Renewable and Sustainable Energy Reviews 44 (2015) 728–737

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Renewable and Sustainable Energy Reviews

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

Lignocellulose biohydrogen: Practical challenges and recent progress

G. Kumar a, P. Bakonyi b,n, S. Periyasamy c, S.H. Kim a, N. Nemestóthy b, K. Bélafi-Bakó b
a
Department of Environmental Engineering, Daegu University, Gyeongsan, Gyeongbuk 712-714, Republic of Korea
b
Research Institute on Bioengineering, Membrane Technology and Energetics, University of Pannonia, Egyetem ut 10, 8200 Veszprém, Hungary
c
Department of Environmental Engineering and Science, Feng Chia University, Taichung 40724, Taiwan

art ic l e i nf o a b s t r a c t

Article history: The objective of this work is to provide update information about the recent progress on lignocellulose
Received 6 August 2014 hydrogen conversion via dark fermentation. Therefore, in this review, the most important fields
Received in revised form associated with lignocellulosic hydrogen fermentation are covered, firstly describing the problems
5 January 2015
associated with raw materials, followed by the discussion of pretreatment and hydrolysis methods
Accepted 6 January 2015
assisting to achieve efficient hydrogen fermentation. Afterwards, issues related to fermentation
inhibitors as well as advanced, integrated bioprocesses for hydrogen production purposes will be
Keywords: presented. Additionally, the paper gives future perspectives of lignocellulose biohydrogen.
Lignocellulose & 2015 Elsevier Ltd. All rights reserved.
Hydrogen
Biohydrogen
Production
Fermentation

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 728
2. Characteristics of lignocellulose biomass – the roles of pretreatment and hydrolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 729
3. Practical difficulties with lignocellulosic pretreatment – inhibitor formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 731
4. Advanced and integrated processes for lignocellulosic biohydrogen production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 732
5. Prospects and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 734
6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 735
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 735
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 735

1. Introduction infrastructures and to produce highly energy efficient as well as

Hydrogen is widely taken into account as a potential solution
for sustainable energy generation not exclusively but especially for
transportation. Driven by the advantages of using hydrogen as a
new age fuel – such as only water vapor is released when it
undergoes combustion – a world-wide progress has been started
with the ultimate objective of promoting the transition into hydrogen
based economy [1].
Nowadays, various countries and several leading companies of
the automotive industry are putting efforts and investing signifi-
cant amount of money to establish national hydrogen fueling
n
Corresponding author. Tel.: þ 36 88 624385; fax: þ 36 88 624292.
E-mail address: bakonyip@almos.uni-pannon.hu (P. Bakonyi).
environmental-friendly fuel cell vehicles [2]. In other words,
hydrogen economy is not only a fine-sounding and theoretical
concept anymore. However, it is important to note that the path
towards its realization is still challenging due to obstacles such as
the large-scale unavailability of ecologically viable production
technologies.
Currently, most of hydrogen used in different industrial appli-
cations or transportation is delivered from non-renewable, carbo-
naceous substances e.g. methane [3]. Therefore, these energy-
intense and polluting methods do not indeed ensure “green”
hydrogen. Hence, to meet the requirements of sustainable devel-
opment, hydrogen should be originated from renewable resources
via environmentally benign and energetically less demanding
techniques. For these purposes, attention is paid to the biotech-
nological approaches aided by specific microbial catalysts that

