hexose)
be achieved by a two-step fermentation where glucose gets converted to Clostridium Thermophilic 1.5 (mol H2.mol- [108]
1
acetate and formate and both these reactions yield 2 mol H2 each as thermolacticum glucose)
Clostridium Thermophilic 1.9 (mol H2.mol- [108]
shown in Equation (2) and Equation (3) [94]. 1
thermobutyricum glucose)
C6 H12 O6 + 2H2 O→2CH3 COO− + 2HCOO− + 4H+ + 2H2 (2) Clostridium Mesophilic 1.9 (mol H2.mol- [109]
1
paraputrificum GlcNAc)
Clostridium thermocellum Thermophilic 1.6 (mol H2.mol- [110]
2HCOOH→2CO2 + 2H2 (3) 1
glucose)
Clostridium bifermentans Mesophilic 0.9 (mmol H2.g- [111]
Alternatively in butyric pathway, butyrate and hydrogen are pro­ 1
Dry Solids)
duced from glucose conversion as shown in Equation (4). In this case, 2 Clostridium sporogenes Mesophilic 32 (mmol H2.g- [112]
mol H2 is produced per mol of glucose consumed [93]. 1
Dry Cells)
Ruminococcus albus Mesophilic 2.37 (mol H2.mol- [113]
C6 H12 O6 + 2H2 O→CH3 CH2 CH 2 COO− + 2HCO3 − + 3H+ + 2H2 (4) 1
glucose)
Enterobacter cloacale Mesophilic 2.1 (mol H2.mol- [114]
The mentioned reactions are all governed by the main reaction 1
glucose)
involving conversion of glucose to pyruvate where 2 mol H2 is produced Citrobacter sporogenes Mesophilic 2.49 (mol H2.mol- [116]
through NADH regeneration [27] as shown in Equation (5). 1
glucose)
Enterobacter aerogenes Mesophilic 1.7 (mol H2.mol- [104]
C6 H12 O6 + 2NAD+ →2CH3 COCOO− + 4H+ + 2NADH (5) 1
glucose)
Mesophilic 2.5 (mol H2.mol- [117]
The Embden-Meyerhof-Parnas (EMP) metabolic pathway is respon­ 1
sucrose)
sible for most bacteria to convert glucose to ATP, NADH, or pyruvate Acetothermicus Thermophilic 4 (mol H2.mol- [118]
1
[95]. Ultimately, the yield of Hydrogen depends on the pyruvate con­ paucivorans glucose)
Spirochaeta thermophila Thermophilic 2.4 (mol H2.mol- [119]
version to acetylCoA. Pyruvate conversion to acetylCoA happens in two 1
glucose)
pathways depending on the enzymatic function of a particular micro­ Spirochaeta sporogenes Thermophilic 2.05 (mol H2.mol- [120]
organism as shown in Equation (6) and Equation (7). 1
glucose)
Acetomicrobium flavidum Thermophilic 3.1 (mol H2.mol- [118]
pyruvate + CoA + Fdox ↔ acetylCoA + CO2 + Fdred (6) 1
glucose)
Thermophilic 4 (mol H2.mol- [121]
The pyruvate pathway as shown in Equation (6) is catalysed by py­ 1
hexose)
ruvate ferredoxin oxyreductase, the ferredoxin acts as coenzyme and Caldicellulosiruptor Thermophilic 6.6 (mol H2.mol- [122]
electron acceptor [96]. In both the acetate and butyric pathway of saccharolyticus 1
sucrose)
acetylCoA metabolism, 1 mol H2 per mol ferredoxin will be produced by Thermotoga elfii Thermophilic 2.8 (mol H2.mol- [122]
1
glucose)
hydrogenase enzyme [27]. In the acetate pathway, due to the reduction
Thermophilic 12 (mol H2.mol- [123]
of NADH to NAD+ during glycolysis, one extra mol H2 is produced 1
hexose)
leading to a total of 4 mol H2 per mol glucose reduced. Likewise, in Thermotoga sporogenes Thermophilic 2.53 (mol H2.mol- [125]
butyric pathway, NADH from the glycolysis is used to oxidize acetylCoA 1
hexose)
to butyrate. Thus only 2 mol H2 is yielded per mol glucose in this path Thermotoga maritima Hyperthermophilic 4 (mol H2.mol- [127]
1
glucose)
[27]. In the presence of certain culture conditions or microorganisms Pyrococcus furiosus Hyperthermophilic 2.97 (mol H2.mol- [128]
such as clostridia, both acetate and butyrate pathways have been 1
cellobiose)
observed to be favourable simultaneously where hydrogen yield be­ Hyperthermophilic 7 (mol H2.mol- [129]
1
tween 2 and 4 mols per mol sugar can be observed [27]. maltose)
Thermotoga neapolitana Hyperthermophilic 3.24 (mol H2.mol- [130]
pyruvate + CoA ↔ acetylCoA + formate (7) 1
glucose)

4
 K.B. Sasidhar et al.
Table 2
Literature Survey of Sequential Hydrogen and Methane Including Inoculum Pre-treatment, Operation Mode, and Operating Conditions for Different Substrates.
Substrate Hydrogen Methane Yield Reference

Inoculum Inoculum Pre- Operation Operating Inoculum Operation Operating H2 CH4 H2/
treatment Conditions Conditions Hythane

Rice AnS from Swine Digester Heat treated Batch pH – 4 AnS from Swine Digester Batch pH – 6.5 125 232 0.35 [19]
(Boiled for 15 min) Temp – 37◦ C Temp – 37◦ C (ml.g- (ml.g-1VS)
1
Time – 7 days VS)
Potato AnS from Swine Digester Heat treated Batch pH – 4 AnS from Swine Digester Batch pH – 6.5 103 237 0.3 [19]
(Boiled for 15 min) Temp – 37◦ C Temp – 37◦ C (ml.g- (ml.g-1VS)
1
Time – 7 days VS)
Lettuce AnS from Swine Digester Heat treated Batch pH – 4 AnS from Swine Digester Batch pH – 6.5 35 148 0.19 [19]
(Boiled for 15 min) Temp – 37◦ C Temp – 37◦ C (ml.g- (ml.g-1VS)
1
Time – 7 days VS)
Lean Meat AnS from Swine Digester Heat treated Batch pH – 4 AnS from Swine Digester Batch pH – 6.5 0 278 0 [19]
(Boiled for 15 min) Temp – 37◦ C Temp – 37◦ C (ml.g- (ml.g-1VS)
1
Time – 7 days VS)
Peanut Oil AnS from Heat treated Batch pH – 4 AnS from Swine Digester Batch pH – 6.5 5 866 0.01 [19]
Swine Digester (Boiled for 15 min) Temp – 37◦ C Temp – 37◦ C (ml.g- (ml.g-1VS)
1
Time – 7 days VS)
Banyan Leaves AnS from Swine Digester Heat treated Batch pH – 4 AnS from Swine Digester Batch pH – 6.5 0 50 0 [19]
(Boiled for 15 min) Temp – 37◦ C Temp – 37◦ C (ml.g- (ml.g-1VS)
1
Time – 7 days VS)
Food Waste AnS from Methane Plant Culture Isolated Batch pH – 6 AnS from Methane Plant Batch pH – 8 73.64 139.24 0.35 [21]
from Sludge Temp – 35◦ C Temp – 37◦ C (ml.g- (ml.g-1VS)
1
(C. butyricum Time – 2 days VS)
dominant)
Sewage sludge AnS from Methane Plant Culture Isolated Batch pH – 6 AnS from Methane Plant Batch pH – 8 38.8 96.9 0.29 [21]
5

from Sludge Temp – 35◦ C Temp – 37◦ C (ml.g- (ml.g-1VS)

1
(C. butyricum Time – 2 days VS)
dominant)
OFMSW AnS from Biogas Plant Heat treated Batch/ pH – 5.5 AnS from Biogas Plant Batch/ pH – 7.5 43 500 0.08 [23]
(Boiled at 100 ◦ C Continuous Temp – 37◦ C Continuous Temp – 37◦ C (ml.g- (ml.g-1VS)
1
for 60 min) VS)
OFMSW AnS from Biogas Plant NA CSTR pH – 5.3 AnS from Biogas Plant CSTR pH – 8.2 NA NA 0.65 [25]
Temp – 55◦ C Temp – 55◦ C
HRT – 3 days HRT – 12 days
Fruit and AnS from WWTP pH controlled at CSTR pH – 4 UASB UASB pH – 6.6–6.8 Nil 261.4 ± 40.8 NA [42]
Vegetable 4.0 Temp – 35◦ C Temp – 35◦ C ml.g-1CODr
Waste HRT – 3 days HRT – 3.5 hrs
Palm Oil Mill Anaerobic Culture from Heat treated CSTR pH – 5.5 Anaerobic Culture from Bottom Microbial pH – 8.2 205 290 0.41 [46]
Effluent Bottom of Anaerobic (Boiled at 90 ◦ C for Temp – 55◦ C of Anaerobic Pond (alkali Electrolysis Cell Temp – 37◦ C (ml.g- (ml.g-1COD)
1
Pond 60 min) HRT – 2 days treated) (MEC) HRT – 8 days COD)

Substrate Hydrogen Methane Yield Reference

Inoculum Inoculum Pre- Operation Operating Inoculum Operation Operating H2 CH4 H2/Hythane
treatment Conditions Conditions

Steam-Exploded UASB Sludge Heat treated Batch pH – 6.5 UASB Sludge Batch pH – 7.5 20.9 39.7 0.34 [47]
Cornstalks (Boiled for 15 min) Temp – 37◦ C Temp – 37◦ C (ml.g− 1 . (ml.g− 1 .
Mixture of Time – 4 days Time – 4 days Substrate) Substrate)

Fuel 317 (2022) 123449

Microbes
augmented
Food Waste and AnS NA CSTR pH – 4.6 AnS CSTR pH – 5.3 129.1 617.6 0.17 [48]
Sewage Sludge Temp – 37◦ C Temp – 37◦ C (ml.g-1VS) (ml.g-1VS)
HRT – 5 days HRT – 10
days
(continued on next page)
 K.B. Sasidhar et al.
Table 2 (continued )
Substrate Hydrogen Methane Yield Reference

Inoculum Inoculum Pre- Operation Operating Inoculum Operation Operating H2 CH4 H2/Hythane
treatment Conditions Conditions

Macro and Micro- AnS from Farm Heat treated Batch pH – 6 AnS from Lab Batch pH – 8 97 224.3 0.30 [49]
Algal Biomass Digester (Boiled at 100◦ C Temp – 37◦ C Digesters Temp – 37◦ C (ml.g-1VS) (ml.g-1VS)
for 30 min) Time – 4 days Time – 26
Microbes days
augmented
Glucose AnS from WWTP Acid Treated Stirred Batch pH – 7.2 AnS from WWTP Batch pH – 7.2 442 530 0.45 [51]
Synthetic (At pH 3 for 24 Reactor Temp – 35◦ C Temp – 35◦ C (mmol.l− 1) (mmol.l− 1)
Medium hrs)
Garbage slurry Enriched Mix Microflora CSTR pH – 5.8–6 AnS from Internal Recirculation pH – NA 2.4 NA NA [54]
1
and Paper Culture from AS enriched by Temp – 60◦ C Thermophilic Packed Bed Reactor Temp – 55◦ C (mol.mol− .
Waste compost cultivation HRT – 1.2 Reactor HRT – 6.8 hexose)
days days
Synthetic Enriched Mix Microflora Batch pH –6 AnS from Thermophilic pH –7 2 NA 0.31 [55]
1
Garbage Slurry Culture from AS enriched by Temp – 60◦ C Thermophilic Downflow Packed Bed Temp – 55◦ C (mol.mol− . (Based on gas
compost cultivation HRT – 0.5 Reactor Reactor HRT – 0.5 hexose) production, not
days days yield)
Wheat Straw Basic Anaerobic Alkali Pre- UASB pH – 5.1 AnS from Potato UASB pH – 7 89 307 0.22 [56]
Hydrolysate Medium treatment Temp – 70◦ C Processing WWTP Temp – 55◦ C (ml.g-1VS) (ml.g-1VS)
(With HRT – 1.2 HRT – 4 days
Bicarbonate) days
Rapeseed Cake Thermophilc AnS NA CSTR pH – 6 Thermophilic AnS CSTR pH – 6 48 320 0.13 [57]
and Glycerol Manure Temp – 55◦ C Manure Temp – 55◦ C (ml.g-1VS) (ml.g-1VS)
HRT – 3 days HRT – 12
days
6

Food Waste AnS from WWTP No pre-treatment CSTR pH – 5.5 AnS from WWTP Fluid Bed Reactor pH – 7.2 205 464 0.31 [60]
Temp – 55◦ C Temp – 35◦ C (ml.g-1VS) (ml.g-1VS)
HRT – 1.3 HRT – 5 days
days
Olive Pulp AnS Heat treated CSTR pH – 4.8 AnS CSTR pH – 7.62 7.2 0.16 NA [61]
1 1
Temp – 35◦ C Temp – 35◦ C (ml.g− .TS) (ml.g− .
HRT – 28.7 HRT – 20 COD)
hrs days
Glucose AnS from Biogas Heat treated & Batch pH – 6 AnS from Biogas Batch pH – 6 2.75 2.13 0.56 [62]
1 1
Plant Chemical Treated Temp – 35◦ C Plant Temp – 35◦ C (mol.mol− . (mol.mol− .
(Boiled for 30 min) glucose) glucose)

Substrate Hydrogen Methane Yield Reference

Inoculum Inoculum Pre-treatment Operation Operating Inoculum Operation Operating H2 CH4 H2/
Conditions Conditions Hythane

Steam Exploded UASB Sludge Heat treated Batch pH – 5.5 UASB Sludge Batch pH – 7.5 228 NA NA [84]
Sugarcane (Heated at 90◦ C for 5 hrs) Temp – 37◦ C Temp – 37◦ C (ml.g-
1
Bagasse Sugar)
Food Waste AS from Aerobic Heat treated CSTR pH – 5.52 AnS from OFMSW CSTR pH – 7.43 12.6 482.1 0.03 [135]
Unit of WWTP (Boiled at 105◦ C for 30 min) Temp – 37◦ C treating digester Temp – 37◦ C (ml.g-1VS) (ml.g-1VS)
HRT – 3 days HRT – 12
days

Fuel 317 (2022) 123449

Food Waste and AS from Aerobic Heat treated CSTR pH – 5.52 AnS from OFMSW CSTR pH – 7.43 8.6 428.3 0.02 [135]
AS Unit of WWTP (Boiled at 105◦ C for 30 min) Temp – 37◦ C treating digester Temp – 37◦ C (ml.g-1VS) (ml.g-1VS)
HRT – 3 days HRT – 12
days
Glucose Synthetic No pre-treatment CSTR AnS from Primary Upflow Reactor NA NA [139]
Medium Digester
(continued on next page)
 K.B. Sasidhar et al.
Table 2 (continued )
Substrate Hydrogen Methane Yield Reference

Inoculum Inoculum Pre-treatment Operation Operating Inoculum Operation Operating H2 CH4 H2/
Conditions Conditions Hythane

AnS from pH – 5.52 pH – 6.9–7.2 1.38

1
Primary Temp – 35◦ C Temp – 28◦ C (mol.mol−
Digester HRT – 0.43 HRT – 2.7 .hexose)
days days
Sucrose AnS from STP Heat treated CSTR pH – 5.2–5.3 AnS from STP Upflow Anaerobic pH – NA 1.62 2.75 0.37 [140]
1 1
(Boiled at 110◦ C for 10 min) Temp – 35◦ C Filter Temp – 35◦ C (mol.mol− (mol.mol−
HRT – 2 days .hexose) .hexose)
Glucose AnS from No pre-treatment CSTR pH – 5.5 AnS from WWTP CSTR pH – 7 0.34 0.12 0.74 [141]
1 1
WWTP Temp – 35◦ C Temp – 35◦ C (mol.mol− (mol.mol−
HRT – 1 day HRT – NA .hexose) .hexose)
Glucose AnS from Acid pre-treatment CSTR pH – 5.5 AnS from WWTP CSTR pH – NA 2.33 0.8 0.74 [142]
1 1
WWTP (Using HCl, HNO3, H2SO4) Temp – 35◦ C Temp – 35◦ C (mol.mol− (mol.mol−
.hexose) .hexose)
Glucose and AnS from Heat treated CSTR pH – 5.5 – 6.5 AnS from WWTP CSTR pH – 7 3.21 3.63 0.47 [143]
1 1
Acetic Acid WWTP (Heated at 70◦ C for 30 min) Temp – 37◦ C Temp – 37◦ C (mol.mol− (mol.mol−
HRT – 8 hrs HRT – 10 .hexose) .hexose)
days
Glucose UASB Sludge Heat treated Sequencing Batch pH – 6 UASB Sludge Sequencing Batch pH – 7 2.12 3.09 0.41 [144]
1 1
(Boiled at 100◦ C for 2 hrs) Biofilm Reactor Temp – 28◦ C Biofilm Reactor Temp – 28◦ C (mol.mol− (mol.mol−
and Acidic treatment (88% HRT – 24 hrs HRT – 24 hrs .hexose) .hexose)
Orthophosphoric Acid for 24
hrs)
Cassava Stillage UASB Sludge No pre-treatment CSTR pH – 5.78 Thermophilic CSTR pH – 6.5 59.76 249 0.19 [145]
Temp – 60◦ C Cassava Digestate Temp – 55◦ C (ml.g-1VS) (ml.g-1VS)
7

HRT – 24 hrs HRT – 4 days

Glucose UASB Sludge Heat treated Batch pH – 7 UASB Sludge Batch pH – 7 185 261 0.41 [146]
(Boiled at 105◦ C for 4 hrs) Temp – 35◦ C Temp – 35◦ C (ml.g- (ml.g-
1 1
COD) COD)

Substrate Hydrogen Methane Yield Reference

Inoculum Inoculum Pre- Operation Operating Inoculum Operation Operating H2 CH4 H2/
treatment Conditions Conditions Hythane

