i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y x x x ( 2 0 1 8 ) 1 e8

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Hydrogen production from xylose by moderate

thermophilic mixed cultures using granules and
biofilm up-flow anaerobic reactors

Prawit Kongjan a,b, Supattra Inchan c, Sukonlarat Chanthong d,

Rattana Jariyaboon a,b, Alissara Reungsang e,f, Sompong O-Thong c,*
a
Chemistry Department of Science, Faculty of Science and Technology, Prince of Songkla University, Pattani, 94000,
Thailand
b
Bio-Mass Conversion to Energy and Chemicals (Bio-MEC) Research Unit, Faculty of Science and Technology, Prince
of Songkla University, Pattani, 94000, Thailand
c
Department of Biology, Faculty of Science, Thaksin University, Phathalung, 93110, Thailand
d
Energy Technology Program, Faculty of Engineering, Prince of Songkla University, Hat Yai, Songkla, 90110,
Thailand
e
Department of Biotechnology, Faculty of Technology, Khon Kaen University, Khon Kaen, 40002, Thailand
f
Research Group for Development of Microbial Hydrogen Production Process, Faculty of Technology, KhonKaen
University, KhonKaen, 40002, Thailand

article info abstract

Article history: Continuous H2 production from xylose by granules and biofilm up-flow anaerobic reactor
Received 5 March 2018 using moderate thermophilic mixed cultures was investigated. The maximum H2 yield of
Received in revised form 251 mL H2/g-xylose with H2production rate of 15.1 L H2/L,d was obtained from granules
29 August 2018 reactor operating at the organic loading rate (OLR) of 60 g-xylose/L,d and hydraulic
Accepted 11 September 2018 retention time (HRT) of 4 h. Meanwhile the highest H2 production rate of 13.3 L H2/L,d with
Available online xxx an H2 yield of 221 mL H2/gexylose was achieved from the biofilm reactor. Both reactors
were dominated by Thermoanaerobacterium species with acetate and butyrate as main
Keywords: fermentation products. The microbial community of the biofilm reactor was composed of
Dark fermentation Thermoanaerobacterium species, while granules reactor was composed of Clostridium sp.,
Biohydrogen Thermoanaerobacterium sp. and Caloramator sp. The granular reactor was more microbial
Xylose diversity and more balance between economic efficiency in term of the hydrogen pro-
Thermophilic mixed culture duction rate and technical efficiency in term of hydrogen yield.
Granular reactor © 2018 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
Biofilm reactor

been utilized for fermentative ethanol by wild S. cerevisiae as

Introduction effectively as cellulosic part of lignocelluloses [1]. Alterna-
tively, a xylose based substrate like hemicellulosic hydroly-
Xylose is the dominant monomeric sugars present the hemi- sate generated simultaneously with cellulose fraction from
cellulosic part of lignocelluloses. However, xylose has not the hydrothermal pretreatment of lignocellulosic biomass has