http://dx.doi.org/10.1016/j.rser.2015.01.042
1364-0321/& 2015 Elsevier Ltd. All rights reserved.
 G. Kumar et al. / Renewable and Sustainable Energy Reviews 44 (2015) 728–737
convert biomass derived materials into molecular hydrogen gas
under anaerobic conditions [4].
Anaerobic hydrogen fermentation was first time reported in the
late 1970 [5] and research of this area has been considerably
expanded. As a result of the extensive investigations in the last
30–40 years, noticeable advancements were accomplished that have
brought the term “biohydrogen” to the spotlight of public awareness.
Currently, among the various conventional ways of generating biohy-
drogen such as biophotolysis, photo fermentation and dark fermenta-
tion, the latter appears as the most feasible one from practical point of
view [4,6]. In recent decades, overwhelming projects have focused on
numerous but equally substantial aspects of dark fermentative hydro-
gen production, including strain selection and development [7,8],
bioreactor engineering [9–11], downstream processes such as biohy-
drogen recovery and purification [12–14], processing fermentation
effluent [15] and feedstock utilization [16–18]. The latter factor,
properties and valorization of fermentation raw materials such as
biomass, which is in the scope of this paper, play a crucial role in
biohydrogen production. It has been demonstrated well that renew-
able biomass can be a potential input material for hydrogen evolution
prominently because of its carbohydrate-rich feature [15–17].
Biomass may be distinguished into various categories [19]. The
so-called “first generation biomass” encompasses mainly food
plants such as corn, wheat, sugar beet, sugar cane, etc. having
easily accessible carbohydrate (e.g. starch) content. Even though
these materials would be ideal from technological standpoint,
their scaled-up employment for hydrogen fermentation likely fails
due to the “fuel vs. food” debate. Consequently, scientists and
engineers rather tend to apply “next generation organic matters”
such as lignocelluloses for hydrogen bioproduction. These abun-
dant materials are recognized as excellent sources for a range of
fermentable sugars, which, unlike in case of first generation
biomass are in a complex form and hardly digestible [20,21]. Thus,
usage of such substrates consisting of cellulose, hemicellulose and
lignin needs careful process design so as to achieve sufficient
hydrogen production capacities. The most substantial stages asso-
ciated with lignocellulosic biofuel generation are as follows [22]:
– Pretreatment of the raw material, when the aim is to break the
recalcitrant heteropolymeric structure of lignocellulose complex.
– Hydrolysis of the cellulose and hemicellulose fractions liber-
ated in order to attain large quantity monomeric sugars.
– Conversion of monomeric carbohydrates into biofuel by effi-
cient microorganisms in adequate bioprocesses.
The present article attempts to survey the recent advancements
made on the above-referenced crucial steps of microbiologically-
assisted lignocellulose hydrogen technology. Firstly, the inherent
traits of lignocellulosic biomass making it difficult to process are
introduced along with pretreatment and hydrolysis considerations.
Afterwards, the strategies for higher performance second generation
biohydrogen fermentation will be discussed, including the mitiga-
tion of fermentation inhibitors as well as integrated fermentation
concepts by touching bioreactor design, strain selection and bioca-
talyst improvement via metabolic engineering. Finally, possible
future perspectives are assessed, as well. Writing this review paper
was driven by the motivation of providing up to date information to
the scientific audience about the current status and technological
developments of lignocellulose biohydrogen production.
2. Characteristics of lignocellulose biomass – the roles of
pretreatment and hydrolysis
Lignocellulosic biomass is the most abundant raw material in the
nature, which covers hardwood, soft wood, grasses, agricultural and
729
forestry residues as well as secondary biofuel wastes. The annual
worldwide yield of lignocellulosic biomass residues is estimated to
exceed 220 billion tons. These materials are composed of holocel-
lulose (referring to cellulose and hemicellulose together and repre-
senting up to 70–80% of lignocellulosic biomass), which is coated
and intimately associated with lignin [23,24]. The former is the
desired portion for hydrogen production that is built up from
long-chain biopolymers, containing exclusively glucose in case of
cellulose and a variety of hexoses and pentoses (i.e. xylose) for
hemicellulose, respectively. On the other hand, presence of lignin is
disadvantageous from the viewpoint of biohydrogen fermentation
since it has notable persistency for biodegradation and only limited
number of species possesses capability for its decomposition.
Hence, due to the protected structure of raw cellulosic biomass,
attempts on its direct conversion into hydrogen are accompanied by
relatively low performances [25].
Therefore, in many cases when lignocellulosic organic matter is
to be utilized, pretreatment methods seem to be necessary. The
aim of any pretreatment is to increase the accessibility towards the
carbohydrate-rich holocellulose. Pretreatment techniques may be
carried out in versatile ways as detailed later on. Nevertheless, all
of them take an effect through the elimination of the “glue”
existing in between cellulose, hemicellulose and lignin, meaning
that the intermolecular or interpolymer linkages have to be
cleaved [26]. In addition, the common goal of lignocellulose
pretreatment is to provide increased surface area, porosity and
reactivity so that it could help the subsequent steps of polysac-
charide hydrolysis and fermentation where the H2 producers
are involved. In recent decade, tremendous investigations have
been performed with the purpose of developing feasible – both
conventional and unconventional – methods for lignocellulosic
biofuel technology [21,27]. Pretreatment usually starts with mech-
anical curing such as milling and grinding that can be followed by
chemical, physicochemical, thermal, biological, etc. procedures to
further destruct the tough structure. This may be achieved by
diluted or concentrated acids such as sulfuric acid, hydrochloric
acid, nitric acid [28] or organic acid e.g. acetic acid [29]. Alter-
natively to acid treatment, soaking of lignocellulosic biomass
in alkali solutions e.g. NaOH, ammonia, Ca(OH)2 could be used
[30–32]. Chemical pretreatment may apply conventional organic
solvents, novel ionic liquids [33,34] and powerful oxidizing agents
such as ozone [35], while the biologically-assisted way exploits
the lignocellulose-unlocking potential of fungi [33,36,37]. Besides,
(hydro)thermal treatment [38], steam-explosion [39], ultra-
sonication [34,40] and microwave irradiation [41,42] and their
combinations are also proven methods to structurally destroy
lignocellulosic materials. General factors apparently influence
pretreatment efficiency are the contact time, temperature and
pressure applied, pH, concentration of chemicals, biomass to
chemical ratio, etc. (Table 1). Moreover, it has been recently
demonstrated that even if various lignocellulosic feedstocks reflect
similar compositions and are pretreated under identical condi-
tions, there could be observable difference in their fermentibility
into bioH2 [28]. Consequently, it is assumable that even though the
biomass used for a long time is routinely collected from the same
source or place, the fine-tuning of the pretreatment process may
be needed over the time because of the seasonal alterations in the
composition of the raw material. Although pretreating substrate
can play a key role to get rid of lignin sealing in order to enhance
microbial hydrogen conversion, it consumes energy and therefore
has a negative contribution to the whole process economy [43].
Hence, considerable attention is paid to environmental-friendly
biological approaches as alternatives to mature chemical technol-
ogies since they possess the benefits of no inhibitory compound
formation, mild operational conditions as well as low energy
demand [36].
730 G. Kumar et al. / Renewable and Sustainable Energy Reviews 44 (2015) 728–737

Table 1
Examples of lignocellulosic biomass pretreatment methods for hydrogen fermentation.