Cassava Stillage and UASB Sludge No pre-treatment Batch/ pH – 6 UASB Sludge Batch/Continuous pH – 7.5 74 350 0.17 [147]
Excess Sludge Continuous Temp – 60◦ C Temp – 60◦ C (ml.g- (ml.g-
1 1
VS) VS)
Food Waste AnS from WWTP Heat treated (Boiled Leaching Bed pH – 6.5 Ans from WWTP UASB pH – 7 310 210 0.60 [148]
for 15 min) Reactor Temp – 37◦ C Temp – 37◦ C (ml.g- (ml.g-
1 1
SRT – 6 days HRT – 0.6 days VS) VS)
Food Waste AnS from Methane Heat treated Semi-Continuous pH – 5 – 5.7 AnS from Methane Biogas Sparging pH – NA 270 287 0.48 [149]
Digester (Heated at 90◦ C for Reactor Temp – 55◦ C Digester type Reactor Temp – 55◦ C (ml.g- (ml.g-
1 1
30 min) SRT – 6 days HRT – 30 days COD) COD)
Food Waste and Sewage Digested Sludge No pre-treatment CSTR pH – 7 Digested Sludge CSTR pH – 6.8–7.2 35.8 365.4 NA [150]
Sludge from WWTP Temp – 35◦ C from WWTP Temp – 35◦ C (ml.g- (ml.g-
1 1
SRT – 1.25 days SRT – 12.5 days TS) COD)
Organic Biowaste AnS from WWTP No pre-treatment CSTR pH – 5.5 AnS from WWTP CSTR pH – 7.6 52 410 0.11 [151]
Temp – 55◦ C Temp – 55◦ C (ml.g- (ml.g-

Fuel 317 (2022) 123449

1 1
HRT – 3.3 days HRT – 12.6 VS) VS)
days
Cheese Whey Powder UASB Sludge Heat treated CSTR pH – 5.9 UASB Sludge UASB pH – 7.5 – 7.8 331 228 0.59 [152]
(Boiled for 40 min) Temp – 37◦ C Temp – 25 − (ml.g- (ml.g-
1 1
HRT – 6 hrs 30◦ C COD) COD)
HRT – 6 hrs
(continued on next page)
 K.B. Sasidhar et al.
Table 2 (continued )
Substrate Hydrogen Methane Yield Reference

Inoculum Inoculum Pre- Operation Operating Inoculum Operation Operating H2 CH4 H2/
treatment Conditions Conditions Hythane

Potato Waste AnS from WWTP NA CSTR pH – 5 AnS from WWTP CSTR pH – 7 30 183 0.14 [153]
Temp – 35◦ C Temp – 35◦ C (ml.g- (ml.g-
1 1
HRT – 6 hrs HRT – 30 hrs TS) TS)
Food Waste and UASB Granules and Basal Media UASB & CSTR pH – 5.5 Inoculum from UASB & CSTR pH – 7.5 33 320 0.09 [154]
Wastewater Basal Media Addition Temp – 35◦ C Previous Digesters Temp – 35◦ C (UASB) (UASB) (UASB &
HRT – 2 days HRT – 2 days (ml.g- (ml.g- CSTR)
1 1
COD) COD)
27 260
(CSTR) (CSTR)
(ml.g- (ml.g-
1 1
COD) COD)
Water Hyacinth Leaves AnS from Marsh Heat treated Batch pH – 6 AnS from Marsh Gas Batch pH – 8 51.7 143.4 0.26 [155]
Gas Plant (Boiled for 30 min) Temp – 35◦ C Plant Temp – 35◦ C (ml.g- (ml.g-
1 1
Time – 2 days Time – 7 days VS) VS)
Aerated Synthetic food UASB Sludge Heat treated Batch pH – 6 UASB Sludge Batch pH – 6 44.4 351.69 0.11 [156]
waste (Heated at 80◦ C for Temp – 35◦ C Temp – 35◦ C (ml.g- (ml.g-
1 1
15 min) VS) VS)
Rice Residue and AnS from Biogas Heat treated Batch pH – 6.5 AnS from Biogas Batch pH – 7.5 223.1 311.5 0.41 [158]
Chlorella pyrenoidosa Plant (Boiled at 100◦ C for Temp – 35◦ C Plant Temp – 35◦ C (ml.g- (ml.g-
1 1
30 min) Time – 12–144 Time – 720 h VS) VS)
Microbes augmented h

Substrate Hydrogen Methane Yield Reference

Inoculum Inoculum Pre- Operation Operating Inoculum Operation Operating H2 CH4 H2/
8

treatment Conditions Conditions Hythane

Agave Tequilana UASB Sludge Heat treated CSTR pH – 5.5 UASB Sludge UASB pH – 7 45% 320 NA [158]
Bagasse Hydrolysate (Boiled at 104◦ C Temp – 35◦ C Temp – 23◦ - (ml.g-
1
for 24 hrs) HRT – 6 hrs 25◦ C VS)
HRT – 14 hrs
Palm-oil decanter and Self-fermentation NA CSTR Temp – 55◦ C UASB Sludge CSTR pH – 7 6 24.9 0.19 [159]
Crude glycerol Time – 4 days Temp – 37◦ C (ml.g- (ml.g-
1 1
Time – 4 days VS) VS)
Cotton Waste AnS from Maize Silage and NA Continuous pH – 6.4 AnS from Maize Silage and Continuous pH – 6.4 0.92 158.3 0.06 [160]
Manure Digester Temp – 38◦ C Manure Digester Temp – 38◦ C (ml.g- (ml.g-
1 1
VS) VS)
Boiled Potato Waste AnS from Maize Silage and NA Continuous pH – 6.23 AnS from Maize Silage and Continuous pH – 6.4 27.5 231.7 0.11 [160]
Manure Digester Temp – 38◦ C Manure Digester Temp – 38◦ C (ml.g- (ml.g-
1 1
VS) VS)
Raw Potato Waste AnS from Maize Silage and NA Continuous pH – 6.4 AnS from Maize Silage and Continuous pH – 6.4 0.8 145.8 0.01 [160]
Manure Digester Temp – 38◦ C Manure Digester Temp – 38◦ C (ml.g- (ml.g-
1 1
VS) VS)
Hydrolysed Wheat AnS from Maize Silage and NA Continuous pH – 6.23 AnS from Maize Silage and Continuous pH – 6.4 31.67 146.7 0.18 [160]
Straw Manure Digester Temp – 38◦ C Manure Digester Temp – 38◦ C (ml.g- (ml.g-
1 1
VS) VS)
OFMSW Mix of Cattle Manure and AnS NA CSTR pH – 5.2 Mix of Cattle Manure and AnS ASBR pH – 7.24 79.4 530 0.13 [161]
from WWTP Temp – 37◦ C from WWTP Temp – 37◦ C (ml.g- (ml.g-

Fuel 317 (2022) 123449

1 1
HRT – 5.3 days HRT – 8 days VS) VS)
Food Waste, Sewage AnS from WWTP Heat treated Batch pH – 5.5 AnS from WWTP Batch pH – 7 140.2 342 0.29 [163]
Sludge and Glycerol (Boiled at 100◦ C Temp – 35◦ C Temp – 35◦ C (ml.g- (ml.g-
1 1
for 30 min) Time – 36 hrs Time – 40 days VS) VS)
Cassava Starch based AnS from pig farm CSTR AnS from pig farm CSTR 0.07 [164]
Polymer
(continued on next page)
 K.B. Sasidhar et al.
Table 2 (continued )
Substrate Hydrogen Methane Yield Reference

Inoculum Inoculum Pre- Operation Operating Inoculum Operation Operating H2 CH4 H2/
treatment Conditions Conditions Hythane

Heat treated pH – 5–6 pH – 7–8 19.9 249.1

(Boiled at 100◦ C Temp – 37◦ C Temp – 37◦ C (ml.g- (ml.g-
1 1
for 30 min) HRT – 5 days HRT – 20 days VS) VS)
Food Waste Mixed AnS from Maize straw No Pre-treatment CSTR pH – 5.5 Mixed AnS from Maize straw CSTR pH – 7.6 135 510 0.21 [165]
treatment unit and AnS from Temp – 55◦ C treatment unit and AnS from Temp – 35◦ C (ml.g- (ml.g-
1 1
WWTP HRT – 5 days WWTP HRT – 9 days VS) VS)

Substrate Hydrogen Methane Yield Reference

Inoculum Inoculum Pre-treatment Operation Operating Inoculum Operation Operating H2 CH4 H2/
Conditions Conditions Hythane

Potato AS from Marsh Gas Plant Heat treated, Culture Batch pH – 6 AS from Marsh Gas Batch pH – 8 271.2 145.1 0.65 [166]
Enriched Chemically(Boiled Temp – 35◦ C Plant Temp – 35◦ C (ml.g- (ml.g-
1 1
for 30 min) TS) TS)
OFMSW AnS from Landfill Heat treated Batch pH – 5.5–6.5 AnS from AD Plant Continuous Temp – 35◦ C 41 301 0.12 [167]
Bioreactor (Boiled at 100◦ C for 60 Temp – 35◦ C HRT – 1 day (ml.g- (ml.g-
1 1
min) Time – 7 days VS) VS)
Palm Oil Mill Anaerobic Culture from Heat treated UASB pH – 5.5 Anaerobic Culture from CSTR pH – 7.5 215 320 0.40 [168]
Effluent Bottom of Anaerobic (Boiled at 90◦ C for 60 min) Temp – 55◦ C Bottom of Anaerobic (ml.g- (ml.g-
1 1
Pond HRT – 2 days Pond Temp – 37 C◦
COD) COD)
HRT – 5 days
9

Palm Oil Mill Anaerobic Culture from Heat treated Batch pH – 5.5 Anaerobic Culture from Batch pH – 7.5 205 310 0.40 [168]
Effluent Bottom of Anaerobic (Boiled at 90◦ C for 60 min) Temp – 55◦ C Bottom of Anaerobic Temp – 37◦ C (ml.g- (ml.g-
1 1
Pond Time – 5 days Pond Time –45 days COD) COD)
Defatted Milk AS from Marsh Gas Plant Heat treated Batch pH – 6 AS from Marsh Gas Batch pH – 8 186.2 209.7 0.47 [169]
Powder (Boiled for 30 min) Temp – 37◦ C Plant Temp – 37◦ C (ml.g- (ml.g-
1 1
TS) TS)
Food Waste Indigenous Microbial NA Semi-Continuous pH – NA UASB Sludge CSTR pH – NA 71 551 0.11 [170]
Culture Rotating Drum Temp – 40◦ C Temp – 40◦ C (ml.g- (ml.g-
1 1
SRT – 96–240 SRT – 16–40 VS) VS)
hrs days
Cheese Whey Indigenous Microbial NA CSTR pH – 5.2 AnS from Digester Periodic Anaerobic pH – 7.1 106 310 0.25 [171]
Culture Temp – 35◦ C Treating Municipal Baffled Reactor Temp – 35◦ C (ml.g- (ml.g-
1 1
HRT –24 hrs Sludge HRT – 44 days COD) COD)
Cheese Whey Indigenous Microbial NA CSTR pH – 5–6 AnS from WWTP CSTR pH – NA 92 136 0.40 [172]
Culture Temp – 35◦ C Temp – 35◦ C (ml.g- (ml.g-
1 1
HRT –24 hrs HRT –20 days COD) COD)
Sweet Indigenous Microbial NA CSTR pH – 4.7 – 5.5 AnS CSTR pH – 7.5 10.4 107 0.09 [173]
Sorghum Culture Temp – 35◦ C Temp – 35◦ C (ml.g- (ml.g-
1 1
Biomass HRT –4 hrs HRT –20 days VS) VS)

Note 1: AnS – Anaerobic Sludge; AS – Aerobic Sludge; CSTR – Continuous Stirred Tank Reactor; UASB – Upflow Anaerobic Sludge Blanket Reactor; WWTP – Wastewater Treatment Plant; STP- Sewage Treatment Plant; NA
– Not Available; Temp – Temperature; HRT – Hydraulic Retention Time; SRT – Solids Retention Time; OFMSW – Organic Fraction of Municipal Solid Waste; TS – Total Solids; VS – Volatile Solids; COD – Chemical Oxygen
Demand. Gas Yields represented on added concentrations of TS/VS/COD/mol.