* Corresponding author.
E-mail address: sompong@tsu.ac.th (S. O-Thong).
https://doi.org/10.1016/j.ijhydene.2018.09.066
0360-3199/© 2018 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Kongjan P, et al., Hydrogen production from xylose by moderate thermophilic mixed cultures using
granules and biofilm up-flow anaerobic reactors, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/
j.ijhydene.2018.09.066
2 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y x x x ( 2 0 1 8 ) 1 e8
the potential for H2 production by fermentative bacteria [2].
Furthermore, H2 production option could lead to having full
energy recovered from lignocellulosic biomass beyond bio-
refinery concept, in which fermentative ethanol and H2 are
produced from cellulose and hemicelluloses respectively,
meanwhile CH4 can be sequentially produced from H2 and
ethanol process effluent by anaerobic digestion [3]. However,
xylose is still more difficult than glucose to be fermented by
hydrogen-producing bacteria [4], thus, more understanding
the microbial physiology and reactor performance of
hydrogen production from xylose is necessary to develop the
platform technology of fermentative hydrogen production
from xylose-rich substrates.
The microbial fermentation gives higher H2 yield and less
variety of the fermentation products at moderate thermo-
philic conditions than mesophilic conditions due to favorable
thermodynamic conditions. Furthermore, thermophilic bac-
teria are degrading a various substrate in particular with
complex carbohydrates [5e7]. Apart from mentioned influ-
ence of operating temperature, organic loading rate (OLR) is
another major factor affecting on hydrogen production
because high OLR usually obtained by either high influent
concentration or short hydraulic retention time (HRT) results
in high hydrogen partial pressure and volatile fatty acids
(VFAs) accumulation which can inhibit hydrogen-producing
bacteria directly. Additionally, low pH as a result of high
VFAs generation could also inhibit hydrogen-producing bac-
teria [8]. Due to low cell densities of thermophiles achieved in
the liquid culture of suspended sludge continuously stirred
tank reactor (CSTR) system, low hydrogen production rate and
risk of hydrogen-producing bacteria washout at low HRT are
reported [9]. Granule based system of up-flow anaerobic
sludge blanket (UASB) reactor and bio-film based system of
anaerobic filter (AF) reactor are employed for improving cell
mass concentration with high hydrogen production rate dur-
ing thermophilic mixed culture fermentation. Both UASB and
AF reactors are capable of resistance to hydraulic organic
overloads, and no requirement of mechanical mixing [5,10,11].
In addition to hydrogen production obtained from granule
based and bio-film based reactors reviewed by Ghimire et al.
[12], UASB reactor provides higher hydrogen production than
bio-film based reactor does, due to better retaining of cell
mass in the system without fragmentation and separation
from the supporting media in bio-film based reactor, which
could have rapid microbial growth inside the reactor. How-
ever, a long start-up period of around 160 days is required for
UASB reactor for allowing granules inside the reactor to be
fully developed [13]. Continuous hydrogen production by
moderate thermophilic mixed cultures using granular reactor
and biofilm reactor was investigated. The performance of both
reactors was investigated comparatively by varying organic
loading rate (OLR) under thermophilic temperature (55
C).
Materials and methods
Experiment set-up and reactor operation
A jacketed glass column with a 220- mL working volume
(Interior diameter 4.5 cm, height 38 cm) was used as the up-
Please cite this article in press as: Kongjan P, et al., Hydrogen production from xylose by moderate thermophilic mixed cultures using
granules and biofilm up-flow anaerobic reactors, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/
j.ijhydene.2018.09.066
flow anaerobic sludge blanket (UASB) reactor for the gran-
ular system or anaerobic filter (AF) reactor for biofilm system
for continuous H2 production from xylose. Both UASB and AF
reactors were control temperature at 55
C by a jacket with hot
water circulation. Inside the UASB reactor or AF reactor was
contained with granules or plastic carriers. Schematic UASB
and AF reactors are shown in Fig. 1 (A and B). Reactors were
started-up by initially feeding with BA medium containing 5 g/
L of xylose at an initial organic loading rate (OLR) of 15 g/L,d,
corresponding to HRT of 8 h according to previously operating
conditions reported by O-Thong et al. [5]. 5 g/L of bicarbonate
buffer and 1 g/l of yeast extract were added to BA medium as
previously used by Angelidaki et al. [14]. Afterward, OLR was
stepwise-increased 30 g/L d by feeding with xylose 10 g/L at an
8 h HRT. The OLR was further increased to 60 and 120 g/L d by
feeding xylose 10 g/L at the HRT of 4 and 2 h, respectively.
After steady state was achieved for 2 times of HRT, OLR was
stepwise-increased to next OLR. The reactors were considered
to be a steady state when H2 production variation less than 5%
[15]. Samples were taken from both reactors for the soluble
metabolites and microbial community analysis.
Monitoring and analysis
Water replacement gas meter monitored the volume of gas
production and the gas composition was analyzed by gas
chromatography GC-8APT, Shimadzu, Japan equipped with a
thermal conductivity detector. Liquid samples were taken
both reactors and analyzed for pH, volatile fatty acids (VFA),
alcohols, lactate, formate, and xylose. VFA was analyzed using
GCe8APF, Shimadzu, Japan equipped with a flame ionization
detector (FID) [16]. For Lactate and formate analysis as previ-
ously described by Kongjan et al. [17], suppressed ion exclu-
sion chromatography comprising with an high performance
liquid chromatography (HPLC) pump (L2100, Hitachi), a HPLC
auto-sampler L2200 Hitachi, a suppressor Dionex AMMS-IEC2,
a column ICE-AS1 (9  250 mm), a conductivity Detector Wa-
ters 432, a prefilter Rheodyne 0,5 mm  3 mm and a column
heating (35
C). Heptafluorobutyric acid solution (4 mM) was
employed for the eluent with a flow rate of 50 mL/h. The
suppressor (25 mM of tetrabutylammonium hydroxide solu-
tion) was applied with a flow rate of 30 mL/h. Xylose was
analyzed by HPLC equipped with a refractive index (RI) de-
tector [15]. Microbial community was analyzed by polymerase
chain reaction-denaturing gradient gel electrophoresis tech-
nique as according to Kongjan et al. [2].
Results and discussion
Granule UASB reactor performances
The granular UASB reactor was continuously fed with xylose
at different OLRs of 15 30, 60, and 120 g/L,d. Monitored pa-
rameters of hydrogen production rate (HPR), effluent xylose
concentration, pH, and metabolite concentrations) during its
continuous operation are shown in Fig. 2. Throughout the
operation period, the hydrogen content in the biogas was in
the range of 34e47%, the rest being carbon dioxide. Addi-
tionally, the reactor has the capacity to self-maintained pH in
 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y x x x ( 2 0 1 8 ) 1 e8 3