Biomass Lignocellulose composition (%) Optimal pretreatment Inoculum Highest H2 yield Highest H2 production Reference
conditions rate
Lignin Cellulose Hemicellulose

Cornstalk 21.81 34.45 27.55 0.6% HCl (90 1C, 2 h) and Heat-pretreated 126.22 mL H2 g  1 DB 9.58 mL H2 g  1 DB h  1 [104]
Trichoderma viride cellulase anaerobic sludge
(50 1C, 72 h, pH 4.8)
Cornstalk 21.81 34.45 27.55 0.6% HCl (90 1C, 2 h) and Heat-pretreated 141.29 mL H2 g  1 DB 12.31 mL H2 g  1 DB h  1 [105]
Trichoderma viride cellulase anaerobic sludge
(50 1C, 72 h, pH 4.8)
Cornstalk 8.65 33.64 24.4 Microbe additives (25 1C, 15 Aerated microbial 176 mL H2 g  1 DB 18 mL H2 g  1 DB h  1 [106]
days) consortium
1 1 1
Beer lees 7.3 20.2 43.54 4% HCl (30 min, boiling) Anaerobic mixed 53.03 mL H2 g DB 4.48 mL H2 g DB h [108]
consortia
1 1 1
Lawn grass 2.9 30.8 43.1 4% HCl (30 min, boiling) Enriched mixed culture 72.21 mL H2 g DB 1.72 mL H2 g DB h [109]
dominated by
C. pasteurianum
Soybean 23.4 39.6 14.6 4% HCl (30 min, boiling) Enriched mixed culture 60.2 mL H2 g  1 DB 1.11 mL H2 g  1 DB h  1 [111]
straw dominated by
C. butiricum
Cornstalk 17.1 49.5 33.6 P. chrysosporium (29 1C, 15 T. thermosaccharolyticum 89.3 mL H2 g  1 DB 0.93 mL H2 g  1 DB h  1 [36]
days) W16
Poplar N.D. N.D. N.D. 2% Viscozyme L (50 1C, 3 h, Enriched mixed culture 44.92 mL H2 g  1 DB 1.09 mL H2 g  1 DB h  1 [116]
leaves pH 4) dominated by
C. pasteurianum
Cornstalk 21.52 38.92 20.87 0.2% HCl (30 min, boiling) Enriched consortia from 149.69 mL H2 g  1 TVS 7.6 mL H2 h  1 [103]
cow dung compost
1 a 1
Sugarcane 4.31 33.63 23.88 0.5% H2SO4 (60 min,121 1C, C. butyricum 44 mL H2 mmol TS 4.7 mL H2 h [107]
bagasse 1.47 bar)
Reed 9.5 45.5 26.5 3% HCl (90 min,121 1C, Enriched microbial 31.6 mL H2 g  1 DBb 1.08 mL H2 h  1 [110]
canary autoclave) culture
grass
(RCG)
Miscanthus 25 38.2 24.3 12% NaOH (70 1C, 4 h) and T. elfii DSM 9442 2280 mL H2c 23.99 mL H2 h  1b [112]
commercial enzymes (45 1C,
72 h, pH 4.8)
Rice straw 22.8 41.4 19.6 10% NH4OH (60 min, 121 1C) T. neapolitana 77.1 mL H2 g  1 DBd 0.19 mL H2 h  1 [113]
and 1% H2SO4 (50 min, 121 1C)
Sweet 17.6 38.5 21.4 10% NaOH (70 1C, 4 h) and C. saccharolyticus 73.6 mL H2 mmol  1 300 mL H2 h  1c [114]
sorghum commercial cellulase (50 1C, C6 sugarse
bagasse 24 h, pH 5)

TVS: total volatile solids; TS: total sugars; DB: dry biomass; N.D.: no data.
a
37 1C, 1 atm.
b
35 1C, 1 atm.
c
65 1C, 1 atm.
d
75 1C, 1 atm.
e
72 1C, 1 atm.