Fuel 317 (2022) 123449

K.B. Sasidhar et al.
The pyruvate pathway as shown in Equation (7) is catalysed by py­
ruvate formate lyase and leads to the formation of formate [27,97]. This
reaction is governed by the metabolism of Enterobacter, E. coli, and a
few clostridia [27,98]. Similar to VFAs, pure cultures lead to more yield
of hydrogen when compared to mixed cultures but are expensive and
require extremely sterile operating conditions. Thus, proper culture se­
lections and proper operating conditions have to be optimized to lead
the metabolism to hydrogen production. Thus, hydrogen production can
be accomplished with a varied microbial community having a wide
palate of substrate, pH, or temperature requirements
[18,26–27,99–100]. The selection of organisms, either mixed culture,
pure culture, or genetically altered organisms, and their respective
operating conditions have to be optimized based on the process
requirements.
2.4.1. Hydrogen production through pure Culture
Many microorganisms have been observed earlier to produce
hydrogen. Researchers argue that pure cultures are more advantageous
compared to mixed cultures especially towards substrate selectivity and
relatively easier alteration of growth [27]. Higher hydrogen yields using
pure cultures have been obtained when compared to mixed cultures
[27]. The hydrogen producing organisms includes strict anaerobes such
as archaea, methanogenic bacteria, clostridia, rumen bacteria, methyl­
otrophs, facultative anaerobes including Citrobacter, Enterobacter, and
E.coli, and a few aerobic consortia such as bacillus and alcaligenes some
of which are shown in Table 1. Strict anaerobes belonging to clostridium
genus such as Clostridium butyricum [101–105,247], C. acetobutyricum
[106], C. beijerinckii [106], C. tyrobutyricum [106–107],
C. thermolacticum [108], C. paraputrificum [109], C. thermocellum [110],
C. bifermentans [111] and Clostridium sp. [112], and rumen bacteria such
as Ruminococcus albus [113], have been observed to produce hydrogen.
Likewise, facultative anaerobes such as Enterobacter cloacale [114–115],
Citrobacter sp. [116], and E. aerogenes [104,117], have also been
recorded to generate hydrogen. In addition to these species, modern
researchers have also stepped onto identifying hydrogen producing or­
ganisms at high temperatures which are thermophilic or hyperthermo­
philic in nature. Thermophiles which have been studied to produce
hydrogen include Acetothermicus paucivorans [118], Spirochaeta ther­
mophila [119], Spirochaeta sp. [120], Acetomicrobium flavidum
[118,121], Caldicellulosiruptor saccharolyticus [122], Thermotoga elfii
[122–123], Thermotoga sp. [124], Thermoanaerobacterium thermo­
saccharolyticum [125]. In addition to this, hyperthermophiles of tem­
perature range 80 ◦ C-110 ◦ C [126], such as Thermotoga maritima [127],
Pyrococcus furiosus [128–129], and Thermotoga neapolitana [130], have
also been considered for hydrogen production. Since increase in tem­
perature favour reaction kinetics, thermophiles and hyperthermophiles
have been studied to increase hydrogen yield [27]. Hyperthermophiles
have been observed to be resistant to hydrogen partial pressure [130]
and undesirable intruders.
2.4.2. Hydrogen production through mixed culture
Mixed culture come in handy when the operating conditions and
control does not need any sterilized medium. This reduces the overall
costs, and a broadens the range of substrates that can be used [131].
Hence, for a full industrial-scale operation, mixed cultures are much
more favourable than pure cultures. Mixed culture though is economi­
cally viable, has a huge disadvantage due to the dominance of non-
hydrogen producing organisms such as methanogens, lactic acid bac­
teria and homoacetogenic bacteria. Thereby, the parent sludge whether
obtained from municipal sewage [48,58,132–137], anaerobic digesters
[21–23,40,42,47,50,63,84,138–158], digestors treating animal manure
[19–20,160–175], compost reactors (from landfills, sand, or ponds),
[46,166–169], or a microbial mixed consortia extracted from specific
wastes [15,110,159,169–173], have to be pre-treated to devoid the
sludge of inhibitory organisms. For accomplishing this, three shocks are
generally adapted to inactivate the methanogens and homo-acetogens.
10
Fuel 317 (2022) 123449
Thermal pre-treatment, acid-base pre-treatment, and chemical pre-
treatment have been used by researchers to inactivate methane pro­
ducing organisms. Detailed pre-treatment employed for hydrogen pro­
ducing inoculum are shown in Table 2. The principal behind the pre-
treatment of inoculum is that upon prolonged exposure to thermal and
chemical shocks, methanogens can be removed from the system [27].
2.4.3. Hydrogen production through genetically modified microorganisms
Hydrogen production through pure culture and mixed culture could
still be limited in certain areas which could be bettered before scaling up
the process. Mixed and pure-culture organisms can function inefficiently
due to substrate complexity, hindrance through inhibitory by-products,
low resistance to hydrogen partial pressure, the presence of oxygen in
the system, and so on. In addition to this, conversion of hexose has a high
yielding limit of only 4 mol H2 per mol hexose consumed even though
hexose contains 12-gram atoms of hydrogen. Researchers have proposed
usage of genetically modified microorganisms through mutagenesis or
genetic engineering, to have advanced selective properties for solving
some of the mentioned bottlenecks. The hydrogen production through
fermentation can be enhanced by (1) augmenting or modifying genes
responsible for the overexpression of enzymes with cellulolytic activities
such as lignase, hemicellulase, and cellulase which can enhance the
glucose availability and conversion, (2) eliminating hydrogen utilizing
hydrogenase enzymes, and (3) augmenting or manipulating genes
responsible for overexpression of hydrogen producing hydrogenase en­
zymes that have themselves been modified to tolerate hydrogen
[174–175]. Modified Clostridium beijerinckii has been studied to enhance
hydrogen production by increasing the cellulolytic activity from ligno­
cellulosic biomass (case1) [175]. Similarly, E. coli has been modified to
produce hydrogen from different cellulose-based substrates including
pentose (case 1) [176]. E. coli strains with supressed succinate and
lactate dehydrogenases activities have also been used with fermentation
directed to pyruvate formate lyase [177] and formate hydrogen lyase
[178] for enhanced biohydrogen production from glucose (case 2).
E. coli strains with overexpression for formate hydrogen lyase have also
be studied (case 3) [178–181].
2.5. Subsequent production methane from hydrogen and VFA
Anaerobic Digestion is a reductive mechanism which is completed by
the production of methane, hydrogen sulphide, or ammonia [182]. In
the anaerobic degradation of complex carbohydrates, cellobiose,
dextrose, formic, acetic, butyric, pyruvic, lactic, and succinic acids are
created as transitional products, and these carbon sources are reduced to
nothing but methane and carbon dioxide [183]. The methanogenic
archaea act in the company of hydrogenase (as hydrogen bearer) to aid
the degradation [13]. The methane yielding pathway from the inter­
mediate product, is governed by Buswell formula as shown in Equation
(8) [183].
( ) ( ) ( )
a b n a b n a b
Cn H a O b + n − − H2 O→ − + CO2 + + − CH4 (8)
4 2 2 4 4 2 8 4
Sequential or two-stage (hydrogen/VFA, and methane) production
has been successful especially due to the decoupling of the two pro­
cesses. Acidic conditions in hydrogen reactors and neutral conditions in
the methane reactors have been established (as seen in Table 2). Like­
wise, thermophilic-mesophilic [46,61], mesophilic-room temperature
[139,152] and hyperthermophilic-thermophilic [54–55] decoupling has
also been studied. However, the system operations are mutually exclu­
sive of each other, there exists a degree of dependency. The studies listed
in Table 2 use the effluent from hydrogen reactors as substrate for the
methanogenic systems. The final product of the hydrogen system is
enriched with VFAs such as acetate and contain H2 and CO2.
Methane production from these systems is governed by the acete­
clastic pathway or the CO2-reducing pathway, the former pathway
K.B. Sasidhar et al.
Fig. 3. Methane production pathways from acetate and carbon dioxide. The
pathway on the left (1–4) is aceteclastic pathway and on the right (5–9) is the
CO2-reduction pathway. Both pathways lead to methane formation (10–12)
from methyl groups of either CH3-H4MPT or CH3-H4SPT. ATP- Adenosine
Triphosphate; ADP- Adenosine Diphosphate; H4MPT – tertrahy­
dromethanopterin; H4SPT – tetrahydrosarcinapterin; Fd – Ferredoxin; CoA –
coenzyme A; CoM – coenzyme M; MF – Methanofuran; F420 – coenzyme F420.
Adopted from Ferry 2011 [184].
yields methyl-tetrahydrosarcinapterin (CH3-H4SPT) and the latter yields
methyl-tetrahydromethanopterin (CH3-H4MPT) [184] as shown in
Fig. 3. The formation of methane from CH3-H4MPT or CH3-H4SPT is
governed by the transfer of the methyl groups to HS-CoM (coenzyme M)
and subsequent reduction to CH4 where HS-CoB (coenzyme B) acts as
the electron donor [184].
3. Challenges in sequential production of biohydrogen and
biomethane
3.1. Sustainability of biohydrogen and biomethane production from
different substrates
Sequential hydrogen and methane production in two-stage system has
been studied since 2003. Over 60 articles on two-stage hydrogen and
methane production dealing with different substrates, different inoculums
and their pre-treatment, and various operating conditions have been
summarized and presented by the authors in Table 2. The majority of
study use food waste or OFMSW as their substrate [19,21,23,25,42,49,54
–55,60,84,89–91,135,148–154,156,160–161,163,165–167,169–173],
followed by sugar-rich wastes [51,61–62,139–147,158,164], lignocellu­
losic and crop wastes [46,47,49,56–57,85–86,155,158,160], and other
wastes such as sewage sludge and palm oil residues [21,46,159,168].
3.1.1. Sugar-Rich wastes
Sugar rich substrates are one of the easily degradable substrates
which have been widely tested in the past for sequential hydrogen and
methane production. Around one-fourth of the studies listed in Table 2,
11
Fuel 317 (2022) 123449
use synthetic sugar-based substrates such as glucose, sucrose, etc., and
real-time wastes such as cassava stillage and olive pulp. All the consid­
ered systems using synthetic sugar medium as substrate are operated in
mesophilic stages with dominant hydrogen producing organism being
Clostridium butyricum. Contrarily, for real-time sugar-based substrates
(cassava wastes), the studies show that thermophilic treatment
increased the hydrogen yield fourfold compared to the mesophilic
treatment [145]. Hydrogen yield of minimum 1.38 mol.mol− 1 hexose
[139] and maximum 2.75 mol.mol− 1 hexose [62], and a methane yield
of minimum 0.12 mol.mol− 1 hexose [141] and a maximum 3.63 mol.
mol− 1 hexose [143] were recorded for sugar-based wastes. Due to the
easier degradability when compared to other substrates, gas yield re­
ported was also higher compared to other substrates. Even though the
sugar degradability and gas yield are high, the availability of sugar-
based biomass is very less compared to OFMSW or lignocellulosic
wastes and providing synthetic medium for high gas yield is not
economically feasible for large-scale plants. Hence, degradation with
other substrates have to be considered for large-scale operations.
3.1.2. Food waste and other organic fractions of municipal solid waste
Food waste and other OFMSW are one of the most abundant wastes
biomasses available, almost 55% of literature considered in this study
(as listed in Table 2), use food waste and OFMSW for Hythane produc­
tion. Food wastes and OFMSW are more complex than the sugar-based
substrates as the latter contains carbohydrates, lipids and proteins
which can impact both hydrolysis and methanogenesis rates. Varied
yields of hydrogen and methane have been obtained from using food
waste and OFMSW depending on substrate complexity, inoculum, and
pre-treatment of substrate and inoculum. Hydrogen yields as low a 0–5
(ml.g-1VS) have been recorded for meat and peanut oil food wastes [19],
and as high as 310 (ml.g-1VS) for food wastes treated in a leaching bed
reactor [148]. Hydrogen yield from food waste was maximum {310 (ml.
g-1VS) [148], 270 (ml.g-1COD) [149], 205 (ml.g-1VS) [60]} when an AnS
was used as seed inoculum and minimum {12.6 (ml.g-1VS) [135]}, when
AS from WWTP was used as seed inoculum. Contrarily, Xie et al., [166]
reported a hydrogen yield of 271.2 ml.g-1TS, and Song et al., 2010
[169], reported a hydrogen yield of 186.1 ml.g-1VS, upon using AS as
seed sludge for degrading potato waste and defatted milk-powder
respectively. This shows that the hydrogen production is controlled by
different parameters for different food wastes and more basic under­
standing is required. Subsequent methane yields were in the range {210
ml.g-1VS [148]} - {500 ml.g-1VS [23]}. Since the authors used accli­
matized seed sludge, and digested hydrogen reactor effluents as sub­
strates, the methane production was not affected as much as the
hydrogen production. However, Luo et al., 2011 [57,248,249], observed
a system failure in a single-stage methane production system subjected
to a high OLR of 4.5 g.VS.l− 1.d-1, whereas a two-stage system yielded
10% higher energy retrieval for the same organic-loading rate.
3.1.3. Lignocellulosic substrates and crop residues
Lignocellulosic biomass is one of the widely available solid wastes
especially in countries such as India and China. Lignin-based substrates
have a rigid three-dimensional structure thus limiting hydrolysis and
hence, the entire AD process. Though reducing lignocellulosic wastes
and crop residues to biofuels is a growing topic in research, only about
16% of the collected literature use such substrates for Hythane pro­
duction. Researchers have used multiple pre-treatments to break the
lignin structure. Pre-Hydrolysed wastes such as hydrolysed wheat straw
and palm oil effluent yielded more Hythane than its non-hydrolysed
counterparts. Hydrolysed wheat straw yielded 89 (ml.g-1VS) of
hydrogen [56] and 31.67 (ml.g-1VS) of hydrogen [160] in two different
studies. Similarly, palm oil effluent yielded 205 (ml.g-1COD) [46] and
215 (ml.g-1COD) [168] of hydrogen. Whereas palm oil decanter and
crude glycerol yielded only 6 (ml.g-1VS) [159] when not hydrolysed.
Substrate pre-treatment such as steam explosion [47,84] or co-digestion
with glycerol [57,159] have also been employed to increase hydrogen
K.B. Sasidhar et al.
Table 3
Acetogenic Reaction Pathways and Their Corresponding Gibb’s Free Energy.
Pathways Gibb’s Energy ΔG◦ Reference
− 1
Lactate to Acetate − 4.2 kJ.mol [190]
Ethanol to Acetate +9.6 kJ.mol− 1 [190]
Butyrate to Acetate +48.1 kJ.mol− 1 [190]
Propionate to Acetate +76.1 kJ.mol− 1 [190]
Methanol to Acetate − 2.9 kJ.mol− 1 [190]
Hydrogen-CO2 to Acetate − 70.3 kJ.mol− 1 [190]
Palmitate to Acetate +345.6 kJ.mol− 1 [190]
yield. Nevertheless, the non-hydrolysed biomasses yield very little
hydrogen when compared to the other wastes used. A meagre hydrogen
yield of 0.92 (ml.g-1VS) was detected for cotton residue [160], and a
maximum hydrogen yield of 51.7 (ml.g-1VS) was observed for water
hyacinth leaves [155] in the non-pre-treated and non-hydrolysed sub­
strates. Subsequently, methane yield varied from 107 ml.g-1VS [173] –
320 ml.g-1VS [57], here as well, it can be noted that the yield was not as
fluctuating as hydrogen production, may be due to the fact that the
substrate was already the digested effluent from acidogenic stage.
3.1.4. Other wastes as substrates
Other organic substrates such as sewage sludge [21], garbage slurry
and paper waste [54] and synthetic garbage slurry [55] were also tested
to produce hydrogen and methane. Thermophilic conditions favoured
hydrogen production in the treatment of garbage slurry [54–55]
whereas, mesophilic conditions were used in treatment of sewage sludge
[21]. Cheng et al., 2016 [21] noted a hydrogen and methane yield of
38.8 (ml.g-1VS), and 96.9 (ml.g-1VS) respectively, while treating sewage
sludge. Ueno et al., [54–55] observed a hydrogen yield of 2.4 (mol.mol-
1
hexose) [54] and 2 (mol.mol-1hexose) [55] while treating synthetic
garbage slurry and paper wastes.
As it can be deduced from Table 2, the biohydrogen and biomethane
production depends on the carbon content in the substrates (wastes)
used. Also, with use of different treatment processes, the production and
yield of Hythane can vary [26]. The aforementioned literatures use fixed
substrate(s), reactor types, and operational parameters for the sequen­
tial hydrogen and methane production. Moreover, the studies have only
conducted laboratory-based experiments and subsequent recommen­
dations for a scale-up has not been provided. Thus, is it necessary to
generalise the operational parameters for scaling-up the process.
3.2. Process parameter optimization
3.2.1. pH
pH control is one of the major requirements for hydrogen and sub­
sequent methane production. As far as hydrogen production is con­
cerned, pH regulates the ion concentration, the growth rate of
microorganisms responsible for hydrogen production, the proton motive
force (PMF), protein synthesis, and membrane potential [185]. pH
controls the metabolism of hydrogen producing organisms [186] and
thus is helps in controlling methanogenic activity. A pH of 5.5–6.8 is
optimum for biohydrogen production as pH out of this range affects the
activity of hydrogenase [187–188]. Almost 82% of the studies depicted
in Table 2, operate in the pH range of 5.5–6.8. Glucose degradation was
studied under a pH of 7.2 [48,146]. Acidic conditions of pH 4 were used
during the degradation of food waste and other OFMSW [19,42,61] for
biohydrogen production, even though a pH of 4.5 was recorded to
inhibit hydrogen production [189]. Methane producing archaea operate
at temperatures from 6.5 to 8.5. Almost 89% of the literature listed in
Table 2, operate in this range. However, acidic intermediates from the
hydrogen system, and inefficient degradation of organic matter, results
in a lower pH, which further dissociates more acidic ions, which in turn
reduces the pH. This continues in a vicious cycle and ultimately inhibits
the methanogenic stage [190]. Thereby pH monitoring and control is
mandatory in methanogenic stage as well. Though manageable in
12
Fuel 317 (2022) 123449
bench-scale units, a pH control in a large-scale plant is tricky by means