Fig. 1 e Schematic experimental set-up for UASB (A) and AF (B) systems.

Fig. 2 e Profiles of hydrogen production rate, effluent xylose concentration, pH, and metabolite concentrations in the UASB
reactor operated at 55
C and the OLR of 15 (I), 30 (II), 60 (III), and 120 (IV) g-xylose/d/L. ETOH: ethanol; AA: acetate; PA:
propionate; BA:n-iso-butyrate; LA: lactate; FA: formate.

Please cite this article in press as: Kongjan P, et al., Hydrogen production from xylose by moderate thermophilic mixed cultures using
granules and biofilm up-flow anaerobic reactors, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/
j.ijhydene.2018.09.066
4 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y x x x ( 2 0 1 8 ) 1 e8

Table 1 e Hydrogen production yield and rate from granular and biofilm reactor operation.
Operating Conditions Hydrogen Yield (HY) (mL H2/g xylose) H2 production rate (HPR) (L H2/L,d)
Xylose (g/L) HRT (h) OLR (g/L,d) UASB AF UASB AF
5 8 15 145 175 2.1 2.6
10 8 30 211 192 6.4 5.8
10 4 60 251 221 15.1 13.3
10 2 120 118 161 14.2 19.4

the optimal range of 4.9e5.7 for hydrogen-producing bacteria.
At the initial OLR of 15 g/L,d, a stable H2 production rate of
2.1 L H2/L,d was achieved simultaneously with complete
xylose degradation. The H2 production rate increased to the
highest value of 15.1 L H2/L,d with hydrogen yield (HY) of
251 mleH2/g-xylose at the OLR of 60 g/L,d (Table 1). At the
same time, xylose in the UASB reactor was fully degraded with
less than 0.5 g/L remaining in the effluent. Moreover, high H2
production was accompanied by the generation of butyrate
and acetate as the dominant metabolites.
However, the increasing of OLR to 120 g/L,d resulted to
system overload, as indicated by a sharp increase of xylose
concentration in the effluent from less than 0.5 g/L to 2.7 g/L
and as light decrease of the hydrogen production rate, while
sharply decreased hydrogen yield of 118 mL-H2/g-VS. At OLR
120 g/L, d and 2 hr-HRT, corresponding to specific flow rate of

Fig. 3 e Profiles of hydrogen production rate, effluent xylose concentration, pH, and metabolite concentrations in the AF
reactor operated at 55
C and the OLR of 15 (I), 30 (II), 60 (III), and 120 (IV) g-xylose/d/L. ETOH: ethanol; AA: acetate; PA:
propionate; BA:n-iso-butyrate; LA: lactate; FA: formate.