Even though a large variety of pretreatments are available to

recover the fermentable sugars, there is no any universal solution to
be suggested for all kinds of lignocellulosic biomass, leading to the
conclusion that a proper approach should be found for the specific
substrate. This statement is supported by the literature studies since
authors have examined and recommended different procedures for
optimal sugar release (Table 1). The reason for the variability could
presumably be ascribed to the diverse composition (different ratios
of cellulose, hemicellulose and lignin in the lignocellulosic matrix) of
the materials used. As it can be observed in Table 1, both hydrogen
yields and production rates vary according to the pretreatments
applied by various research laboratories in the range of 31.6–
176 mL H2 g  1 dry biomass and 0.19–300 mL H2 h  1, respectively.
It is to note that comparative evaluation of literature results could be
challenging since the results are presented on hardly comparable
basis (lack of information to calculate one unit to another) or with
different terminology. Nevertheless, Fig. 1 plots hydrogen productiv-
Fig. 1. H2 productivity vs. H2 yield obtained with pretreated lignocelluloses.
ity vs. hydrogen yield using studies in Table 1 whose data could be
converted to common units. The H2 productivity and yield are
widely accepted process indicators to assess the efficiency of This interpretation is supposed to highlight that each and every
the bioreaction. In Fig. 1, hydrogen productivity (vertical axis) is study in the field should aim to be qualified in an “attractive region”
defined as the time derivative of hydrogen yield (horizontal axis). – e.g. designated by the trend line fitted in Fig. 1 – where sufficiently
 G. Kumar et al. / Renewable and Sustainable Energy Reviews 44 (2015) 728–737 731

high values of both key parameters could coexist. This is a crucial
issue to be tackled for biohydrogen technology since notable
cumulative hydrogen generation – which may be correlated with
feedstock utilization efficacy – alone is not satisfactory, only if it is
fulfilled in a reasonable (short) time. When this problem is tend to
be resolved via continuous improvements it will result in the
expansion of upper bound performances that assists the successful
implementation of scaled-up systems and possible commercial
applications in the future.
As a result of pretreatment, the raw material is partly solubi-
lized, the carbohydrate fractions are more or less accessible and a
so-called pre-hydrolysate is obtained [44]. This material stream
can usually be characterized by a higher soluble chemical oxygen
demand to total chemical oxygen demand ratio because of
carbohydrate, protein, etc. release and hence it is far more feasible
for the whole cell biocatalysts to accomplish the task of molecular
hydrogen gas production [45]. However, this pre-hydrolysate
mixture is still composed of insoluble polymers, which need to
be split in order to be consumed and biotransformed by hydrogen
producing strains. This step, referred as hydrolysis, may be con-
ducted either by chemical and biological routes [46–48]. The
former relies e.g. on acidic or alkaline solutions, while the latter
is dependent on enzymes. Cellulose- and hemicellulose-decom-
posing biocatalysts – called as cellulases and hemicellulases – are
extracellular, potentially synergetic [49,50] and created by broad
range of microorganisms. Hydrolysis may be performed by using
purified, commercially available enzymes, however, it adds an
extra cost to the hydrogen production technology and restricts
economic viability. Hence, on-site hydrolytic protein generation by
carefully selected inoculum source can be a crucial issue to obtain
satisfactory hydrogen bioproduction from complex feedstock [51].
For this reason, it might be advisable to search for bacterial
sources demonstrating plant cell wall opening capability. Such
diverse microbial community with lignocellulose-deconstructing
affinity is located in the rumen of certain animals living on cellu-
losic organic matter [52]. Therefore, usage of seed inocula derived
from the manure of these creatures is expectedly a good way to
accelerate substrate solubilization and attain more promising H2
fermentation turnouts. Moreover, thermophilic and hyperthermo-
philic bacteria isolated from hot springs and from other natural
niches can also be characterized by fibrolytic properties and
considerable holocellulose hydrolyzing capability [53].
Though hydrolysis and subsequent fermentation are tradition-
ally carried out in different tanks, which is called as Separate
Hydrolysis and Fermentation (SHF), the combination of bacterial
populations expressing hydrolytic as well as hydrogen fermenting
activity has led to the development of advanced, integrated
technologies where depolymerization of cellulose/hemicellulose
and bioH2 generation take place successively at the same space
and time. These joint applications such as Simultaneous Sacchar-
ification and Fermentation (SSF) and Consolidated Bioprocess (CBP)
will be discussed later.
3. Practical difficulties with lignocellulosic pretreatment –
inhibitor formation
As presented in the previous sections, pretreatment could be a
key to unlock carbohydrate content of lignocellulosic organic
matter. However, pretreatment in most cases induces the forma-
tion of technologically unfavorable compounds, known as fermen-
tation inhibitors (Table 2). This phenomenon is complex and may
be coupled with the loss of fermentable sugars [54–56]. Inhibitory
substances might be present in the pre-hydrolysate and are
considered as hydrolysis and fermentation barriers, severely
depressing microbiological hydrogen production and concomi-
tantly process performance. These by-products of pretreatments
can be classified as phenolic components (originated from lignin
degradation), furan derivatives such as furfural and 5-hydro-
xymethylfurfural (generated from the transformation of C5 and
C6 sugars, respectively) and weak acids e.g. acetic acid, formic acid,
etc. [45].
During pretreatment, the ultimate aim is always to get better
accessibility to carbohydrate fractions and in parallel, to avoid the
formation of undesirable inhibitors, as secondary products. How-
ever, it is quite clear that the production of fermentation inhibitors
cannot be fully prevented but they can be more or less restricted
through the adequate control and optimization of the pretreat-
ment circumstances (e.g. exposure time, temperature, pressure,
concentration of chemicals and so on) to ensure an acceptable
sugar to inhibitor ratio [54,56]. Table 2 presents fermentation
inhibitor data found by various researchers. As it can be concluded,
only bio-pretreatment was not accompanied by inhibitor forma-
tion, in all the other cases at least one pretreatment by-product
could be detected.
It is important to note that these inhibitory components cause toxic
effect on cell growth and metabolic activity which may be explained
by distinct mechanisms. For instance, the most poisonous phenolic
agents injure cell membranes, furan-derivatives interact with enzymes
involved in fermentative bioreaction and undissociated carboxylic

Table 2
Fermentation inhibitor profiles of treated lignocellulosic biomass used for hydrogen fermentation.