of external acid addition. Thereby, higher OLR and lower retention
times could be used for maintaining acidic environment in hydrogen
stage without microbial washout. Subsequently, increasing the retention
time in the methanogenic phase, could reduce the load and increase pH
to required conditions.
3.2.2. Temperature
Temperature plays a vital role in controlling the methanogenic
archaea and promoting the hydrogen producing organisms. Almost 55%
of the literature surveyed by the authors use heat shock as a pre-
treatment for the hydrogen producing sludge. But there is not much
clarity on the effect of temperature on the hydrogen and methane pro­
ducing organisms. Hydrogen producing organisms operate in room
temperature (15 ◦ C-27 ◦ C) [144], mesophilic (27 ◦ C-40 ◦ C), thermo­
philic (40 ◦ C-80 ◦ C), and hyperthermophilic (greater than80 ◦ C) tem­
peratures (as listed in Table 1 and Table 2), whereas methanogens are
sensitive to temperatures beyond 55 ◦ C [191]. Methanogens are more
stable in mesophilic than in thermophilic conditions. Hence, the
methane reactor can be operated in room temperature or mesophilic
temperature as done by almost 72% of the researchers from the surveyed
literature. Thermophilic and hyperthermophilic conditions for hydrogen
enhancement were noted mainly due to the suppression of methanogens
and availability of hydrogen producing organisms at high temperatures.
Nonetheless, the energy yield from hydrogen production in large-scale
plants need to be greater than the energy supplied in order to have a
net positive energy. Thereby, a centre-point has to be derived between
the energy supplied for thermophilic operation, and the energy obtained
from the process, for any given substrate.
3.2.3. Retention time
Retention time is a vital parameter which affects the microbial
metabolism in the reactors. The retention time increases the contact
time between the substrate and the microbial organisms which aid the
degradation. Retention time can be classified into HRT and SRT the
former is a function of reactor volume, and the latter is a function of the
predominant microbial community in the reactor. VFA production is
governed by HRT [192] and a higher HRT provides sufficient time for
the acidogenic bacteria to hydrolyse and solubilise the organics, which
in-turn results in higher VFA production [193]. Though higher HRT is
required for VFA production, too much HRT will result in VFA accu­
mulation which is detrimental to both hydrogen and methane produc­
tion. A study conducted on the fermentation of food waste demonstrated
that VFA concentrations increased with increase in HRT from 96 to 192
h, but no further increase was noted when HRT increased to 288 h [194].
Contrary to VFA production, biohydrogen production decreases with
increased HRT. It was observed in recent research that biohydrogen
yield increased when the HRT was decreased from 20 to 12 h [195].
Longer HRT can favour the acidogenic to methanogenic pathway
resulting in lesser biohydrogen production. Factors influencing HRT are
inoculum and substrate type, pH and loading rate which holds true for
Table 4
Operation and Maintenance Cost for Purification Technologies.
Treatment Purification Investment Cost for Annual Upgradation
Technology Cost (€/N.m3 150–2000 m3/h Cost for 150–2000 m3/
gas) flow rate plants (in h flow rate plants (in
Million €) 1000 €/year)
Water 0.13 [219] 0.5–2.5 [228] 10–175 [228]
Scrubbing
Chemical 0.17 [219] 0.5–2.8 [228] 50–500 [228]
Treatment
PSA 0.40 [219] NA NA
Membrane 0.12 [219] NA NA
Separation
Cryogenic 0.44 [219] 0.5–2.5 [228] 60–875 [228]
Separation
K.B. Sasidhar et al.
Table 5
Yield and Cost Associated with Different Hydrogen Production Methods.
Treatment Technology Hydrogen Yield Hydrogen Production Reference
Cost (USD.kg− 1)
Steam Methane 74% 2.1 [231]
Reforming
Biomass Gasification 46% 2 [231]
Electrolysis 75% 2.3 [231]
Water Splitting 20% 3.7 [231]
Dark Fermentation 4 mol H2/mol NA [231]
(Biohydrogen) glucose
both hydrogen and methane producing systems. Since methanogens
have slow growth rate compared to acidogens, the decoupled SRT of the
system should be long enough for the methanogens to consume the VFA
[39]. Since the acidogens grow faster compared to the methanogens, a
shorter SRT is sufficient to perform hydrolysis. The SRT of hydrogen
reactor is about 2–5 days whereas the methane reactor has an SRT of
about 30–90 days.
3.2.4. Organic loading rate
Organic loading rate as the name suggests is defined as the rate of
substrates fed into the system. This depends on the substrate composi­
tion, and reactor configuration. The authors did not find any literature
on the explicit correlation between the yield of hydrogen and OLR.
Nevertheless, the trend of acid and hydrogen production with change in
OLR can be foreseen. Lactic acid concentration increased from 29 to 36
g.l− 1, when the OLR was raised from 14 to 18 gTS.l− 1.d-1 [196] and with
rise in OLR to 22 gTS.l− 1.d-1, the acid concentration dropped to 22 g.l− 1,
indicating a reduced rate of hydrolysis with an increase in OLR [190].
Biohydrogen production also follows a similar trend to VFAs. It was
observed that with an initial increase of OLR from 4 to 22 gCOD.l− 1.d-1,
the biohydrogen production rate increased to 0.196 mol.l− 1.d-1. At a
higher loading rate of 30 gCOD.l− 1.d-1, the biohydrogen production rate
dropped to 0.196 mol.l− 1.d-1 [197]. Similarly, at higher organic loading
rates, hydrogen production is inhibited by the production and saturation
of VFAs in the system. In line with this theory, biohydrogen production
rate decreased as the OLR of glucose increased beyond 2 g.l− 1 [198].
Similarly, the methane reactors also fail at very high OLRs when the rate
of hydrolysis is limited [39]. The high OLR reduces increases the rate of
hydrolysis initially which leads to a drop in pH due to the accumulation
of propionic acid. The limitation of OLR in a methanogenic system can
thus be avoided by using a two-stage system as it reduces the chance of
acidification [199–201]. OLR and HRT are two parameters which go
hand-in-hand, defining value for one parameter requires value assign­
ment for the other. Thus, each of it can be controlled by carefully
altering the other as per requirement.
3.2.5. Partial pressure of hydrogen
Both hydrogen and methane production depend on the partial
pressure of hydrogen in liquid phase. The increase in hydrogen con­
centration in the liquid phase favours production of other intermediates
such as ethanol, acetone, butanol, lactate, etc., [202] thereby reducing
biohydrogen concentration in the gas phase. It can be seen from Table 3,
that out of all the hydrogen production pathways from VFA, only the
lactate pathway is spontaneous. Only when hydrogen partial pressure
reduces (below 10-4 ATM), these reactions become exergonic to produce
hydrogen [190].
3.3. Bottlenecks due to process inhibitors
3.3.1. Competitive organisms
Methanogenesis happens in two steps namely, acetotrophic (Equa­
tion (9)) and hydrogenotrophic pathways (Equation (10)). The hydro­
genotrophic methanogens utilize CO2 and H2 to yield methane. H2 can
also combine with the bicarbonate generated in the acetotrophic
13
Fuel 317 (2022) 123449
pathway (Equation (9)) to yield acetate this is known as homo-
acetogenesis. Both these pathways though productive for methane
generation, highly inhibits the hydrogen production and yield.
CH3 COO− + H2 O→CH4 + HCO−3 (9)
CO2 + 4H2 →CH4 + 2H2 O (10)
CO2 is reduced to methane by using hydrogen as the electron donor.
In addition to this, hydrogen can be consumed as ana electron donor by
alcohol producing organisms. NADH is consumed by the propionate
producing Clostridium propionicum and Clostridium homopropionicum
during lactate degradation [203–204].
Sulphate reducing bacteria (SRB) are troublesome to both hydrogen
and methane producing organisms. SRB use the different carbon sources
as electron donors to reduce sulphate into sulphide. Just like the
methanogens, SRB consume hydrogen as electron donor to perform
reduction [205]. SRB outcompete methanogens for H2 uptake [205] and
thereby reduces the yield of both hydrogen and methane. In addition to
this, the reduced compound sulphide, is detrimental to methanogens
and to the SRB as well. Thereby, SRB present in hydrogen or methane
reactor reduces the Hythane yield drastically.
3.3.2. Nutrient composition
Nitrogen, phosphorus, and sulphur are some of the most essential
macro-nutrients required for the degradation of organic compounds.
Proteins, nucleic acids, and enzymes contain majority of nitrogen.
However, higher concentrations of nitrogen results in the formation of
free ammonia which are detrimental to both hydrogen-producing or­
ganisms and methanogens. Optimum nitrogen concentration for
hydrogen production was said to be 0.1 gN.l− 1 [206]. Likewise, phos­
phate provides the necessary buffer for the fermentation and subsequent
anaerobic digestion. It has a major role in glycolysis and energy transfer
from ATP, NADH and FAD+ [207]. However, methanogens are inhibi­
ted by phosphorus at high concentrations. A study revealed that upon
increased phosphorus concentration, VFA production increased to
500%, and bacterial growth rate reduced by 35%, accompanied by the
formation of filamentous microorganisms. The system did not respond
to reduction in phosphorus concentration, indicating an irreversible
damage of biomass [208]. Sulphur is a necessary macronutrient for
anaerobic digestion and the optimum level of sulphur in the system
should be in the range of 1–25 mgS.l− 1 [205]. High concentrations of
sulphate have found to cause several operational failures including
propionate-degrading ability inhibition [209].
Similar to the macro-nutrients, the micro-nutrients are extremely
crucial for the bacterial growth and for boosting the enzymatic activities
[210]. Yet, higher concentration of these nutrients is also repressive to
the hydrogen and methane production. Magnesium aids in the build-up
of proteins and in the enzymatic functions for the anaerobic digestion
[210]. However, a concentration of 20 mg.l− 1 was discovered to be
inhibitory to hydrogen generation [211] and a concentration of 400 mg.
l− 1 stopped the methanogenic growth rate in a Methanosarcinae-domi­
nated UASB reactor [212]. Similarly, an Mg2+ concentration beyond
600 mg.l− 1 was found to be repressive to biohydrogen production [213].
Other nutrients such as Na2+, Ca2+, and Fe2+, which help in bacterial
growth, cell synthesis, and cell retention [26], have shown detrimental
properties at higher concentrations. The inhibitory concentrations for
hydrogen production, have been recorded as 2000 mg.l− 1, 100 mg.l− 1,
and 100 mg.l− 1, of Na2+, Ca2+, and Fe2+ ions respectively [203,213].
The specific methanogenic activity of the inoculum treating starch
reduced by 50% upon concentrations of other heavy metals such as zinc
(105 mg.l− 1), nickel (120 mg.l− 1), copper (180 mg.l− 1), cadmium
(greater than400 mg.l− 1) and chromium (310 mg.l− 1) [214].
3.3.3. Aromatics and other organics from Pre-treatment processes
Phenolic products and furan derivatives used in the pre-treatment of
substrates have been found to be inhibitory to hydrogen production
K.B. Sasidhar et al.
[203]. Whereas organic compounds such as benzenes, phenols, alcohols,
aliphatic compounds, and aldehydes, which are alkyl, halogenated or N-
substituted, have been found to be inhibitory to the entire AD process
[205]. Chlorophenols disrupt the proton gradient in cells and hinders
with the energy transduction of cells [205]. A pentachlorophenol con­
centration as stunted as 0.5–10 mg.l− 1 has been recorded to cause in­
hibition to both acidogens and methanogens [205]. On the other hand, a
furfural concentration of 2–4 mg.l− 1 has been found to decrease
hydrogen production from 29% to 63% [215]. Research shows that
higher biomass concentration supposedly provides higher process sta­
bility against such toxic shocks form organics [216].
3.3.4. Alcohol and VFA composition
As discussed earlier, AD of organics yields VFA and alcohols as in­
termediate products before the formation of hydrogen or methane.
Long-chain fatty acids (LCFAs) are produced in the acidogenic phase of
the AD. Since the growth-rate of methanogenic bacteria are very low
when compared to that of acidogenic bacteria, there exists possibility of
VFA accumulation which decreases the pH [26,39,190]. This discrep­
ancy in pH results in the growth-hindrance of hydrogen producing or­
ganisms [26]. Since the cell walls of the LCFA resemble that of a gram-
positive organism, it inhibits the gram-negative methanogens [205]. In
addition to this, sorption of LCFAs onto the biomass results in sludge
flotation and hence, subsequent washout of the sludge [205]. By varying
the operational parameters such as the OLR or HRT, this issue can be
leapfrogged to a major extent.
3.3.5. Other In-situ inhibitors
In addition to the above-mentioned inhibitors, certain in-process or
in-situ inhibitors also affect the hydrogen and methane production.
These are mostly due to the compounds present in the substrate itself
such as lignin and ammonia which poses harm to the fermentation/AD
process. In addition to lowering the rate of hydrolysis, and subsequently
the gas productions, Lignin-rich wastes are capable of forming de­
rivatives with aldehyde groups which acts as toxic compounds to
methanogens [205]. Ammonia on the other hand, which are produced
due to the reduction of proteins and other nitrogen-rich wastes such as
urea, are found to be beneficial to methanogens at concentrations below
200 mg.l− 1 [217]. However, an ammoniacal nitrogen concentration of
1.7 to 14 g.l− 1 have been proclaimed to be detrimental to both hydrogen
producing and methane producing organisms [205]. Passive release of
ammonia into the microbial cells, reduces the cell stability due to the
imbalance in sodium and potassium exchange, which ultimately results
in pH reduction [26] and causes reduced Hythane production and yield.
4. Challenges in gas purification and storage
4.1. Challenges in hydrogen and methane purification
Refining hydrogen and methane from the biogas mixture is a major
necessity, as only the pure gases can be used for energy generation.
Methane and hydrogen separation/purification have been achieved
through various operations. However, the authors did not find purifi­
cation techniques specific to biohydrogen from dark fermentation.
4.1.1. Purification through water or solvent-scrubbing
Water or solvent-scrubbing in methane reactors, work in the same
principal that both CO2 and H2S, are more soluble in the solvents than
methane. Similarly, hydrogen also is insoluble in water and scrubbing-
solvent. Thus, this technique could also be observed for hydrogen.
Selexol is the commonly used industrial solvent for the purification of
methane from CO2 and H2S. When compared to hydrogen, CH4, CO2,
and H2S solubilizes 5, 76, and 670 times in selexol respectively [218].
Selexol removes water and halogenated hydrocarbons in addition to the
gaseous impurities. It requires lesser solvent and pumping pressure, for
operation [219]. The major disadvantage of using selexol is the
14
Fuel 317 (2022) 123449
formation of elemental sulphur hence, gas stripping has to be carried out
with inert gas [219].
4.1.2. Chemical absorption
Absorption to chemicals is based on the principal that reversible
bonds between the solute and solvent can be formed [219]. Chemical
absorption can be carried out at low pressure and offers high reaction
efficiency with 100% H2S removal when compared to water-scrubbing
[219]. These advantages make this treatment feasible for industrial-
scale treatment. The main drawback of chemical absorption is the in­
vestment in procuring the chemicals and the necessity to treat the
chemical wastes. In practice, alkaline salt solutions such as NaOH, KOH,
Ca(OH)2, or aqueous alkanolamine solutions such as mono-, di-, or tri-
ethanolamine are used as solvents [219].
4.1.3. Pressure-swing-adsorption (PSA)
PSA uses gases under high pressure to separate a component of gas
from the mixture owing to its molecular characteristics and its affinity to
an adsorbent material [219]. PSA uses zeolites or activated carbon
[220–221] as adsorbents at near-ambient temperatures [221] at high
pressure and consequently shifts to low pressure to desorb the gas [220].
PSA has been used to obtain hydrogen purity of 99.999% and a recovery
of 70–90% at near-ambient temperature [221–222]. Similarly, methane
recovery of 97% has also been observed using PSA [219]. Main disad­
vantage of PSA is that an H2S removal step is required prior PSA, and the
trail gas from the technology also needs further post-treatment [219].
4.1.4. Membrane separation
Membrane separation of gases have been employed commercially for
purification of gases such as methane and hydrogen from biogas.
Membranes such as metallic membranes, polymer membranes, carbon-
based membranes, and metal organic frameworks have been used to
purify hydrogen from different gas mixtures [222]. However, bio­
hydrogen has been effectively separated from the fermentation-gas by
using a Gas Separation Membrane Bioreactor (GSMBR) where the
fermentation-gas mixture containing 80%:20% of CO2:H2, was purified
to 90%:10% of H2:CO2 in the retentate [223]. Likewise, an increase of
10–15% in the hydrogen production was observed on using a silica
membrane [224]. These promising studies however have been carried
out in lab-scale reactors. The real-time gas mixture contains more trace
elements such as H2S, in addition to the H2, CO2 and N2 in the gas
mixture. Prolonged exposure to such elements will be a key factor in
adjudging the membrane durability and stability which will help in
deciding the feasibility in gas separation. Similarly to hydrogen purifi­
cation, methane purification has also been widely carried out by the use
of membranes using GSMBR techniques and gas–liquid-adsorption
techniques. However, traces of methane could be observed in the waste
gas-streams [219]. Though the process is compact, and the energy
required for operation is less, the gas yield and purity are low, and there
is a possibility of membrane fouling, which increases the cost of oper­
ation [219].
4.1.5. Cryogenic purification
Cryogenic purification works on the principle that different gases
liquefy at different temperature. A high purity of hydrogen [222] and
methane [219] can be obtained by cryogenic purification. The main
disadvantage of using a cryogenic system is the high capital and oper­
ational costs required for the instalment and running of energy intense