Please cite this article in press as: Kongjan P, et al., Hydrogen production from xylose by moderate thermophilic mixed cultures using
granules and biofilm up-flow anaerobic reactors, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/
j.ijhydene.2018.09.066
 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y x x x ( 2 0 1 8 ) 1 e8
Fig. 4 e Metabolic pathway of xylose dark fermentation for
biohydrogen production.
500 m$props_value{literPattern}/L h, sludge granules could
not retain in sludge bed zone but the lifting of all parts of the
bed and gas pulsing eruption often occurred were observed.
Subsequently, washout of poor-settling sludge was observed,
resulting in significantly low xylose consumption and
hydrogen production yield. The sudden flotation of granular
sludge would be caused by a too high upward force derived by
substrate flow-rate and gas pulsing. Parawira et al. [18] pre-
viously reported similar phenomenon when operating UASB
to treat potato waste leachate at OLRs above 6.0 g-COD/L,d
could give poor segregation between granular and flocculant
sludges leading to sudden sludge flotation and reactor failure.
Thus, operating a UASB reactor fed with xylose at OLR 60 g-VS/
L,d could be the optimum for both economic efficiency in
term of hydrogen production rate and technical efficiency in
term of hydrogen yield. Chan and Lin [13] reported increasing
OLR of UASB reactor for hydrogen production from 63 g-VS/
d,L to 84 g-VS/d,L and 126 g-VS/d,L caused sharply drop of
hydrogen yield from 1.5 mmol-H2/mol-sucrose to 0.9 mmol-
H2/mol-sucrose and 0.4 mmol-H2/mol-sucrose, respectively.
AF reactor performance
The biofilm reactor was also continuously fed xylose with AF
reactor at different OLRs of 15 30, 60, and 120 g/L,d. Hydrogen
content in the gas phase was 35e47% and no methane was
detected. The pH was self-maintained within the range of
5.0e5.6 throughout the operation period as well. Fig. 3 shows
the profiles of hydrogen production rate, effluent xylose con-
centration, pH, and metabolite concentrations during the
Please cite this article in press as: Kongjan P, et al., Hydrogen production from xylose by moderate thermophilic mixed cultures using
granules and biofilm up-flow anaerobic reactors, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/
j.ijhydene.2018.09.066
5
continuous operation. The H2production rate of 2.6L H2/L,d
and the yield of 175 mL-H2/g-xylose were achieved under the
steady-state condition by feeding with the OLR of 15 g/L,d.
The highest hydrogen yield in a biofilm reactor was
221 mleH2/g-xylose at the OLR of 60 g/L,d with an H2 pro-
duction rate of 13.3 L H2/L,d. Xylose in the AF reactor was fully
degraded with less than 0.5 g/L remaining in the effluent at the
OLR of 60 g/L,d. Butyrate and acetate were also the main
soluble fermentation products generated during the operation
of the AF reactor fed with xylose solution.
Increasing OLR up to 120 g/L,d resulted in increasing of
hydrogen production rate up to 19.4L H2/L,d but lowerH2yield
of 161 mL-H2/g-xylose achieved. Meanwhile, the remaining
xylose of 1.9 g/L in the effluent was considerably observed,
indicating incomplete degradation occurred. This demon-
strates that the AF reactor is more tolerant to high OLR than
the UASB reactor at high OLR of 120 g/L,d. Anaerobic filters
(AF) reactor used in this investigation is a typical type of up-
flow anaerobic packed bed reactor, which is stationary
packed with inert carriers to provide mechanical support for
the growth of biofilm [19]. Even high influent flow rate with 2
hr-HRT or 110 mL/h, having 120 g/L,d OLR, all plastic carriers
attached with microbial biomass could be at a standstill.
Obviously, the concentration of bacterial biomass attaching
on plastic carriers inside AF reactor should be higher than that
of bacterial biomass containing inside UASB reactor, which
were in flotation and losing sludge flocculants at the same OLR
of 120 g/L,d, leading to achieving higher xylose degradation
and hydrogen production from AF reactor. However, under
normal feed conditions, the performance of AF reactor is
generally not as good as that of UASB reactor, as previously
reported by Kotsopoulos et al. [10], Zheng et al. [11], Parawira
et al. [18], and Kongjan et al. [20]. This is due to the low surface
area of the plastic carrier in AF to be used for cell immobili-
zation compared to the surface area of granular sludge in
UASB reactor [20]. AF is more likely to be encountered with
clogging and channeling than UASB reactor [18].
The microbial consortium responsible for xylose
degradation
Xylose dark fermentation employing hydrogen-producing
bacteria using could theoretically produce 3.33 mol-H2/mol-
xylose along with 1.67 mol-acetate/mole-xylose (Eq. (1)), but
lower hydrogen yield of 1.67 mol-H2/mol-xylose could be
produced alternatively along with 0.83 mol-butyrate/mol-H2
(Eq. (2)) as previously stated by Kongjan et al. [2].
C5H10O5 þ 1.67H2O / 1.67C2H3O2 þ 1.67Hþ þ 3.33 H2 þ 1.67
CO2 DG0 ¼ 195.5 kJ/mol (1)
C5H10O5 / 0.83C4H7O2 þ 0.83Hþ þ 1.67 H2 þ 1.67 CO2
DG0 ¼ 233:9 kJ/mol (2)
During xylose metabolism via glycolytic pathway adapted
from Zhu and Yang [21] (Fig. 4), hydrogen formation or proton
reduction is catalyzed by enzyme hydrogenase and use elec-
tron from intracellular electron carriers of reduced ferredoxin
(Fdred) and/or NADH, depending on hydrogen partial pressure
6 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y x x x ( 2 0 1 8 ) 1 e8