Biomass Pretreatment conditions Inhibitors (g L  1) Reference

Furan compounds Weak acids Phenolic compounds

5-HMF Furfural Acetic acid Formic acid

Barley straw 0% H2SO4 (170 1C, 60 min) 0.1 0.4 2.1 N.D. N.D. [56]
Corn stover 1.69% H2SO4 (121 1C, 117 min) 0.41 1.35 N.D. 0.04 [54]
Cornstalk P. chrysosporium (29 1C, 15 days) N.D. N.D. N.D. N.D. N.D. [36]
Sunflower stalk 4% HCl (170 1C, 60 min) 0.13 1.15 0.31 0.6 0.02 [55]
L. japonica Thermal treatment (150–180 1C, 20 min) N.D. 1.09–3.18 N.D. N.D. N.D. [38]
Oil palm trunk 1.57% H2SO4 (7.62 min, N.D. N.D. 2.61 N.D. N.D. [41]
450 W microwave oven)
Oil palm empty fruit brunch 6% H2SO4 (120 1C, 15 min) 0.55 0.97 N.D. N.D. N.D. [44]
Miscanthus giganteus Hydrothermal treatment (150–180 1C, 0.063–0.98 1.54–6.96 3.72–7.52 N.D. N.D. [115]
6–35 min, 0.2–6.69 bar)
Sugarcane bagasse 2% H2SO4 (121 1C, 60 min) N.D. 0.19 1.49 N.D. N.D. [59]