equipment such as compressors and heat exchangers [219]. Impurities
such as CO2 and H2O have to be removed prior the process to avoid
blockage of equipment [222].
4.2. Challenges in hydrogen storage
Hydrogen storage has been established in six pathways namely,
compression in high pressure cylinders, liquefaction in cryogenic tanks,
K.B. Sasidhar et al.
adsorption or physisorption, using complex compounds such as metallic
hydrides, and forming complex with metals and water [225–226].
Hydrogen purification and storage go hand-in-hand as most technolo­
gies for purification, couples with storage. Compressing hydrogen at 20
MPa is the simplest yet costliest method for storing hydrogen in tanks
[225–226]. Hydrogen can be compressed with a standard piston-type
mechanism with increased pressure. Increasing pressure, increases the
volumetric density, but decreases the gravimetric density [225]. Since
hydrogen has a meagre density (0.084 kg.m− 3), exorbitant pressure is
needed for compression, which increases the energy cost [225–226]
thereby making this technology inapt for industrial-scale storage.
Hydrogen purified from cryogenic process can be stored as liquid
hydrogen in cryogenic tanks. Since hydrogen has a critical temperature
of 33 K, it is generally stored in open systems with a pressure of 104 bar
[225]. The major disadvantage is the cost of purification, and the precise
thermal sealing so that the liquid hydrogen does not boil off [225].
Physisorption of hydrogen onto a solid adsorbent layer (such as zeolite)
happens at is said to increase with increase in temperature and pressure
[225]. At a temperature of 573 K and 10 MPa pressure, 0.08% mass of
hydrogen was desorbed [225]. Metal hydrides and complex hydrides are
recent technological advancements which are of great interest in the
research field for hydrogen storage. These have the potential to safely
and compactly store large quantities of hydrogen [225]. These work
under ambient pressure and temperature where the gravimetric
hydrogen density is less than 3% mass [225]. However, the stability,
reversibility, and sorption kinetics of complex hydrides have to be
studied. A tank containing 4 kg hydrogen in a metal hydride is about
300 kg in weight and has a volume of 60 l [225]. On using it for auto­
motive applications, the body of the vehicle can be modified as metal
hydride to store hydrogen, thereby reducing the weight of an additional
storage component.
4.3. Challenges associated with production cost
The cost of production of hydrogen from electrolysis, gasification or
other non-biological systems has not been very industry-friendly due to
the intricacies in the production and storage of hydrogen. Almost 80% of
the world’s hydrogen, is produced from the conversion of natural gas
and oil [26]. These processes usually include the cost of carbon-capture
and storage from the fossil fuel used for hydrogen production in addition
to the actual cost of production thus making it more expensive. On the
other hand, the process of biohydrogen from dark fermentation of
organic waste and wastewater does not produce any carbon intensive
exhausts except CO2. Studies show that the produced CO2 can be
recirculated back into the reactor as a buffer to enhance hydrogen
production [227]. The cost of hydrogen production through steam
reformation of methane, gasification of coal and biomass, or electrolysis
have been well defined over extensive research [26]. Nevertheless, a
defined cost for biohydrogen production has not been recorded yet. This
maybe related largely to the fact that different process and different
substrates yield different quantities of biohydrogen and biomethane,
and hence a single production cost cannot be defined. However, the
biohydrogen production, purification, and bottling, follows the same
line as that of biomethane. The main cost associated with biohydrogen
and biomethane production is the capital cost involved in the con­
struction of reactors and its mechanical components. The anaerobic
tanks maybe CSTR, UASB, Packed Bed Reactors, Photo-Bioreactors, or
Membrane Bioreactors some of which have been used in a few research
works stated in Table 2.
Purification costs associated with biogas, can also be applied to
biohydrogen as discussed earlier, and the respective costs associated
with each process are tabulated in Table 4. The operational costs
involved in reactor function are split as 0.08€/kWh for electricity,
0.046€/kWh for heat, 2€/ N.m3 gas [228]. The price associated with
biomethane (0.6 £/kg gas, and 0.059 £/kWh) was observed to be the
least among diesel (1.01 £/kg gas, and 0.9 £/kWh), biodiesel (0.8 £/kg
15
Fuel 317 (2022) 123449
gas, and 0.078 £/kWh), bioethanol (0.65 £/kg gas, and 1.1 £/kWh), and
CNG (0.95 £/kg gas, and 0.065 £/kWh) [229]. The cost of Hythane fuel
was noted to increase by 15% when compared to biomethane on using a
20:80% (H2:CH4 volumetric) mixture [230]. Even after the increased
price, Hythane costs (0.69 £/kg gas, and 0.067 £/kWh) would be lesser
than diesel, biodiesel, bioethanol, and CNG. It is to be noted that the
15% high price was obtained by combining an industrially produced
hydrogen and methane and the production value of biohydrogen should
be considered for a better comparison. Table 5 summarises the cost and
system yield associated with various hydrogen production technologies.
4.4. Reactor configurations for hythane production
Production of biohythane in large-scale industrial plants will be
considered in coming days, as Hythane is a lean fuel with minimal
emission characteristics and complies with green-energy standards. In­
dustries such as Eden Innovations Limited [30] have already cashed in
on the idea. The research works covered in this study already strongly
indicate that the usage of a two-stage system is advantageous over a
single scale hydrogen or methane production. Thus, the best digester
design would be is to first separate the acidogenic reactor and the
methanogenic reactors. The digestors treating the wastes can be classed
into two types centred on the TS concentration of the system. i) A system
capable of treating more than 15% TS, is classified as a solid-waste
digester and the corresponding mechanism is solid-waste digestion
[232–233]. The solid-waste digestion is said to be advantageous than the
wet-stage digestion as it requires less reactor volume, lesser energy, and
lesser feedstock volume, and has higher biological decomposition rates
when compared to high-rate wastewater treatment systems [234]. On
the other hand, certain disadvantages have also been recorded in the
usage of solid-waste digestion. Few of those include need of high HRT,
and accumulation of end products, leading to methanogenic inhibition.
The solid-waste digestion can be effected by CSTR or Leaching-Bed
Reactor where the high solids concentration can be efficiently fer­
mented to biohydrogen [17]. ii) The next set of digestion unit can be
classified as wet anaerobic digestion, where the total solids concentra­
tion can be ranged from 2% to 12% [17]. In such system, the first-stage
hydrogen reactor or acidogenic reactor can be a CSTR. For systems
treating wastes with less than 2% TS a high-rate UASB reactor could be
used [17]. Despite the TS in the first-stage (hydrogen-production), the
second-stage (methane-production) can be effected using a CSTR,
Anaerobic Membrane Bioreactor (AnMBR), or a high-rate UASB reactor
[17]. Since the substrate of the methanogenic system would be the
digested effluent of the hydrogenic system, a wet anaerobic digestion
unit is sufficient for digestion. UASB reactors and AnMBR are energy
intensive than a conventional CSTR, hence, either of these units can be
used based on the energy availability, and the effluent standards
required to be met.
5. Potential of hythane as renewable alternative fuel
5.1. Fuel properties and potential of hythane in commercial use
Purification and storage of biohydrogen and biomethane is the key
factor to biohythane blending. Once impurities such as CO2 and H2S
have been removed from the system, a H2/(H2 + CH4) blend of
0.01–0.74 can be obtained as observed in Table 2. However, this value is
subjected to change depending on the operational parameters in large-
scale digesters. Moreover, the two-stage fermentation can be used to
obtain hydrogen and methane in separate systems as separate fuel, thus
in addition to a blended Hythane, hydrogen and methane can also be
used as individual resources if necessary. Hydrogen has an energy
density of 145 MJ.kg− 1 when compared to methane which has only 55.6
MJ.kg− 1 at ambient pressure. LPG (50 MJ.kg− 1), Petrol (46 MJ.kg− 1),
and diesel (45 MJ.kg− 1) have almost only one-third of the energy density
as of hydrogen. This indicates that hydrogen produces almost three
K.B. Sasidhar et al.
times more energy than the conventional fuels at ambient pressure.
Theoretically, Hythane produced from the two-stage digestion of
biomass and manure can provide four-times the energy consumed from
natural gas in a country such as China [17].
In addition to this, Hythane has been tested successfully since 1980s
[33] as a fuel for automotive engines in lab-scale [31–34,235–239].
Hydrogen blending to methane is attractive because of three main rea­
sons. The first being its high energy density compared to that of
methane. The second being the energy required for ignition; hydrogen
needs only one twelfth of the energy required to ignite methane. The
minimum ignition energy needed to spark hydrogen is 0.017 mJ and
that of methane is 0.2 mJ [17]. The third being the flammable limit in
air; hydrogen has a limit of 4–75% (volume/volume) when compared to
5–15% (volume/volume) for methane [17] which increases the instant
and sustained combustion. Thermal efficiency, and performance of
Hythane in an AVL research engine was observed to be in between the
efficiency from using hydrogen and methane [33]. Addition of hydrogen
to methane reduced the fuel consumption (of methane by 22%) and
pollutant emissions [32]. Contrarily, using a CFR engine at 700–900
rpm, a hydrogen to methane mix of 60%:40% by volume, decreased the
thermal efficiency by 2%, and reduced the break-specific fuel con­
sumption (BSFC) to 14% [31]. On adding hydrogen to methane, the
second-law efficiency was noticed to increase as the irreversibility
during combustion decreased [235]. At low and medium engine loads,
the break-thermal efficiency raised with a rise in hydrogen portion, and
remained constant at higher engine loads, and a 20% hydrogen addition
to methane (by volume) produced optimum results [236]. Similarly,
under 20% H2 addition, the BSFC was observed to decrease, and the
overall efficiency was observed to increase [230].
5.2. Effect of hythane on environment
Combining hydrogen with methane has significantly increased the
engine performances in terms of efficiency and fuel consumption. The
corresponding combustion gases which are exhausted were also noted to
be lesser than that of the gas mixtures emanating from engines using
only methane (natural gas). A 60:40% ratio of hydrogen to methane (by
volume), resulted in a CO2 reduction to 26%, CO reduction to 40%, and
unburnt hydrocarbon to 60%, whereas NOx (as NO) increased by 30%
[31]. A reduction of 23% CO2 and 73% NO was observed at 40%
hydrogen replacement addition (by volume) whereas CO, and hydro­
carbons remained unchanged [32]. A 20% hydrogen replacement in an
SI engine, resulted in a significant reduction of unburnt hydrocarbons
than using CNG [34]. In another study, with the same 20% volumetric
addition of hydrogen, a significant reduction in unburnt hydrocarbon
and CO2 was observed, whereas the NOx emissions increased with high
engine load [236]. A hydrogen replacement of more than 20% resulted
in higher NOx emissions at high engine loads [236]. Similarly, a 30%
hydrogen (by volume) blend produced significantly lower NOx emis­
sions than CNG [237]. Correspondingly, an engine operating with full
load conditions at 1300, 1500, 1800, and 2200 RPM, a volumetric
hydrogen blend of 29%, effects in substantial reduction in hydrocarbon
and CO2 production [238]. The higher NOx produced was also
controlled by increasing the excess air ratio (λ) from 1.4 to 2, where the
emissions were no higher than the ones emitted by pure CNG [237]. In
another study, Hythane of 15% hydrogen blend (by volume) exhibited
13–32% reduction in NOx and 5–13% reduction in hydrocarbons [239].
A hydrogen to methane ratio of 20:80% (by volume), has been found to
effectively reduce hydrocarbon, CO2, and CO emissions [230]. Likewise,
a study which analysed the greenhouse-gas emissions from the tail-pipe
of vehicle observed a significant reduction of methane from 10 mg.l− 1 to
6 mg.l− 1 on using a 20% H2 volumetric replacement to methane [240].
The corresponding N2O also reduced from 8 mg.l− 1 to 4 mg.l− 1 on
shifting from methane to Hythane. Thus, Hythane can be considered as a
lean fuel when compared to biomethane and CNG.
16
Fuel 317 (2022) 123449
6. Recommendations
From the literatures considered in this review article, the following
recommendations can be drafted for the future research to further
optimize and enhance the process.
i. More than one-third of studies considered in two-stage bio­
hythane production were batch tests. A continuous system using
the suggested reactor configurations or similar set-ups can be
studied for more understanding of the system.
ii. More studies are required on co-digestion of different solid and
liquid substrates. Though certain aspects of optimization such as
C/N ratio optimization, nutrient content optimization exist, it is
not evident on what constitutes a better co-digestion ratio, or
which substrates can be co-digested together, and what would be
the effect on the microbial population. Understanding this would
play a key role in designing and optimizing a generic system
which is not substrate specific.
iii. This study provides very little insight on biohydrogen production,
purification, and storage costs owing to less research in this area.
The costs provided in this study for the operation and mainte­
nance of the system is a comparative cost based on the existing
technologies for biomethane and industrial hydrogen purification
systems. Thereby, more studies are required in this area to eval­
uate the costs associated with industrial-scale biohythane
production.
iv. The carbon dioxide gas acidifies the system on recirculation,
thereby providing operational stability to the reactor. Thus, the
effect of recirculating and sparging the gas (biohydrogen and
carbon dioxide in the acidogenic reactor, and methane and car­
bon dioxide in the methanogenic reactor), on reactor stability,
and gas purification is an area of research which has to be looked
into.
v. The articulation of microbial community and their corresponding
biokinetics for substrate-specific conditions and co-digestion
conditions have to be looked onto. It was observed that the
Clostridium genus played a huge role as pure cultures for
hydrogen production. The involvement of this community and
other communities on biohydrogen and consequently the bio­
methane reactors using complex organic wastes, would be a
value-added information towards understanding the fundamen­
tals of biohydrogen and biomethane production.
vi. Process in-situ blending of hydrogen and methane into a single
Hythane fuel, can be looked into, to avoid storage costs of fuels as
two separate entities with different pressure, temperature, and
density.
7. Conclusion
With countries adopting zero-emission technologies to counter
global warming, biohythane will be one of the most sought-after fuels in
the near future. And especially because of its dual function as waste-
treatment technology, and energy production technology, sequential
hydrogen and methane production from organic wastes will be a
preferred pathway to biohythane production. This study covers the
stoichiometric and biological pathway of hydrogen production, using
pure cultures, mixed-cultures, and genetically modified organisms to
understand the possibility of augmentation in the large-scale systems.
This literature also covers an exhaustive range of study on two-stage
hydrogen and methane production from a range of mono- and co-
digested substrates, wherein the operational parameters such as pH,
temperature, retention time, OLR, and hydrogen partial pressure have
been critically discussed. This study further encompasses the bottlenecks
in the gas production due to competitive organisms, nutrient composi­
tion of the substrates, alcohols, VFAs, and Aromatic chemicals from pre-
treatment of substrate. The next sections sum up the available
K.B. Sasidhar et al.
purification technologies of hydrogen and methane, their corresponding
storage technologies, and the costs associated with production and pu­
rification. Finally, the potential of biohythane as a commercial fuel and
its environmental impacts have been discussed. The knowledge obtained
from the existing research studies need to be extrapolated to design a
sustainable and profitable generic model for biohythane production for
similar substrate characteristics.
CRediT authorship contribution statement
K.B. Sasidhar: Writing – review & editing, Data curation, Formal
analysis. P. Senthil Kumar: Conceptualization, Validation, Supervision.
Leilei Xiao: Conceptualization, Validation, Formal analysis.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgement
The authors would like to thank Sri Sivasubramaniya Nadar College
of Engineering for their support towards this work. This work was
supported by Youth Innovation Promotion Association, CAS (2021213).
References
[1] United Nations, Department of Economic and Social Affairs, Population Division
(2013). World Population Prospects: The 2012 Revision, Highlights and Advance
Tables. Working Paper Number ESA/P/WP.228. url: https://population.un.org/
wpp/Publications/Files/WPP2012_HIGHLIGHTS.pdf.
[2] Moodley P, Trois C. Lignocellulosic biorefineries: the path forward. In Sustainable
Biofuels 2021 Jan 1 (pp. 21-42). Academic Press. doi: https://doi.org/10.1016/
B978-0-12-820297-5.00010-4.
[3] Foroutan R, Peighambardoust SJ, Mohammadi R, Peighambardoust SH,
Ramavandi B. Application of walnut shell ash/ZnO/K2CO3 as a new composite
catalyst for biodiesel generation from Moringa oleifera oil. Fuel 2022;311:
122624. https://doi.org/10.1016/j.fuel.2021.122624.
[4] Foroutan R, Mohammadi R, Ramavandi B. Waste glass catalyst for biodiesel
production from waste chicken fat: Optimization by RSM and ANNs and toxicity
assessment. Fuel 2021;1(291):120151. https://doi.org/10.1016/j.
fuel.2021.120151.
[5] Foroutan R, Mohammadi R, Razeghi J, Ramavandi B. Biodiesel production from
edible oils using algal biochar/CaO/K2CO3 as a heterogeneous and recyclable
catalyst. Renewable Energy 2021;1(168):1207–16. https://doi.org/10.1016/j.
renene.2020.12.094.
[6] Khalid A, Arshad M, Anjum M, Mahmood T, Dawson L. The anaerobic digestion
of solid organic waste. Waste Manage 2011;31(8):1737–44. https://doi.org/
10.1016/j.wasman.2011.03.021.
[7] Matsakas L, Gao Q, Jansson S, Rova U, Christakopoulos P. Green conversion of
municipal solid wastes into fuels and chemicals. Electron J Biotechnol 2017;1
(26):69–83. https://doi.org/10.1016/j.ejbt.2017.01.004.
[8] McCarty PL. Anaerobic waste treatment fundamentals. Public works. 1964;95:9.
[9] Chen C, Guo W, Ngo HH. Advances in granular growth anaerobic membrane
bioreactor (G-AnMBR) for low strength wastewater treatment. J Energy Environ
Sustainability 2016;1:77–83. http://jees.in/uploads/one81-87.pdf.
[10] Angelidaki I, Ellegaard L, Ahring BK. Applications of the anaerobic digestion
process. Biomethanation 2003;II:1–33. https://doi.org/10.1007/3-540-45838-7_
1.
[11] Lastella G, Testa C, Cornacchia G, Notornicola M, Voltasio F, Sharma VK.
Anaerobic digestion of semi-solid organic waste: biogas production and its
purification. Energy Convers Manage 2002;43(1):63–75. https://doi.org/
10.1016/S0196-8904(01)00011-5.
[12] Lata K, Rajeshwari KV, Pant DC, Kishore VV. Volatile fatty acid production during
anaerobic mesophilic digestion of tea and vegetable market wastes. World J
Microbiol Biotechnol 2002;18(6):589–92. https://doi.org/10.1023/A:
1016314903817.
[13] Wainaina S, Lukitawesa, Kumar Awasthi M, Taherzadeh MJ. Bioengineering of
anaerobic digestion for volatile fatty acids, hydrogen or methane production: A
critical review. Bioengineered 2019;10(1):437–58.
[14] Kleerebezem R, Joosse B, Rozendal R, Van Loosdrecht MC. Anaerobic digestion
without biogas? Rev Environ Sci Bio/Technol 2015;14(4):787–801. https://doi.
org/10.1007/s11157-015-9374-6.
[15] Marone A, Izzo G, Mentuccia L, Massini G, Paganin P, Rosa S, et al. Vegetable
waste as substrate and source of suitable microflora for bio-hydrogen production.
17
Fuel 317 (2022) 123449
Renewable Energy 2014;68:6–13. https://doi.org/10.1016/j.
renene.2014.01.013.
[16] Piera M, Martínez-Val JM, Montes MJ. Safety issues of nuclear production of
hydrogen. Energy Convers Manage 2006;47(17):2732–9. https://doi.org/
10.1016/j.enconman.2006.02.002.
[17] Liu Z, Zhang C, Lu Y, Wu X, Wang L, Wang L, et al. States and challenges for high-
value biohythane production from waste biomass by dark fermentation
technology. Bioresour Technol 2013;135:292–303. https://doi.org/10.1016/j.
biortech.2012.10.027.
[18] Guo XM, Trably E, Latrille E, Carrere H, Steyer JP. Hydrogen production from
agricultural waste by dark fermentation: a review. Int J Hydrogen Energy 2010;
35(19):10660–73. https://doi.org/10.1016/j.ijhydene.2010.03.008.
[19] 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(6):575–85. https://doi.org/10.1080/
15567030903117653.
[20] Rena, Mohammed Bin Zacharia K, Yadav S, Machhirake NP, Kim S-H, Lee B-D,
et al. Bio-hydrogen and bio-methane potential analysis for production of bio-
hythane using various agricultural residues. Bioresour Technol 2020;309:123297.
https://doi.org/10.1016/j.biortech.2020.123297.
[21] Cheng J, Ding L, Lin R, Liu M, Zhou J, Cen K. Physicochemical characterization of
typical municipal solid wastes for fermentative hydrogen and methane co-
production. Energy Convers Manage 2016;1(117):297–304. https://doi.org/
10.1016/j.enconman.2016.03.016.
[22] Cheng J, Ding L, Lin R, Yue L, Liu J, Zhou J, et al. Fermentative biohydrogen and
biomethane co-production from mixture of food waste and sewage sludge: Effects
of physiochemical properties and mix ratios on fermentation performance. Appl
Energy 2016;184:1–8. https://doi.org/10.1016/j.apenergy.2016.10.003.
[23] Liu D, Liu D, Zeng RJ, Angelidaki I. Hydrogen and methane production from
household solid waste in the two-stage fermentation process. Water Res 2006;40
(11):2230–6. https://doi.org/10.1016/j.watres.2006.03.029.
[24] Monlau F, Barakat A, Trably E, Dumas C, Steyer JP, Carrère H. Lignocellulosic
materials into biohydrogen and biomethane: impact of structural features and
pretreatment. Critical Rev Environ Sci Technol 2013;43(3):260–322. https://doi.
org/10.1080/10643389.2011.604258.
[25] Giuliano A, Zanetti L, Micolucci F, Cavinato C. Thermophilic two-phase anaerobic
digestion of source-sorted organic fraction of municipal solid waste for bio-
hythane production: effect of recirculation sludge on process stability and
microbiology over a long-term pilot-scale experience. Water Sci Technol 2014;69
(11):2200–9. https://doi.org/10.2166/wst.2014.137.
[26] Khan MA, Ngo HH, Guo W, Liu Y, Zhang X, Guo J, et al. Biohydrogen production
from anaerobic digestion and its potential as renewable energy. Renewable
Energy 2018;129:754–68. https://doi.org/10.1016/j.renene.2017.04.029.
[27] Ntaikou I, Antonopoulou G, Lyberatos G. Biohydrogen production from biomass
and wastes via dark fermentation: a review. Waste Biomass Valorization 2010;1
(1):21–39. https://doi.org/10.1007/s12649-009-9001-2.
[28] Shanmugam S, Mathimani T, Rene ER, Edwin Geo V, Arun A, Brindhadevi K, et al.
Biohythane production from organic waste: recent advancements, technical
bottlenecks and prospects. Int J Hydrogen Energy 2021;46(20):11201–16.
https://doi.org/10.1016/j.ijhydene.2020.10.132.
[29] Shanmugam S, Sekar M, Sivaramakrishnan R, Raj T, Ong ES, Rabbani AH, et al.
Pretreatment of second and third generation feedstock for enhanced biohythane
production: challenges, recent trends and perspectives. Int J Hydrogen Energy
2021;46(20):11252–68. https://doi.org/10.1016/j.ijhydene.2020.12.083.
[30] Chris Abatelli. Eden Innovations That Work. [Internet]. Denver, USA: Eden
Innovations Limited; [2016; 2021 Aug]. Available from: https://edeninnovations.
com/innovations/.
[31] Bauer C. 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
(1):55–70.
[32] Bauer C. Effect of hydrogen addition on the performance of methane-fueled
vehicles. Part II: driving cycle simulations. Int J Hydrogen Energy 2001;26(1):
71–90.
[33] Nagalingam B, Duebel F, Schmillen K. Performance study using natural gas,
hydrogen-supplemented natural gas, and hydrogen in AVL research engine. Int J
Hydrogen Energy 1983;8(9):715–20. https://doi.org/10.1016/0360-3199(83)
90181-7.
[34] Ma F, Wang Y, Liu H, Li Y, Wang J, Ding S. Effects of hydrogen addition on cycle-
by-cycle variations in a lean burn natural gas spark-ignition engine. Int J
Hydrogen Energy 2008;33(2):823–31. https://doi.org/10.1016/j.
ijhydene.2007.10.043.
[35] Abbas HF, Daud WW. Hydrogen production by methane decomposition: a review.
Int J Hydrogen Energy 2010;35(3):1160–90. https://doi.org/10.1016/j.
ijhydene.2009.11.036.
[36] Henze M, van Loosdrecht MC, Ekama GA, Brdjanovic D, editors. Biological
wastewater treatment. IWA publishing; 2008 16: Biological Wastewater
Treatment; 415-456.
[37] Adekunle KF, Okolie JA. A review of biochemical process of anaerobic digestion.
Adv Biosci Biotechnol 2015;06(03):205–12. https://doi.org/10.4236/
abb.2015.63020.
[38] Gerardi MH, editor. Wastewater Microbiology Series. Hoboken, NJ, USA: John
Wiley & Sons, Inc.; 2003.
[39] Khan MA, Ngo HH, Guo WS, Liu Y, Nghiem LD, Hai FI, et al. Optimization of
process parameters for production of volatile fatty acid, biohydrogen and
methane from anaerobic digestion. Bioresour Technol 2016;219:738–48. https://
doi.org/10.1016/j.biortech.2016.08.073.
K.B. Sasidhar et al.
[40] Mohan SV, Mohanakrishna G, Goud RK, Sarma PN. Acidogenic fermentation of
vegetable-based market waste to harness biohydrogen with simultaneous
stabilization. Bioresour Technol 2009;100(12):3061–8. https://doi.org/10.1016/
j.biortech.2008.12.059.
[41] Kandylis P, Bekatorou A, Pissaridi K, Lappa K, Dima A, Kanellaki M, et al.
Acidogenesis of cellulosic hydrolysates for new generation biofuels. Biomass
Bioenergy 2016;91:210–6. https://doi.org/10.1016/j.biombioe.2016.05.006.
[42] Wu Y, Wang C, Liu X, Ma H, Wu J, Zuo J, et al. A new method of two-phase
anaerobic digestion for fruit and vegetable waste treatment. Bioresour Technol
2016;211:16–23. https://doi.org/10.1016/j.biortech.2016.03.050.
[43] Bar-Even A. Does acetogenesis really require especially low reduction potential?
Biochim Biophys Acta (BBA) - Bioenergetics 2013;1827(3):395–400.
[44] Gujer W, Zehnder AJ. Conversion processes in anaerobic digestion. Water Sci
Technol 1983;15(8–9):127–67. https://doi.org/10.2166/wst.1983.0164.
[45] Yu J, Zhao Y, Liu B, Zhao Y, Wu J, Yuan X, et al. Accelerated acidification by
inoculation with a microbial consortium in a complex open environment.
Bioresour Technol 2016;1(216):294–301. https://doi.org/10.1016/j.
biortech.2016.05.093.
[46] Krishnan S, Md Din MF, Taib SM, Nasrullah M, Sakinah M, Wahid ZA, et al.
Accelerated two-stage bioprocess for hydrogen and methane production from
palm oil mill effluent using continuous stirred tank reactor and microbial
electrolysis cell. J Cleaner Prod 2019;229:84–93. https://doi.org/10.1016/j.
jclepro.2019.04.365.
[47] Lu Y, Lai Q, Zhang C, Zhao H, Ma K, Zhao X, et al. Characteristics of hydrogen and
methane production from cornstalks by an augmented two-or three-stage
anaerobic fermentation process. Bioresour Technol 2009;100(12):2889–95.
https://doi.org/10.1016/j.biortech.2009.01.023.
[48] Siddiqui Z, Horan NJ, Salter M. Energy optimisation from co-digested waste using
a two-phase process to generate hydrogen and methane. Int J Hydrogen Energy
2011;36(8):4792–9.
[49] Ding L, Cheng J, Xia A, Jacob A, Voelklein M, Murphy JD. Co-generation of
biohydrogen and biomethane through two-stage batch co-fermentation of macro-
and micro-algal biomass. Bioresour Technol 2016;1(218):224–31. https://doi.
org/10.1016/j.biortech.2016.06.092.
[50] Perera KRJ, Ketheesan B, Arudchelvam Y, Nirmalakhandan N. Fermentative
biohydrogen production II: Net energy gain from organic wastes. Int J Hydrogen
Energy 2012;37(1):167–78.
[51] Ruggeri B, Tommasi T, Sassi G. Energy balance of dark anaerobic fermentation as
a tool for sustainability analysis. Int J Hydrogen Energy 2010;35(19):10202–11.
[52] Ke S, Shi Z, Fang HH. Applications of two-phase anaerobic degradation in
industrial wastewater treatment. Int J Environ Pollut 2005;23(1):65–80. https://
doi.org/10.1504/IJEP.2005.006396.
[53] Li WW, Yu HQ. From wastewater to bioenergy and biochemicals via two-stage
bioconversion processes: a future paradigm. Biotechnol Adv 2011;29(6):972–82.
https://doi.org/10.1016/j.biotechadv.2011.08.012.
[54] Ueno Y, Fukui H, Goto M. Operation of a two-stage fermentation process
producing hydrogen and methane from organic waste. Environ Sci Technol 2007;
41(4):1413–9. https://doi.org/10.1021/es062127f.
[55] Ueno Y, Tatara M, Fukui H, Makiuchi T, Goto M, Sode K. Production of hydrogen
and methane from organic solid wastes by phase-separation of anaerobic process.
Bioresour Technol 2007;98(9):1861–5. https://doi.org/10.1016/j.
biortech.2006.06.017.
[56] Kongjan P, Sompong O, 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(5):4028–35. https://doi.org/10.1016/j.biortech.2010.12.009.
[57] 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(18):8700–6. https://doi.org/10.1016/j.
biortech.2011.02.012.
[58] Prajapati KB, Singh R. Bio-electrochemically hydrogen and methane production
from co-digestion of wastes. Energy. 2020;1(198):117259. https://doi.org/
10.1016/j.energy.2020.117259.
[59] Chu CF, Ebie Y, Xu KQ, Li YY, Inamori Y. Characterization of microbial
community in the two-stage process for hydrogen and methane production from
food waste. Int J Hydrogen Energy 2010;35(15):8253–61. https://doi.org/
10.1016/j.ijhydene.2009.12.021.
[60] Chu CF, 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(18):4739–46. https://doi.org/10.1016/j.
ijhydene.2008.06.060.
[61] Koutrouli EC, Kalfas H, Gavala HN, Skiadas IV, Stamatelatou K, Lyberatos G.
Hydrogen and methane production through two-stage mesophilic anaerobic
digestion of olive pulp. Bioresour Technol 2009;100(15):3718–23. https://doi.
org/10.1016/j.biortech.2009.01.037.
[62] Xie B, Cheng J, Zhou J, Song W, Cen K. Cogeneration of hydrogen and methane
from glucose to improve energy conversion efficiency. Int J Hydrogen Energy
2008;33(19):5006–11. https://doi.org/10.1016/j.ijhydene.2008.07.048.
[63] Zhu Y, Li J, Tan M, Liu L, Jiang L, Sun J, et al. Optimization and scale-up of
propionic acid production by propionic acid-tolerant Propionibacterium
acidipropionici with glycerol as the carbon source. Bioresour Technol 2010;101
(22):8902–6. https://doi.org/10.1016/j.biortech.2010.06.070.
[64] Wu Q, Guo W, Yang S, Luo H, Peng S, Ren N. Enhancement of volatile fatty acid
production using semi-continuous anaerobic food waste fermentation without pH
control. RSC Adv 2015;5(126):103876–83. https://doi.org/10.1039/
C5RA21162J.
18
Fuel 317 (2022) 123449
[65] Coral J, Karp SG, de Souza Vandenberghe LP, Parada JL, Pandey A, Soccol CR.
Batch fermentation model of propionic acid production by Propionibacterium
acidipropionici in different carbon sources. Appl Biochem Biotechnol 2008;151
(2):333–41. https://doi.org/10.1007/s12010-008-8196-1.
[66] Bhatia SK, Yang YH. Microbial production of volatile fatty acids: current status
and future perspectives. Rev Environ Sci Bio/Technol 2017;16(2):327–45.
https://doi.org/10.1007/s11157-017-9431-4.
[67] De Gioannis G, Muntoni A, Polettini A, Pomi R. A review of dark fermentative
hydrogen production from biodegradable municipal waste fractions. Waste
Manage 2013;33(6):1345–61. https://doi.org/10.1016/j.wasman.2013.02.019.
[68] Zhou M, Yan B, Wong JW, Zhang Y. Enhanced volatile fatty acids production from
anaerobic fermentation of food waste: A mini-review focusing on acidogenic
metabolic pathways. Bioresour Technol 2018;1(248):68–78. https://doi.org/
10.1016/j.biortech.2017.06.121.
[69] Ghimire A, Trably E, Frunzo L, Pirozzi F, Lens PNL, Esposito G, et al. Effect of
total solids content on biohydrogen production and lactic acid accumulation
during dark fermentation of organic waste biomass. Bioresour Technol 2018;248:
180–6. https://doi.org/10.1016/j.biortech.2017.07.062.
[70] Zhang W, Li X, Zhang T, Li J, Lai S, Chen H, et al. High-rate lactic acid production
from food waste and waste activated sludge via interactive control of pH
adjustment and fermentation temperature. Chem Eng J 2017;328:197–206.
https://doi.org/10.1016/j.cej.2017.06.174.
[71] Nayak J, Pal P. Transforming waste cheese-whey into acetic acid through a
continuous membrane-integrated hybrid process. Ind Eng Chem Res 2013;52(8):
2977–84. https://doi.org/10.1021/ie3033729.
[72] Pal P, Nayak J. Acetic acid production and purification: critical review towards
process intensification. Sep Purif Rev 2017;46(1):44–61. https://doi.org/
10.1080/15422119.2016.1185017.
[73] Ehsanipour M, Suko AV, Bura R. Fermentation of lignocellulosic sugars to acetic
acid by Moorella thermoacetica. J Ind Microbiol Biotechnol 2016;43(6):807–16.
https://doi.org/10.1007/s10295-016-1756-4.
[74] Atasoy M, Owusu-Agyeman I, Plaza E, Cetecioglu Z. Bio-based volatile fatty acid
production and recovery from waste streams: current status and future
challenges. Bioresour Technol 2018;1(268):773–86. https://doi.org/10.1016/j.
biortech.2018.07.042.
[75] Du G, Liu L, Chen J. White biotechnology for organic acids. In: Industrial
Biorefineries & White Biotechnology. Elsevier; 2015. p. 409–44. https://doi.org/
10.1016/B978-0-444-63453-5.00013-6.
[76] Zhang A, Yang ST. Propionic acid production from glycerol by metabolically
engineered Propionibacterium acidipropionici. Process Biochem 2009 Dec 1;44
(12):1346–51. https://doi.org/10.1016/j.procbio.2009.07.013.
[77] Goswami V, Srivastava A. Propionic acid production in an in-situ cell retention
bioreactor. Appl Microbiol Biotechnol 2001;56(5):676–80. https://doi.org/
10.1007/s002530000582.
[78] Luna-Flores CH, Palfreyman RW, Krömer JO, Nielsen LK, Marcellin E. Improved
production of propionic acid using genome shuffling. Biotechnol J 2017;12(2):
1600120. https://doi.org/10.1002/biot.201600120.
[79] Liu L, Guan N, Zhu G, Li J, Shin HD, Du G, et al. Pathway engineering of
Propionibacterium jensenii for improved production of propionic acid. Sci Rep
2016 Jan 27;6(1):1–9. https://doi.org/10.1038/srep19963.
[80] Baroi GN, Baumann I, Westermann P, Gavala HN. Butyric acid fermentation from
pretreated and hydrolysed wheat straw by an adapted Clostridium tyrobutyricum
strain. Microb Biotechnol 2015;8(5):874–82. https://doi.org/10.1111/1751-
7915.12304.
[81] Jiang L, Wang J, Liang S, Cai J, Xu Z, Cen P, et al. Enhanced butyric acid tolerance
and bioproduction by Clostridium tyrobutyricum immobilized in a fibrous bed
bioreactor. Biotechnol Bioeng 2011;108(1):31–40. https://doi.org/10.1002/
bit.22927.
[82] Ravinder T, Ramesh B, Seenayya G, Reddy G. Fermentative production of acetic
acid from various pure and natural cellulosic materials by Clostridium
lentocellum SG6. World J Microbiol Biotechnol 2000;16(6):507–12. https://doi.
org/10.1023/A:1008966205306.
[83] Gottumukkala LD, Sukumaran RK, Mohan SV, Valappil SK, Sarkar O, Pandey A.
Rice straw hydrolysate to fuel and volatile fatty acid conversion by Clostridium
sporogenes BE01: bio-electrochemical analysis of the electron transport
mediators involved. Green Chem 2015;17(5):3047–58. https://doi.org/10.1039/
C5GC00310E.
[84] Thungklin P, Sittijunda S, Reungsang A. Sequential fermentation of hydrogen and
methane from steam-exploded sugarcane bagasse hydrolysate. Int J Hydrogen
Energy 2018 May 24;43(21):9924–34. https://doi.org/10.1016/j.
ijhydene.2018.04.056.
[85] Sołowski G, Konkol I, Cenian A. Production of hydrogen and methane from
lignocellulose waste by fermentation. A review of chemical pretreatment for
enhancing the efficiency of the digestion process. J Cleaner Prod 2020;10(267):
121721. https://doi.org/10.1016/j.jclepro.2020.121721.
[86] Jönsson LJ, Martín C. Pretreatment of lignocellulose: formation of inhibitory by-
products and strategies for minimizing their effects. Bioresour Technol 2016;1
(199):103–12. https://doi.org/10.1016/j.biortech.2015.10.009.
[87] Lu Y, Slater FR, Mohd-Zaki Z, Pratt S, Batstone DJ. Impact of operating history on
mixed culture fermentation microbial ecology and product mixture. Water Sci
Technol 2011;64(3):760–5. https://doi.org/10.2166/wst.2011.699.
[88] Kleerebezem R, van Loosdrecht MC. Mixed culture biotechnology for bioenergy
production. Curr Opin Biotechnol 2007;18(3):207–12. https://doi.org/10.1016/j.
copbio.2007.05.001.