N.D.: no data.
732 G. Kumar et al. / Renewable and Sustainable Energy Reviews 44 (2015) 728–737
acids have the ability to enter the intracellular space where they shift
the pH into an unfavorable range [57]. Nevertheless, the magnitude of
these harmful impacts is basically dependent on two factors, such as
concentration of the inhibitors and the specific tolerance of micro-
organisms towards them.
Since it is complicated to avoid the appearance of hindering
components introduced, detoxification processes could be applied
[58] covering neutralization, washing and removal e.g. by subject-
ing the pre-hydrolysate to ion exchange resin [59], applying
advanced oxidation processes [60], etc. In the removal of fermen-
tation inhibitors, an interesting concept, referred as Integrated
Biohydrogen Refinery [61] may stand as a way forward. This
strategy involves so-called electro-extractive fermentation [62]
which, in principles, combines bacterial hydrogen production with
a membrane process: electrodialysis. This application enables the
user to regulate pH by separation of organic acids – formed as
secondary metabolic products – from the culture media and hence
prolong efficient hydrogen production activity. Technically, how-
ever, this integrated, electrokinetic detoxification could be an
equally useful opportunity to discharge some of the poisonous
materials loaded to the fermentation liquor with the pretreated
(and hydrolyzed) biomass feedstock [63]. Even though separation
of inhibitors from (pre-)hydrolysates is promising to subsequently
obtain higher hydrogen fermentation efficiencies, it means an
extra cost for the production.
4. Advanced and integrated processes for lignocellulosic
biohydrogen production
After pretreatment stage hydrolysis of the oligomer substances
released is essential to attain higher monomeric sugar quantities
[64,65]. This hydrolysis can be realized either prior to the hydro-
gen fermenter or directly inside that. The former is the most
conventional case and known as SHF [66], as specified in the
previous section. The advantage of SHF is that both hydrolysis and
fermentation can be conducted under their optimal circumstances
e.g. temperature. On the other hand, hydrolysis may be inhibited
due to accumulation of monomeric sugars as end-products in the
liquor, which is assigned as the major drawback of this technology.
This bottleneck can be overcome by using integrated applications
as possible ways forward. The common in these approaches is that
they are devoted to accomplish hydrolysis and fermentation in a
single apparatus. The simplest integrated concept is the SSF, which
has been reported as a promising alternative having a remarkable
contribution to improved bacterial hydrogen evolution [24,36].
Furthermore, SSF is considered as an economic solution because of
the reduced process time demand and less equipment investment.
Recently, a new approach, called Simultaneous Saccharification,
Filtration and Fermentation (SSFF) has been introduced for the
intensification of biological hydrogen generation [59], where an
extra filtration step is inserted, as well.
Alternatively to SSF described, bioH2 production in CBP –
sometimes mentioned as direct microbial conversion – is an
emerging option, presenting a highly integrated, one-stage design
for the utilization of lignocellulosic biomass [67]. The aim of CBP is
the direct and satisfactory conversion of cellulosic materials into
biofuels that could lead to more competitive and economically
feasible technologies and hence, bring the biofuel dream into
reality.
Consolidated Bioprocessing owns all the advantageous qualities
of SSF and in addition, it further cuts the investment cost due to
the even lower installation (e.g. number of bioreactors) needs. In
this construction, specific microbial communities get to work,
generate (hemi)cellulolytic enzyme cocktail, accomplish the
hydrolysis of pretreated biomass moreover simultaneously and
in-situ convert the C5- and C6-monosaccharides released into
hydrogen gas. This comprehensive process design is highly attrac-
tive from economic point of view since it considers all the
ingredients of holocellulose biopolymer and in this a way it
exploits better the potential of the raw material. Nevertheless,
CBP has to have more trade-offs in comparison with SSF since
optimum conditions for the higher number of individual bioreac-
tions taking place together can differ significantly.
Some researchers have already emphasized the application of
CBP for biohydrogen production purposes. In a consolidated
biprocess, the presence of acclimatized consortia or purposefully
chosen pure- and functional cultures with (synergetic) holocellu-
lose degrading and H2 producing capability could appear as the
most crucial criteria [16,17,45,51,68,69].
Table 3 summarizes literature studies conducted in various
bioprocesses. It can be extracted from Table 3 that CBP applies
mesophilic as well as thermophilic and extreme thermophilic
strains [70,71]. The reason of turning to thermostable biocatalysts
could be that they presumably exhibit higher and more exhaustive
hydrolysis in comparison to others working at lower temperatures
e.g. under mesophilic regulations. Furthermore, recent compara-
tive evaluation suggested the better heat shock-resistance of
continuous cellulosic H2 fermentation carried out at thermophilic
temperature in comparison with mesophilic regimes [74].
A further advantage of (extreme) thermophilic bacteria might be
their natural ability of producing H2 with higher yields compared
to their mesophilic counterparts [75]. However, the volumetric H2
production rates are lower than those of mesophilic bacteria due
to slower proliferation and lower cell densities. A possible way to
ensure higher cell mass concentrations is the employment
of membrane bioreactors, representing an attractive set-up for
fermentative hydrogen production [10]. During thermophilic
hydrogen generation, the energy consumption of maintaining high
temperature should also be taken into account to judge the
feasibility of the process [4,76]. Besides meso- and thermophilic
pure cultures, co-cultures [72] and systematically enriched popu-
lations [66,73] are also potential candidates able to efficiently
catalyze lignocellulose bioconversion.
Furthermore, apart from the isolation and enrichment of
efficiently working microorganisms, more viable lignocellulose-
based biohydrogen production could be accomplished by the
construction of mutant strains through metabolic engineering
[7,8]. Metabolic engineering is a powerful tool and represents a
hot field nowadays. Its objectives could be the generation of non-
native compounds and also improvement of production perfor-
mance regarding certain substances by redirecting the metabolic
fluxes via the modification of the organisms' genome. In other
words, metabolic engineering in dark H2 production involves the
redirection of carbon flow to reach nearly theoretical yields from
an extended range of feasible substrates. In particular, the ultimate
aim of strain engineering is to exceed the Thauer-limit that equals
to the highest, 4 mol H2/mol glucose yield [15]. Nevertheless, it has
been reported that it could be possible to produce 12 mol H2/mol
glucose and 10 mol H2/xylose via synthetic enzymatic pathways
[77,78]. In addition, the design and manipulation of cellulosome
(an intricate multi-enzyme complex that catalyzes cellulose
decomposition) has been recently focused by metabolic engineering
approach, as well [79].
The raw materials subjected to hydrogen production in direct
(one-step) conversion systems are listed in Table 3 and cover i.e.
marine algae [25], raw corn stalk [68], wastewater mixed with sea
plant [80], agricultural wastes [81–83], energy plants [84] and
waste papers [85]. As it can be observed in Table 3, the obtainable
hydrogen yields from untreated feedstocks range between 13.65
and 310 mL H2 g  1 dry biomass. In addition, Fig. 2 illustrates a
comparison of scientific investigations reporting on hydrogen
 G. Kumar et al. / Renewable and Sustainable Energy Reviews 44 (2015) 728–737 733

Table 3
Examples of biohydrogen production conducted in various bioprocess designs.