[89] Wainaina S, Parchami M, Mahboubi A, Horváth IS, Taherzadeh MJ. Food waste-
derived volatile fatty acids platform using an immersed membrane bioreactor.
K.B. Sasidhar et al.
Bioresour Technol 2019;1(274):329–34. https://doi.org/10.1016/j.
biortech.2018.11.104.
[90] Akinbomi J, Taherzadeh MJ. Evaluation of fermentative hydrogen production
from single and mixed fruit wastes. Energies. 2015;8(5):4253–72. https://doi.
org/10.3390/en8054253.
[91] Kim SH, Han SK, Shin HS. Feasibility of biohydrogen production by anaerobic co-
digestion of food waste and sewage sludge. Int J Hydrogen Energy 2004;29(15):
1607–16. https://doi.org/10.1016/j.ijhydene.2004.02.018.
[92] Nandi R, Sengupta S. Microbial production of hydrogen: an overview. Crit Rev
Microbiol 1998;24(1):61–84. https://doi.org/10.1080/10408419891294181.
[93] Thauer RK, Jungermann K, Decker K. Energy conservation in chemotrophic
anaerobic bacteria. Bacteriol Rev 1977;41(1):100–80. https://doi.org/10.1128/
br.41.1.100-180.1977.
[94] Sawers RG. Formate and its role in hydrogen production in Escherichia coli.
Biochem Soc Trans 2005;33(1):42–6.
[95] Peretó J. In: Encyclopedia of Astrobiology. Berlin, Heidelberg: Springer Berlin
Heidelberg; 2011. p. 485. https://doi.org/10.1007/978-3-642-11274-4_503.
[96] Uyeda K, Rabinowitz JC. Pyruvate-Ferredoxin Oxidoreductase: IV. Studies on the
reaction mechanism. J Biol Chem. 1971;246(10):3120-5. doi: https://doi.org/
10.1016/S0021-9258(18)62203-3.
[97] Knappe J, Blaschkowski HP, Grobner P, Schmttt T. Pyruvate formate-lyase of
Escherichia coli: the acetyl-enzyme intermediate. Eur J Biochem 1974;50(1):
253–63. https://doi.org/10.1111/j.1432-1033.1974.tb03894.x.
[98] Gottschalk G. Bacterial fermentations. In Bacterial metabolism 1986 (pp. 208-
282). Springer, New York, NY. doi: https://doi.org/10.1007/978-1-4612-1072-6_
8.
[99] Wang J, Wan W. Factors influencing fermentative hydrogen production: a review.
Int J Hydrogen Energy 2009;34(2):799–811. https://doi.org/10.1016/j.
ijhydene.2008.11.015.
[100] Hallenbeck PC. Fundamentals of the fermentative production of hydrogen. Water
Sci Technol 2005;52(1–2):21–9. https://doi.org/10.2166/wst.2005.0494.
[101] Chong ML, Rahim RA, Shirai Y, Hassan MA. Biohydrogen production by
Clostridium butyricum EB6 from palm oil mill effluent. Int J Hydrogen Energy
2009;34(2):764–71. https://doi.org/10.1016/j.ijhydene.2008.10.095.
[102] Kataoka N, Miya A, Kiriyama K. Studies on hydrogen production by continuous
culture system of hydrogen-producing anaerobic bacteria. Water Sci Technol
1997;36(6–7):41–7. https://doi.org/10.2166/wst.1997.0573.
[103] Saint-Amans S, Girbal L, Andrade J, Ahrens K, Soucaille P. Regulation of carbon
and electron flow in Clostridium butyricum VPI 3266 grown on glucose-glycerol
mixtures. J Bacteriol 2001;183(5):1748–54. https://doi.org/10.1128/
JB.183.5.1748-1754.2001.
[104] Yokoi H, Saitsu A, Uchida H, Hirose JU, Hayashi S, Takasaki Y. Microbial
hydrogen production from sweet potato starch residue. J Biosci Bioeng 2001;91
(1):58–63. https://doi.org/10.1016/S1389-1723(01)80112-2.
[105] Prapinagsorn W, Sittijunda S, Reungsang A. Co-digestion of napier grass and its
silage with cow dung for bio-hydrogen and methane production by two-stage
anaerobic digestion process. Energies. 2018;11(1):47. https://doi.org/10.3390/
en11010047.
[106] Lin P-Y, Whang L-M, Wu Y-R, Ren W-J, Hsiao C-J, Li S-L, et al. Biological
hydrogen production of the genus Clostridium: metabolic study and mathematical
model simulation. Int J Hydrogen Energy 2007;32(12):1728–35. https://doi.org/
10.1016/j.ijhydene.2006.12.009.
[107] Jo JH, Lee DS, Park D, Park JM. Biological hydrogen production by immobilized
cells of Clostridium tyrobutyricum JM1 isolated from a food waste treatment
process. Bioresour Technol 2008;99(14):6666–72. https://doi.org/10.1016/j.
biortech.2007.11.067.
[108] Collet C, Adler N, Schwitzguébel JP, Péringer P. Hydrogen production by
Clostridium thermolacticum during continuous fermentation of lactose. Int J
Hydrogen Energy 2004;29(14):1479–85. https://doi.org/10.1016/j.
ijhydene.2004.02.009.
[109] Evvyernie D, Yamazaki S, Morimoto K, Karita S, Kimura T, Sakka K, et al.
Identification and characterization of Clostridium paraputrificum M-21, a
chitinolytic, mesophilic and hydrogen-producing bacterium. J Biosci Bioeng
2000;89(6):596–601. https://doi.org/10.1016/S1389-1723(00)80063-8.
[110] Levin DB, Islam R, Cicek N, Sparling R. Hydrogen production by Clostridium
thermocellum 27405 from cellulosic biomass substrates. Int J Hydrogen Energy
2006;31(11):1496–503. https://doi.org/10.1016/j.ijhydene.2006.06.015.
[111] Wang CC, Chang CW, Chu CP, Lee DJ, Chang BV, Liao CS. Producing hydrogen
from wastewater sludge by Clostridium bifermentans. J Biotechnol 2003;102(1):
83–92. https://doi.org/10.1016/S0168-1656(03)00007-5.
[112] Taguchi F, Mizukami N, Saito-Taki T, Hasegawa K. Hydrogen production from
continuous fermentation of xylose during growth of Clostridium sp. strain No. 2.
Can. J. Microbiol. 1995;41(6):536–40.
[113] Iannotti EL, Kafkewitz D, Wolin MJ, Bryant MP. Glucose fermentation products of
Ruminococcus albus grown in continuous culture with Vibrio succinogenes:
changes caused by interspecies transfer of H2. J Bacteriol 1973;114(3):1231–40.
https://doi.org/10.1128/jb.114.3.1231-1240.1973.
[114] Kumar N, Ghosh A, Das D. Redirection of biochemical pathways for the
enhancement of H2 production by Enterobacter cloacae. Biotechnol Lett 2001;23
(7):537–41. https://doi.org/10.1023/A:1010334803961.
[115] Kumar N, Das D. Continuous hydrogen production by immobilized Enterobacter
cloacae IIT-BT 08 using lignocellulosic materials as solid matrices. Enzyme
Microb Technol 2001;29(4–5):280–7. https://doi.org/10.1016/S0141-0229(01)
00394-5.
19
Fuel 317 (2022) 123449
[116] Oh YK, Seol EH, Kim JR, Park S. Fermentative biohydrogen production by a new
chemoheterotrophic bacterium Citrobacter sp. Y19. Int J Hydrogen Energy 2003;
28(12):1353–9. https://doi.org/10.1016/S0360-3199(03)00024-7.
[117] Tanisho S, Ishiwata Y. Continuous hydrogen production from molasses by the
bacterium Enterobacter aerogenes. Int J Hydrogen Energy 1994;19(10):807–12.
https://doi.org/10.1016/0360-3199(94)90197-X.
[118] Dietrich G, Weiss N, Winter J. Acetothermus paucivorans, gen. nov., sp. nov., a
strictly anaerobic, thermophilic bacterium from sewage sludge, fermenting
hexoses to acetate, CO2 and H2. Syst Appl Microbiol 1988;10(2):174–9. https://
doi.org/10.1016/S0723-2020(88)80033-X.
[119] Janssen PH, Morgan HW. Glucose catabolism by Spirochaeta thermophila RI 19.
B1. J Bacteriol 1992;174(8):2449–53. https://doi.org/10.1128/jb.174.8.2449-
2453.1992.
[120] Rainey FA, Janssen PH, Wild DJ, Morgan HW. Isolation and characterization of an
obligately anaerobic, polysaccharolytic, extremely thermophilic member of the
genus Spirochaeta. Arch Microbiol 1991;155(4):396–401. https://doi.org/
10.1007/BF00243461.
[121] Soutschek E, Winter J, Schindler F, Kandler O. Acetomicrobium flavidum, gen.
nov., sp. nov., a thermophilic, anaerobic bacterium from sewage sludge, forming
acetate, CO2 and H2 from Glucose. Syst Appl Microbiol 1984;5(3):377–90.
[122] Van Niel EW, Budde MA, De Haas GG, Van der Wal FJ, Claassen PA, Stams AJ.
Distinctive properties of high hydrogen producing extreme thermophiles,
Caldicellulosiruptor saccharolyticus and Thermotoga elfii. Int J Hydrogen Energy
2002;27(11–12):1391–8. https://doi.org/10.1016/S0360-3199(02)00115-5.
[123] De Vrije TD, De Haas GG, Tan GB, Keijsers ER, Claassen PA. Pretreatment of
Miscanthus for hydrogen production by Thermotoga elfii. Int J Hydrogen Energy
2002;27(11–12):1381–90. https://doi.org/10.1016/S0360-3199(02)00124-6.
[124] Janssen PH, Morgan HW. Heterotrophic sulfur reduction by Thermotoga sp. strain
FjSS3.B1. FEMS Microbiol Lett 1992;96(2-3):213–7.
[125] Sompong O, Prasertsan P, Karakashev D, Angelidaki I. Thermophilic fermentative
hydrogen production by the newly isolated Thermoanaerobacterium
thermosaccharolyticum PSU-2. Int J Hydrogen Energy 2008;33(4):1204–14.
https://doi.org/10.1016/j.ijhydene.2007.12.015.
[126] Vieille C, Zeikus GJ. Hyperthermophilic enzymes source, uses, and molecular
mechanisms for thermostability. Microbiol Mol Biol Rev 2001;65(1):1–43.
https://doi.org/10.1128/MMBR.65.1.1-43.2001.
[127] Schröder C, Selig M, Schönheit P. Glucose fermentation to acetate, CO2 and H2 in
the anaerobic hyperthermophilic eubacterium Thermotoga maritima:
involvement of the Embden-Meyerhof pathway. Arch Microbiol 1994;161(6):
460–70. https://doi.org/10.1007/BF00307766.
[128] Kengen SW, Stams AJ. Formation of L-alanine as a reduced end product in
carbohydrate fermentation by the hyperthermophilic archaeon Pyrococcus
furiosus. Arch Microbiol 1994;161(2):168–75. https://doi.org/10.1007/
BF00276479.
[129] Schäfer T, Schönheit P. Maltose fermentation to acetate, CO2 and H2 in the
anaerobic hyperthermophilic archaeon Pyrococcus furiosus: evidence for the
operation of a novel sugar fermentation pathway. Arch Microbiol 1992;158(3):
188–202. https://doi.org/10.1007/BF00290815.
[130] Nguyen TA, Han SJ, Kim JP, Kim MS, Sim SJ. Hydrogen production of the
hyperthermophilic eubacterium, Thermotoga neapolitana under N2 sparging
condition. Bioresour Technol 2010;101(1):S38–41. https://doi.org/10.1016/j.
biortech.2009.03.041.
[131] Valdez-Vazquez I, Ríos-Leal E, Esparza-García F, Cecchi F, Poggi-Varaldo HM.
Semi-continuous solid substrate anaerobic reactors for H2 production from
organic waste: mesophilic versus thermophilic regime. Int J Hydrogen Energy
2005;30(13–14):1383–91. https://doi.org/10.1016/j.ijhydene.2004.09.016.
[132] Chen CC, Lin CY, Lin MC. Acid–base enrichment enhances anaerobic hydrogen
production process. Appl Microbiol Biotechnol 2002;58(2):224–8. https://doi.
org/10.1007/s002530100814.
[133] Chang JS, Lee KS, Lin PJ. Biohydrogen production with fixed-bed bioreactors. Int
J Hydrogen Energy 2002;27(11–12):1167–74. https://doi.org/10.1016/S0360-
3199(02)00130-1.
[134] Ren N, Li J, Li B, Wang Y, Liu S. Biohydrogen production from molasses by
anaerobic fermentation with a pilot-scale bioreactor system. Int J Hydrogen
Energy 2006;31(15):2147–57. https://doi.org/10.1016/j.ijhydene.2006.02.011.
[135] Baldi F, Pecorini I, Iannelli R. Comparison of single-stage and two-stage anaerobic
co-digestion of food waste and activated sludge for hydrogen and methane
production. Renewable Energy 2019;1(143):1755–65. https://doi.org/10.1016/j.
renene.2019.05.122.
[136] Deheri C, Acharya SK. Effect of calcium peroxide and sodium hydroxide on
hydrogen and methane generation during the co-digestion of food waste and cow
dung. J Cleaner Prod 2021;10(279):123901. https://doi.org/10.1016/j.
jclepro.2020.123901.
[137] Farghaly A, Tawfik A. Simultaneous hydrogen and methane production through
multi-phase anaerobic digestion of paperboard mill wastewater under different
operating conditions. Appl Biochem Biotechnol 2017;181(1):142–56. https://doi.
org/10.1007/s12010-016-2204-7.
[138] Okamoto M, Miyahara T, Mizuno O, Noike T. Biological hydrogen potential of
materials characteristic of the organic fraction of municipal solid wastes. Water
Sci Technol 2000;41(3):25–32. https://doi.org/10.2166/wst.2000.0052.
[139] Kraemer JT, Bagley DM. Continuous fermentative hydrogen production using a
two-phase reactor system with recycle. Environ Sci Technol 2005;39(10):
3819–25. https://doi.org/10.1021/es048502q.
[140] Kyazze G, Dinsdale R, Guwy AJ, Hawkes FR, Premier GC, Hawkes DL.
Performance characteristics of a two-stage dark fermentative system producing
K.B. Sasidhar et al.
hydrogen and methane continuously. Biotechnol Bioeng 2007;97(4):759–70.
https://doi.org/10.1002/bit.21297.
[141] Cooney M, Maynard N, Cannizzaro C, Benemann J. Two-phase anaerobic
digestion for production of hydrogen–methane mixtures. Bioresour Technol 2007;
98(14):2641–51. https://doi.org/10.1016/j.biortech.2006.09.054.
[142] Lee MJ, Song JH, Hwang SJ. Enhanced bio-energy recovery in a two-stage
hydrogen/methane fermentation process. Water Sci Technol 2009;59(11):
2137–43. https://doi.org/10.2166/wst.2009.236.
[143] Hafez H, Nakhla G, El Naggar H. An integrated system for hydrogen and methane
production during landfill leachate treatment. Int J Hydrogen Energy 2010;35
(10):5010–4.
[144] Mohan SV, Mohanakrishna G, Sarma PN. Integration of acidogenic and
methanogenic processes for simultaneous production of biohydrogen and
methane from wastewater treatment. Int J Hydrogen Energy 2008;33(9):
2156–66. https://doi.org/10.1016/j.ijhydene.2008.01.055.
[145] Luo G, Xie L, Zou Z, Wang W, Zhou Q, Shim H. Anaerobic treatment of cassava
stillage for hydrogen and methane production in continuously stirred tank reactor
(CSTR) under high organic loading rate (OLR). Int J Hydrogen Energy 2010;35
(21):11733–7. https://doi.org/10.1016/j.ijhydene.2010.08.033.
[146] Giordano A, Cantù C, Spagni A. Monitoring the biochemical hydrogen and
methane potential of the two-stage dark-fermentative process. Bioresour Technol
2011;102(6):4474–9. https://doi.org/10.1016/j.biortech.2010.12.106.
[147] Wang W, Xie L, Chen J, Luo G, Zhou Q. Biohydrogen and methane production by
co-digestion of cassava stillage and excess sludge under thermophilic condition.
Bioresour Technol 2011 Feb 1;102(4):3833–9. https://doi.org/10.1016/j.
biortech.2010.12.012.
[148] Han SK, Shin HS. Performance of an innovative two-stage process converting food
waste to hydrogen and methane. J Air Waste Manag Assoc 2004 Feb 1;54(2):
242–9. https://doi.org/10.1080/10473289.2004.10470895.
[149] 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 Jan 1;101(1):
S42–7. https://doi.org/10.1016/j.biortech.2009.03.037.
[150] Zhu H, Parker W, Conidi D, Basnar R, Seto P. Eliminating methanogenic activity
in hydrogen reactor to improve biogas production in a two-stage anaerobic
digestion process co-digesting municipal food waste and sewage sludge. Bioresour
Technol 2011 Jul 1;102(14):7086–92. https://doi.org/10.1016/j.
biortech.2011.04.047.
[151] Cavinato C, Bolzonella D, Fatone F, Cecchi F, Pavan P. Optimization of two-phase
thermophilic anaerobic digestion of biowaste for hydrogen and methane
production through reject water recirculation. Bioresour Technol 2011 Sep 1;102
(18):8605–11. https://doi.org/10.1016/j.biortech.2011.03.084.
[152] Cota-Navarro CB, Carrillo-Reyes J, Davila-Vazquez G, Alatriste-Mondragón F,
Razo-Flores E. Continuous hydrogen and methane production in a two-stage
cheese whey fermentation system. Water Sci Technol 2011 Jul 1;64(2):367–74.
https://doi.org/10.2166/wst.2011.631.
[153] Zhu H, Stadnyk A, Béland M, Seto P. Co-production of hydrogen and methane
from potato waste using a two-stage anaerobic digestion process. Bioresour
Technol 2008 Jul 1;99(11):5078–84. https://doi.org/10.1016/j.
biortech.2007.08.083.
[154] DiStefano TD, Palomar A. Effect of anaerobic reactor process configuration on
useful energy production. Water Res 2010 Apr 1;44(8):2583–91. https://doi.org/
10.1016/j.watres.2010.01.010.
[155] Cheng J, Xie B, Zhou J, Song W, Cen K. Cogeneration of H2 and CH4 from water
hyacinth by two-step anaerobic fermentation. Int J Hydrogen Energy 2010 Apr 1;
35(7):3029–35. https://doi.org/10.1016/j.ijhydene.2009.07.012.
[156] Rafieenia R, Girotto F, Peng W, Cossu R, Pivato A, Raga R, et al. Effect of aerobic
pre-treatment on hydrogen and methane production in a two-stage anaerobic
digestion process using food waste with different compositions. Waste Manage
2017;59:194–9. https://doi.org/10.1016/j.wasman.2016.10.028.
[157] Sun C, Xia A, Fu Q, Huang Y, Lin R, Murphy JD. Effects of pre-treatment and
biological acidification on fermentative hydrogen and methane co-production.
Energy Convers Manage 2019 Apr;1(185):431–41. https://doi.org/10.1016/j.
enconman.2019.01.118.
[158] Corona VM, Razo-Flores E. Continuous hydrogen and methane production from
Agave tequilana bagasse hydrolysate by sequential process to maximize energy
recovery efficiency. Bioresour Technol 2018 Feb;1(249):334–41. https://doi.org/
10.1016/j.biortech.2017.10.032.
[159] Kanchanasuta S, Sillaparassamee O. Enhancement of hydrogen and methane
production from co-digestion of palm oil decanter cake and crude glycerol using
two stage thermophilic and mesophilic fermentation. Int J Hydrogen Energy 2017
Feb 2;42(5):3440–6. https://doi.org/10.1016/j.ijhydene.2017.01.032.
[160] Sołowski G, Konkol I, Cenian A. Methane and hydrogen production from cotton
waste by dark fermentation under anaerobic and micro-aerobic conditions.
Biomass Bioenergy 2020 Jul;1(138):105576. https://doi.org/10.1016/j.
biombioe.2020.105576.
[161] Farhat A, Miladi B, Hamdi M, Bouallagui H. Fermentative hydrogen and methane
co-production from anaerobic co-digestion of organic wastes at high loading rate
coupling continuously and sequencing batch digesters. Environ Sci Pollut Res
2018 Oct;25(28):27945–58. https://doi.org/10.1007/s11356-018-2796-2.
[162] Ganesh R, Torrijos M, Sousbie P, Lugardon A, Steyer JP, Delgenes JP. Single-
phase and two-phase anaerobic digestion of fruit and vegetable waste:
comparison of start-up, reactor stability and process performance. Waste Manage
2014 May 1;34(5):875–85. https://doi.org/10.1016/j.wasman.2014.02.023.
[163] Silva FM, Mahler CF, Oliveira LB, Bassin JP. Hydrogen and methane production in
a two-stage anaerobic digestion system by co-digestion of food waste, sewage
20
Fuel 317 (2022) 123449
sludge and glycerol. Waste Manage 2018 Jun;1(76):339–49. https://doi.org/
10.1016/j.wasman.2018.02.039.
[164] Cremonez PA, Sampaio SC, Teleken JG, Meier TW, Frigo EP, de Rossi E, et al.
Effect of substrate concentrations on methane and hydrogen biogas production by
anaerobic digestion of a cassava starch-based polymer. Ind Crops Prod 2020;151:
112471. https://doi.org/10.1016/j.indcrop.2020.112471.
[165] Algapani DE, Qiao W, Ricci M, Bianchi D, Wandera SM, Adani F, et al. Bio-
hydrogen and bio-methane production from food waste in a two-stage anaerobic
digestion process with digestate recirculation. Renewable Energy 2019;130:
1108–15. https://doi.org/10.1016/j.renene.2018.08.079.
[166] Xie B, Cheng J, Zhou J, Song W, Liu J, Cen K. Production of hydrogen and
methane from potatoes by two-phase anaerobic fermentation. Bioresour Technol
2008 Sep 1;99(13):5942–6. https://doi.org/10.1016/j.biortech.2007.10.048.
[167] Yeshanew MM, Paillet F, Barrau C, Frunzo L, Lens PNL, Esposito G, et al. Co-
production of hydrogen and methane from the organic fraction of municipal solid
waste in a pilot scale dark fermenter and methanogenic biofilm reactor. Front
Environ Sci 2018 Jun;6. https://doi.org/10.3389/fenvs.2018.00041.
[168] Krishnan S, Singh L, Sakinah M, Thakur S, Nasrul M, Otieno A, et al. An
investigation of two-stage thermophilic and mesophilic fermentation process for
the production of hydrogen and methane from palm oil mill effluent. Environ
Prog Sustainable Energy 2017;36(3):895–902. https://doi.org/10.1002/
ep.12537.
[169] Song W, Cheng J, Zhou J, Xie B, Su H, Cen K. Cogeneration of hydrogen and
methane from protein-mixed food waste by two-phase anaerobic process. Int J
Hydrogen Energy 2010;35(7):3141–6.
[170] Wang X, Zhao YC. A bench scale study of fermentative hydrogen and methane
production from food waste in integrated two-stage process. Int J Hydrogen
Energy 2009 Jan 1;34(1):245–54. https://doi.org/10.1016/j.
ijhydene.2008.09.100.
[171] Antonopoulou G, Stamatelatou K, Venetsaneas N, Kornaros M, Lyberatos G.
Biohydrogen and methane production from cheese whey in a two-stage anaerobic
process. Ind Eng Chem Res 2008 Aug 6;47(15):5227–33. https://doi.org/
10.1021/ie071622x.
[172] 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 Aug
1;100(15):3713–7. https://doi.org/10.1016/j.biortech.2009.01.025.
[173] Antonopoulou G, Gavala HN, Skiadas IV, Angelopoulos K, Lyberatos G. Biofuels
generation from sweet sorghum: fermentative hydrogen production and
anaerobic digestion of the remaining biomass. Bioresour Technol 2008 Jan 1;99
(1):110–9. https://doi.org/10.1016/j.biortech.2006.11.048.
[174] Nath K, Das D. Improvement of fermentative hydrogen production: various
approaches. Appl Microbiol Biotechnol 2004 Oct;65(5):520–9. https://doi.org/
10.1007/s00253-004-1644-0.
[175] Claassen PAM, van Lier JB, Lopez Contreras AM, van Niel EWJ, Sijtsma L,
Stams AJM, et al. Utilisation of biomass for the supply of energy carriers. Appl
Microbiol Biotechnol 1999;52(6):741–55. https://doi.org/10.1007/
s002530051586.
[176] Ghosh D, Hallenbeck PC. Fermentative hydrogen yields from different sugars by
batch cultures of metabolically engineered Escherichia coli DJT135. Int J
Hydrogen Energy 2009 Oct 1;34(19):7979–82. https://doi.org/10.1016/j.
ijhydene.2009.08.004.
[177] Yoshida A, Nishimura T, Kawaguchi H, Inui M, Yukawa H. Enhanced hydrogen
production from glucose using ldh-and frd-inactivated Escherichia coli strains.
Appl Microbiol Biotechnol 2006 Nov;73(1):67–72. https://doi.org/10.1007/
s00253-006-0456-9.
[178] Maeda T, Sanchez-Torres V, Wood TK. Enhanced hydrogen production from
glucose by metabolically engineered Escherichia coli. Appl Microbiol Biotechnol
2007 Dec;77(4):879–90. https://doi.org/10.1007/s00253-007-1217-0.
[179] Sauter M, Böhm R, Böck A. Mutational analysis of the operon (hyc) determining
hydrogenase 3 formation in Escherichia coli. Mol Microbiol 1992;6(11):1523–32.
https://doi.org/10.1111/j.1365-2958.1992.tb00873.x.
[180] Lloyd JR, Thomas GH, Finlay JA, Cole JA, Macaskie LE. Microbial reduction of
technetium by Escherichia coli and Desulfovibrio desulfuricans: Enhancement via
the use of high-activity strains and effect of process parameters. Biotechnol
Bioeng 1999;66(2):122–30. https://doi.org/10.1002/(SICI)1097-0290(1999)66:
2<122::AID-BIT5>3.0.CO;2-Y.
[181] Sanchez-Torres V, Maeda T, Wood TK. Protein engineering of the transcriptional
activator FhlA to enhance hydrogen production in Escherichia coli. Appl Environ
Microbiol 2009 Sep 1;75(17):5639–46. https://doi.org/10.1128/AEM.00638-09.
[182] Achinas S, Euverink GJ. Theoretical analysis of biogas potential prediction from
agricultural waste. Resour-Effic Technol 2016 Sep 1;2(3):143–7. https://doi.org/
10.1016/j.reffit.2016.08.001.
[183] Symons GE, Buswell AM. The methane fermentation of carbohydrates1, 2. J Am
Chem Soc 1933 May;55(5):2028–36. https://doi.org/10.1021/ja01332a039.
[184] Ferry JG. Fundamentals of methanogenic pathways that are key to the
biomethanation of complex biomass. Curr Opin Biotechnol 2011 Jun 1;22(3):
351–7. https://doi.org/10.1016/j.copbio.2011.04.011.
[185] Chang H, Zou Y, Hu R, Feng H, Wu H, Zhong N, et al. Membrane applications for
microbial energy conversion: a review. Environ Chem Lett 2020;18(5):1581–92.
https://doi.org/10.1007/s10311-020-01032-7.
[186] Kumar G, Zhen G, Sivagurunathan P, Bakonyi P, Nemestóthy N, Bélafi-Bakó K,
et al. Biogenic H2 production from mixed microalgae biomass: impact of pH
control and methanogenic inhibitor (BESA) addition. Biofuel Res J 2016 Jul 1;3
(3):470–4.
K.B. Sasidhar et al.
[187] Gonzales RR, Kim SH. Dark fermentative hydrogen production following the
sequential dilute acid pretreatment and enzymatic saccharification of rice husk.
Int J Hydrogen Energy 2017 Nov 9;42(45):27577–83. https://doi.org/10.1016/j.
ijhydene.2017.08.185.
[188] Liu IC, Whang LM, Ren WJ, Lin PY. The effect of pH on the production of
biohydrogen by clostridia: thermodynamic and metabolic considerations. Int J
Hydrogen Energy 2011 Jan 1;36(1):439–49. https://doi.org/10.1016/j.
ijhydene.2010.10.045.
[189] Ruggeri B, Tommasi T, Sanfilippo S. BioH2 & BioCH4 through anaerobic
digestion: from research to full-scale applications. Springer; 2015 Feb 2.
[190] Van Lier JB, Mahmoud N, Zeeman G. Anaerobic wastewater treatment. Biological
wastewater treatment: principles. modelling and design. 2008;415–56. https://
doi.org/10.2166/9781789060362_0701.
[191] Zinder SH, Anguish T, Cardwell SC. Effects of temperature on methanogenesis in a
thermophilic (58 C) anaerobic digestor. Appl Environ Microbiol 1984 Apr;47(4):
808–13. https://doi.org/10.1128/aem.47.4.808-813.1984.
[192] Kim W, Shin SG, Lim J, Hwang S. Effect of temperature and hydraulic retention
time on volatile fatty acid production based on bacterial community structure in
anaerobic acidogenesis using swine wastewater. Bioprocess Biosyst Eng 2013 Jun;
36(6):791–8. https://doi.org/10.1007/s00449-013-0905-7.
[193] Bengtsson S, Hallquist J, Werker A, Welander T. Acidogenic fermentation of
industrial wastewaters: Effects of chemostat retention time and pH on volatile
fatty acids production. Biochem Eng J 2008 Jul 1;40(3):492–9. https://doi.org/
10.1016/j.bej.2008.02.004.
[194] Lim S-J, Kim BJ, Jeong C-M, Choi J-D-R, Ahn YH, Chang HN. Anaerobic organic
acid production of food waste in once-a-day feeding and drawing-off bioreactor.
Bioresour Technol 2008;99(16):7866–74. https://doi.org/10.1016/j.
biortech.2007.06.028.
[195] Abe JO, Popoola AP, Ajenifuja E, Popoola OM. Hydrogen energy, economy and
storage: review and recommendation. Int J Hydrogen Energy 2019 Jun 7;44(29):
15072–86. https://doi.org/10.1016/j.ijhydene.2019.04.068.
[196] Tang J, Wang X, Hu Y, Zhang Y, Li Y. Lactic acid fermentation from food waste
with indigenous microbiota: Effects of pH, temperature and high OLR. Waste
Manage 2016 Jun;1(52):278–85. https://doi.org/10.1016/j.
wasman.2016.03.034.
[197] Zhang S, Lee Y, Kim TH, Hwang SJ. Effects of OLRs and HRTs on hydrogen
production from high salinity substrate by halophilic hydrogen producing
bacterium (HHPB). Bioresour Technol 2013 Aug;1(141):227–32. https://doi.org/
10.1016/j.biortech.2012.12.056.
[198] Mohan SV, Bhaskar YV, Krishna PM, Rao NC, Babu VL, Sarma PN. Biohydrogen
production from chemical wastewater as substrate by selectively enriched
anaerobic mixed consortia: influence of fermentation pH and substrate
composition. Int J Hydrogen Energy 2007 Sep 1;32(13):2286–95. https://doi.
org/10.1016/j.ijhydene.2007.03.015.
[199] 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 Dec;1(173):256–65. https://doi.org/10.1016/
j.biortech.2014.09.039.
[200] Jariyaboon R, Sompong O, Kongjan P. Bio-hydrogen and bio-methane potentials
of skim latex serum in batch thermophilic two-stage anaerobic digestion.
Bioresour Technol 2015 Dec;1(198):198–206. https://doi.org/10.1016/j.
biortech.2015.09.006.
[201] Zhong J, Stevens DK, Hansen CL. Optimization of anaerobic hydrogen and
methane production from dairy processing waste using a two-stage digestion in
induced bed reactors (IBR). Int J Hydrogen Energy 2015 Dec 7;40(45):15470–6.
https://doi.org/10.1016/j.ijhydene.2015.09.085.
[202] Kim DH, Han SK, Kim SH, Shin HS. Effect of gas sparging on continuous
fermentative hydrogen production. Int J Hydrogen Energy 2006 Dec 1;31(15):
2158–69. https://doi.org/10.1016/j.ijhydene.2006.02.012.
[203] Bundhoo MZ, Mohee R. Inhibition of dark fermentative bio-hydrogen production:
a review. Int J Hydrogen Energy 2016 May 4;41(16):6713–33. https://doi.org/
10.1016/j.ijhydene.2016.03.057.
[204] Saady NM. Homoacetogenesis during hydrogen production by mixed cultures
dark fermentation: unresolved challenge. Int J Hydrogen Energy 2013 Oct 8;38
(30):13172–91. https://doi.org/10.1016/j.ijhydene.2013.07.122.
[205] Chen Y, Cheng JJ, Creamer KS. Inhibition of anaerobic digestion process: a
review. Bioresour Technol 2008 Jul 1;99(10):4044–64. https://doi.org/10.1016/
j.biortech.2007.01.057.
[206] Liu J, Luo J, Zhou J, Liu Q, Qian G, Xu ZP. Inhibitory effect of high-strength
ammonia nitrogen on bio-treatment of landfill leachate using EGSB reactor under
mesophilic and atmospheric conditions. Bioresour Technol 2012 Jun;1(113):
239–43. https://doi.org/10.1016/j.biortech.2011.11.114.
[207] Chaudhry R, Varacallo M. Biochemistry, Glycolysis. [Updated 2020 Sep 13]. In:
StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021 Jan-.
Available from: https://www.ncbi.nlm.nih.gov/books/NBK482303/.
[208] Mancipe-Jiménez DC, Costa C, Márquez MC. Methanogenesis inhibition by
phosphorus in anaerobic liquid waste treatment. Waste Treatment and Recovery.
2017 Apr 1;2(1):1–8. https://doi.org/10.1515/lwr-2017-0001.
[209] O’Flaherty V, Colleran E. Effect of sulphate addition on volatile fatty acid and
ethanol degradation in an anaerobic hybrid reactor. I: process disturbance and
remediation. Bioresour Technol 1999 May 1;68(2):101–7. https://doi.org/
10.1016/S0960-8524(98)00145-X.
[210] Sinha P, Pandey A. An evaluative report and challenges for fermentative
biohydrogen production. Int J Hydrogen Energy 2011 Jul 1;36(13):7460–78.
https://doi.org/10.1016/j.ijhydene.2011.03.077.
21
Fuel 317 (2022) 123449
[211] Bao MD, Su HJ, Tan TW. Dark fermentative bio-hydrogen production: Effects of
substrate pre-treatment and addition of metal ions or L-cysteine. Fuel 2013 Oct;1
(112):38–44. https://doi.org/10.1016/j.fuel.2013.04.063.
[212] Schmidt JE, Ahring BK. Effects of magnesium on thermophilic acetate-degrading
granules in upflow anaerobic sludge blanket (UASB) reactors. Enzyme Microb
Technol 1993 Apr 1;15(4):304–10. https://doi.org/10.1016/0141-0229(93)
90155-U.
[213] Srikanth S, Mohan SV. Regulatory function of divalent cations in controlling the
acidogenic biohydrogen production process. RSC Adv 2012;2(16):6576–89.
https://doi.org/10.1039/C2RA20383A.
[214] Fang HH, Hui HH. Effect of heavy metals on the methanogenic activity of starch-
degrading granules. Biotechnol Lett 1994 Oct;16(10):1091–6. https://doi.org/
10.1007/BF01022409.
[215] Haroun BM, Nakhla G, Hafez H, Nasr FA. Impact of furfural on biohydrogen
production from glucose and xylose in continuous-flow systems. Renewable
Energy 2016 Aug;1(93):302–11. https://doi.org/10.1016/j.renene.2016.02.072.
[216] Uberoi V, Bhattacharya SK. Toxicity and degradability of nitrophenols in
anaerobic systems. Water Environ Res 1997 Mar;69(2):146–56. https://doi.org/
10.2175/106143097X125290.
[217] Liu T, Sung S. Ammonia inhibition on thermophilic aceticlastic methanogens.
Water Sci Technol 2002 May;45(10):113–20. https://doi.org/10.2166/
wst.2002.0304.
[218] Bell DA, Towler BF, Fan M. Coal gasification and its applications. William
Andrew; 2010 Dec 8. doi: https://doi.org/10.1016/B978-0-8155-2049-8.10006-
3.
[219] Zhao Q, Leonhardt E, MacConnell C, Frear C, Chen S. Purification technologies for
biogas generated by anaerobic digestion. Available from: Compressed
Biomethane, CSANR, Ed. 2010;24. http://www.build-a-biogas-plant.com/PDF/Bi
ogasPurificationTech2010.PDF.
[220] Cavenati S, Grande CA, Rodrigues AE. Upgrade of methane from landfill gas by
pressure swing adsorption. Energy Fuels 2005 Nov 16;19(6):2545–55. https://
doi.org/10.1021/ef050072h.
[221] Sircar S, Golden TC. Purification of hydrogen by pressure swing adsorption. Sep
Sci Technol 2000 Jan 5;35(5):667–87. https://doi.org/10.1081/SS-100100183.
[222] Du Z, Liu C, Zhai J, Guo X, Xiong Y, Su W, et al. A Review of Hydrogen
Purification Technologies for Fuel Cell Vehicles. Catalysts. 2021 Mar;11(3):393.
https://doi.org/10.3390/catal11030393.
[223] Teplyakov VV, Gassanova LG, Sostina EG, Slepova EV, Modigell M, Netrusov AI.
Lab-scale bioreactor integrated with active membrane system for hydrogen
production: experience and prospects. Int J Hydrogen Energy 2002 Nov 1;27
(11–12):1149–55. https://doi.org/10.1016/S0360-3199(02)00093-9.
[224] Liang TM, Cheng SS, Wu KL. Behavioral study on hydrogen fermentation reactor
installed with silicone rubber membrane. Int J Hydrogen Energy 2002 Nov 1;27
(11–12):1157–65. https://doi.org/10.1016/S0360-3199(02)00099-X.
[225] Züttel A. Hydrogen storage methods. Naturwissenschaften 2004 Apr;91(4):
157–72. https://doi.org/10.1007/s00114-004-0516-x.
[226] Zhou L. Progress and problems in hydrogen storage methods. Renew Sustain
Energy Rev 2005 Aug 1;9(4):395–408. https://doi.org/10.1016/j.
rser.2004.05.005.
[227] Kumar Gupta S, Kumari S, Reddy K, Bux F. Trends in biohydrogen production:
major challenges and state-of-the-art developments. Environ Technol 2013 Jul 1;
34(13–14):1653–70. https://doi.org/10.1080/09593330.2013.822022.
[228] Scholz M, Frank B, Stockmeier F, Falß S, Wessling M. Techno-economic analysis of
hybrid processes for biogas upgrading. Ind Eng Chem Res 2013 Nov 27;52(47):
16929–38. https://doi.org/10.1021/ie402660s.
[229] Patterson T, Esteves S, Dinsdale R, Guwy A. An evaluation of the policy and
techno-economic factors affecting the potential for biogas upgrading for transport
fuel use in the UK. Energy policy. 2011 Mar 1;39(3):1806–16. https://doi.org/
10.1016/j.enpol.2011.01.017.
[230] Akansu SO, Dulger Z, Kahraman N, Veziroǧlu TN. Internal combustion engines
fueled by natural gas—hydrogen mixtures. Int J Hydrogen Energy 2004 Nov 1;29
(14):1527–39. https://doi.org/10.1016/j.ijhydene.2004.01.018.
[231] Velazquez-Abad A, Dodds P. Production of Hydrogen. Encyclopedia of
Sustainable Technologies. 2017 Jul;4:293–304. https://doi.org/10.1016/B978-0-
12-409548-9.10117-4.
[232] Forster-Carneiro T, Pérez M, Romero LI. Influence of total solid and inoculum
contents on performance of anaerobic reactors treating food waste. Bioresour
Technol 2008 Oct 1;99(15):6994–7002. https://doi.org/10.1016/j.
biortech.2008.01.018.
[233] Abbassi-Guendouz A, Brockmann D, Trably E, Dumas C, Delgenès J-P, Steyer J-P,
et al. Total solids content drives high solid anaerobic digestion via mass transfer
limitation. Bioresour Technol 2012;111:55–61. https://doi.org/10.1016/j.
biortech.2012.01.174.
[234] De Bere L. Anaerobic digestion of solid waste: state-of-the-art. Water Sci Technol
2000 Feb;41(3):283–90. https://doi.org/10.2166/wst.2000.0082.
[235] Rakopoulos CD, Scott MA, Kyritsis DC, Giakoumis EG. Availability analysis of
hydrogen/natural gas blends combustion in internal combustion engines. Energy.
2008 Feb 1;33(2):248–55. https://doi.org/10.1016/j.energy.2007.05.009.
[236] Wang J, Huang Z, Fang Yu, Liu B, Zeng Ke, Miao H, et al. Combustion behaviors of
a direct-injection engine operating on various fractions of natural gas–hydrogen
blends. Int J Hydrogen Energy 2007;32(15):3555–64. https://doi.org/10.1016/j.
ijhydene.2007.03.011.
[237] Hoekstra RL, Van Blarigan P, Mulligan N. NOx emissions and efficiency of
hydrogen, natural gas, and hydrogen/natural gas blended fuels. Available from
SAE Trans 1996 Jan;1:761–73. http://www.jstor.org/stable/44729090.
K.B. Sasidhar et al.
[238] Bysveen M. Engine characteristics of emissions and performance using mixtures
of natural gas and hydrogen. Energy 2007 Apr 1;32(4):482–9. https://doi.org/
10.1016/j.energy.2006.07.032.
[239] Larsen JF, Wallace JS. Comparison of emissions and efficiency of a turbocharged
lean-burn natural gas and hythane-fueled engine. doi: https://doi.org/10.1115/
1.2815553.
[240] Graham LA, Rideout G, Rosenblatt D, Hendren J. Greenhouse gas emissions from
heavy-duty vehicles. Atmos Environ 2008;42(19):4665–81. https://doi.org/
10.1016/j.atmosenv.2008.01.049.
[241] Jiafeng Yu, Jian Liu, P. Senthil Kumar, Yunwei Wei, Meng Zhou, Dai-Viet N.Vo,
Leilei Xiao, Promotion of methane production by magnetite via increasing
acetogenesis revealed by metagenome-assembled genomes, Biores Technol, 345
(2022) 126521.
[242] Xiao Leilei, Sun Ran, Zhang Peng, Zheng Shiling, Tan Yang, Li Jiajia,
Zhang Yuechao, Liu Fanghua. Simultaneous intensification of direct acetate
cleavage and CO2 reduction to generate methane by bioaugmentation and
increased electron transfer. Chem Eng J 2019;378:122229.
[243] Xiao L, Lichtfouse E, Kumar S, Wang Q, Liu F. Biochar promotes methane
production during anaerobic digestion of organic waste. Environ Chem Lett 2021;
19:3557–64.
[244] Xiao L, Wang Y, Lichtfouse E, Li Z, Kumar S, Liu J, Feng D, Yang Q, Liu F. Effect of
antibiotics on the microbial efficiency of anaerobic digestion of wastewater: A
review. Front Microbiol 2021;11:611613.
22
Fuel 317 (2022) 123449
[245] Xiao Leilei, Zheng Shiling, Lichtfouse Eric, Luo Min, Tan Yang, Liu Fanghua.
Carbon nanotubes accelerate acetoclastic methanogenesis: From pure culture to
anaerobic soil. Soil Biol Biochem 2020;150:107938.
[246] Xiao Leilei, Liu Fanghua, Lichtfouse Eric, Zhang Peng, Li Fangbai. Methane
production by acetate dismutation stimulated by Shewanella oneidensis and
carbon materials: An alternative to classical CO2 reduction. Chem Eng J 2020;
389:124469.
[247] Zhang Yuechao, Liu Fanghua, Hengduo Xu, Xiao Leilei. Extraction of electrons by
magnetite and ferrihydrite from hydrogen-producing clostridium bifermentans by
strengthening the acetate production pathway. Sci China Technol Sc 2019;62:
1719–25.
[248] Li Jiajia, Xiao Leilei, Zheng Shiling, Zhang Yuechao, Luo Min, Tong Chuan,
Hengduo Xu, Tan Yang, Liu Jinchao, Wang Oumei, Liu Fanghua. A new insight
into the strategy for methane production affected by conductive carbon cloth in
wetland soil: Beneficial to acetoclastic methanogenesis instead of CO2 reduction.
Sci Total Environ 2018;643:1024–30.
[249] Xiao Leilei, Liu Fanghua, Liu Jinchao, Li Jiajia, Zhang Yuechao, Jiafeng Yu,
Wang Oumei. Nano-Fe3O4 particles accelerating electromethanogenesis on an
hourlong timescale in wetland soil. Environ Sci: Nano 2018;5:436–45.
[250] Zhang Yuechao, Liu Fanghua, Hao Qinqin, Xiao Leilei. Target-oriented
recruitment of Clostridium to promote biohydrogen production by nano-
ferrihydrite. Fuel 2020;276:118049.
[251] Xiao Leilei, Lichtfouse Eric, Kumar Senthil. Advantage of conductive materials on
interspecies electron transfer-independent acetoclastic methanogenesis: A critical
review. Fuel 2021;306:121577.