Process Biomass Inoculum Temperature (1C) Reactor type Highest H2 yield Reference

SSF Corn stalk T. thermosaccharolyticum W16 60 Batch 89.3 mL H2 g  1 dry biomass [36]
SHF Wheat straw Anaerobic mixed culture 35 Batch 9.87 mL H2 mmol  1 glucosea [24]
SSF Wheat straw Anaerobic mixed culture 35 Batch 22.4 mL H2 mmol  1 glucose [24]
CBP Wheat straw Anaerobic mixed culture 35 Batch 0.9 mL H2 mmol  1 glucose [24]
SSFF Sugarcane bagasse E. aerogenes 30 Batch N.D. [59]
CBP Corn stalk C. butyricum 36 Batch 92.9 mL H2 g  1 dry biomass [68]
CBP Rice straw Waste water sludge 55 Batch 24.8 mL H2 g  1 dry biomass [81]
CBP Microalgae Enriched functional consortia 30 Batch 25.1 mL H g  1 dry biomass powderb [69]
CBP L. japonica Anaerobic mixed culture 35 Batch 71.4 mL H2 g  1 dry biomass [25]
CBP L. japonica Anaerobic mixed culture 35 ASBR 20 mL H2 mmol  1 hexosea [25]
CBP Water hyacinth and Beverage waste water Pig slurry 45 Batch 13.65 mL H2 g  1 dry feedstock [80]
CBP Distillers grain C. thermocellum 60 Batch 29.2 mL H2 g  1 dry biomass [82]
CBP Barley hulls C. thermocellum 60 Batch 29.2 mL H2 g  1 dry biomass [82]
CBP Contaminated barley hulls C. thermocellum 60 Batch 28.9 mL H2 g  1 dry biomass [82]
CBP Mixture of corn husk, hut shell, rice husk Buffalo dung compost 37 Batch 65.78 mL H2 g  1 TVS [83]
CBP Wheat straw C. saccharolyticus 70 Batch 44.68 mL H2 g  1 dry biomass [84]
CBP 5 Waste papers R. albus 37 Batch 42.8–282.7 mL H2 g  1 dry biomass [85]
CBP Switchgrass C. saccharolyticus 65 Batch 310 mL H2 g  1 dry biomass [71]
CBP Deoiled Jatropha waste Anaerobic mixed culture 55 ASBR 8.7 mL H2 g  1 VS [86]
CBP L. japonica Anaerobic digester sludge 35 ASBR 61.3 mL H2 g  1 dry biomass [87]

TVS: total volatile solids; VS: volatile solids; TS: total sugars; SSF: Simultaneous Saccharification and Fermentation; SHF: Separate Hydrolysis and Fermentation;
SSFF: Simultaneous Saccharification, Filtration and Fermentation; CBP: Consolidated Bioprocessing.
a
35 1C, 1 atm.
b
30 1C, 1 atm.

Untreated Lignocellulose

Pretreated Lignocellulose

Mean: 87 Mean: 86

[104] [105] [106] [108] [109] [111] [36] [116] [110] [113] [68] [81] [69] [25] [80] [82] [82] [84] [85] [85] [71] [87]

Fig. 2. H2 yields for pretreated and untreated lignocelluloses.

yields (convertible to the same units) from pretreated (Table 1)
and untreated (Table 3) lignocellulosic biomass. It is to point out
that the results widely vary in the same group and besides, a quite
remarkable deviation can be noted between the two clusters
(pretreated, non-treated). However, the average group-specific
H2 yields for pretreated and directly fermented biomass are
87 mL H2 g  1 dry biomass and 86 mL H2 g  1 dry biomass, respec-
tively. Thus, it would appear (based on 22 data of 20 independent
studies presented in Fig. 2) that biohydrogen yields of raw,
complex organic materials could be in the same order of magni-
tude as those of pretreated lignocelluloses. However, when
comparing these research outcomes it is to keep in mind that an
in-depth evaluation is rather difficult to make due to the unstan-
dardized experimental circumstances (i.e. different source and
features of starting materials, temperature, pH, etc.) and therefore
conclusions to draw are limited. The assessment to reveal the most
technologically feasible process for lignocellulose biohydrogen
generation can be attempted by using a single substrate of
particular interest (when the change in raw matter composition
is not a factor to deal with) in all the competing alternatives (SHF,
SSF, CBP) e.g. as to be found in [24] pertaining wheat straw
(Table 3).
As it can also be seen in Table 3, investigations on biohydrogen
production using biomass as raw materials were mostly carried
out in batch systems, only a limited number of studies applied
continuous operation where ASBR (Fig. 3b) was proven as a
feasible reactor configuration for feedstock possessing remarkable
amount of solids [25,86,87]. The dominancy of batch studies
734 G. Kumar et al. / Renewable and Sustainable Energy Reviews 44 (2015) 728–737
Fig. 3. The scheme of the most common continuous bioreactors treating lignocellulose biomass.
Fig. 4. The most important technological aspects of successful lignocellulosic biohydrogen fermentation.
shows that the research on lignocellulosic biohydrogen has been
pursued in laboratory-scale conditions and further progress is
needed to reach pilot/demonstration-scale. Furthermore, testing
complex organic matter in continuous operation bioreactors is of
remarkable interest [90] since biodegradation features e.g. soluble
metabolic product pattern and corresponding process indicators,
namely H2 production rate and yield probably can differ in
comparison to the ones observed in discontinuous reactors.
Besides, transition of batch processes to continuous ones has to
consider reactor design. Many types of bioreactors have been
developed for continuous dark fermentative hydrogen technology,
demonstrating feasibility to handle liquid streams such as hydro-
lysate of lignocellulose materials, various waste waters, etc. For
example, continuously stirred tank reactor (CSTR) (Fig. 3a) and
upflow anaerobic sludge blanket reactor (UASBR) (Fig. 3c) to treat
wheat straw hydrolysate [117,118], trickling bioreactor (TBR)
(Fig. 3d) to be fed with oat straw hydrolysate [119,120] or conti-
nuously external circulating bioreactor to process complex mix-
ture of rice straw and food industry wastewater [121].
However, when lignocelluloses are to be used in solid form
during Consolidated Bioprocessing, common fermenter systems
might need retrofitting for proper adaption to high solid contents.
For example, the so-called Intermittent-CSTR (I-CSTR) has shown
potential for the management of organic solid wastes [88,89].
Additionally, anaerobic membrane bioreactors (AnMBRs) might
also play a role towards the realization of more sufficient biohy-
drogen fermentation when working with solid and hard-to-digest
lignocellulose biomass. The benefit of AnMBRs is related to the fact
the bioreactors attached with membranes are able to ensure
separate hydraulic and solid detention times. When a purposefully
increased solid residence time is maintained it results in higher
concentrations of hydrogen producing strains as well as a pro-
longed “biomass and strain” contact, leading potentially to more
efficient biodegradation and subsequently, a better gas evolution
performance [10]. Besides, it has been recently shown that
conventional AnMBRs could be integrated with gas separation
membranes to attractively facilitate downstream processing by
in-situ recovering and purifying biohydrogen from the complex
fermentation gas [122]. For specific technical/structural features of
each bioreactor mentioned above, the reader should refer to the
authentic sources.
The hydrogen production strategy represented by CBP is related
to the idea of biorefinery, purposing the complete and environ-
mental benign conversion and stabilization of the total biomass
input [91,92] and contributing to a higher valorization of the
residues. For example, considering the biorefinery concept, the
effluent of hydrogen fermentation – being rich in high energy
content organic substances – may be fed to complementary proce-
dures such as anaerobic digestion to obtain biomethane [93,94],
bioelectrochemical systems [95] e.g. microbial fuel cell [96] and
microbial electrohydrogenesis cell [97] or photo-fermentation [98].
On the other hand, it is also viable when the hydrogen producing
bioreactor serves as a second stage to treat waste streams
(i.e. distillery wastewater) of lignocellulose-based liquid biofuel
(e.g. bioethanol) generation process [99].
5. Prospects and future directions
Lignocellulosic biohydrogen generation can be considered as an
emerging technology that has maturity on smaller-scales whilst –
to the authors' knowledge – pilot- or demonstration-scale results
are not yet available. Nevertheless, to reach scaled-up levels,
further research is required (1) to establish energy-efficient and
biocatalyst-friendly (limited inhibitor formation) pretreatment
methods and (2) to find or engineer robust (industrially viable)
microorganisms with sustainable hydrolytic and/or H2 fermenting
capability, as suggested by Fig. 4. From economic point of view, the
development of integrated bioprocesses should be pursued
because of the reduced capital expenditures. However, finding
 G. Kumar et al. / Renewable and Sustainable Energy Reviews 44 (2015) 728–737
the most feasible bioprocess, or in other words, the selection
between technological alternatives represented by SHF, SSF and
CBP might need comprehensive and case-specific optimization
and critical assessment since enhanced process efficiencies – even
in less-integrated applications – may compensate higher invest-
ment costs. In this regard, the advancements made in the future
should help to establish attractive systems that satisfy the neces-
sity of simultaneously high lignocellulosic hydrogen yield and
productivity, as implied by Fig. 1. This can be a significant
constraint to take steps forward and attract interest for larger-
scale implementations. As for the feedstock, beyond the second
generation lignocelluloses such as forestry- and agricultural
wastes, there is a special interest to assess the applicability of
new, third generation raw materials (microalgal biomass) for
hydrogen fermentation [100]. Microalgae could be favored sources
since they are easy and quick to cultivate, accumulate lipids, which
can be transformed into biodiesel. Generally, the extraction of oils
requires killing the algae and thereafter the carbohydrate-rich cell
residues can be subjected to anaerobic fermentation where they
undergo H2-forming bioreactions [101,102]. The use of lignocellu-
losic feedstocks for biohydrogen production may be more feasible
option compared to wastewaters because of their abundant
quantity and easier collection/storage. Regardless of lignocellulose
biomass source, investigations should be expanded to continuous
systems in order to provide much more elaborative results and
experiences that could facilitate the realization of scaled-up
biohydrogen technology.
6. Conclusions
In this paper, recent progress on lignocellulose biohydrogen
fermentation was reviewed with special regard to biomass pre-
treatment, hydrolysis, issues of inhibitor formation and advanced,
integrated fermentation systems. Although comprehensive research
has been performed applying a wide range of raw materials, micro-
organisms and the results are promising, it could be concluded that
further efforts – as outlined in section of “prospects and future
directions” – are needed to make the technology feasible in larger
scales.
Acknowledgments
The research was supported by TÁMOP 4.2.2/A-11/1/KONV-
2012-0071 financed by the European Union and the European
Social Fund. The János Bolyai Research Scholarship of the Hungarian
Academy of Sciences is also gratefully acknowledged.
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