Fig. 5 e DGGE profiles of microbial community responsible for hydrogen production from xylose at different OLR.

in the fermentative system. Maximum hydrogen partial pres-
sures (PH2) of 0.3 atm and 6  104 atm (60 Pa) could thermo-
dynamically limit proton reduction by associating with Fdred
and NADH, respectively [22]. Oxidative decarboxylation of py-
ruvate to acetyl Co-A catalyzed by pyruvate: ferredoxin
oxidoreductase (PFOR) could result in Fdred which is eventually
used for hydrogen production. NADH generated from xylose
degradation to pyruvate catalyzed by NADH: Fd oxidoreduc-
tase (NFOR) could be optionally oxidized through either addi-
tional hydrogen formation under PH2 lower than 60 Pa with
acetate metabolite or acetyl-CoA reduction to butyrate.
Microbial community analysis showed that both granular
and biofilm reactors have similarity dominated species
responsible for hydrogen production from xylose. Granular and
biofilm reactors were dominated by Thermoanaerobacter-
iumthermosaccharolyticum and Thermoanaerobacteriumacidotoler-
ans.Microbial community in biofilm reactors was composed of
Thermoanaerobacteriumthermosaccharolyticum, Thermoanaer-
obacterium THPB-2, and Thermoanaerobacteriumacidotolerans.
Microbial community in granular reactors was composed of
Clostridium sp. Clostridium roseum, Thermoanaerobacter-
iumthermosaccharolyticum, Thermoanaerobacterium THPB-2,
Thermoanaerobacteriumacidotolerans and Caloramatorfervidus
(Fig. 5). Thermoanaerobacterium species are well-known moder-
ate thermophilic hydrogen producers [23]. Those strictly
anaerobic spore-forming bacteria are found in environments

Table 2 e Different continuous system for Hydrogen production from xylose.

Reactor type Seed sludge Operating Conditions Yield(mol-H2/mol-sugar)/ Ref.
[(Temp(ºC)/HRT(hr)/Initial H2-Rate (L/L,d)
xylose concentration (g/L)]
CSTR Anaerobic digester sludge from 37/8/10 1.56/7.9 [25]
wastewater treatment
FBR Activated sludge 55/6/7.5 1.2/6.77 [26]
CSTR-Cell immobilized Clostridium sp. T2 37/10/10 1.16/1.69 [27]
on mycelia pellets
UASB Seed sludge from anaerobic digester of 35/6/28 2.98/239 [28]
local wastewater
CSTR Enriched cultures from xylose batch 70/72/1 1.36/62 [17]
fermentation
UASB Enriched cultures from xylose batch 55/4/10 1.68/15.1 This study
fermentation
AF Enriched cultures from xylose batch 55/4/10 1.48/13.3 This study
fermentation

CSTR: Continuously Stirred Tank Reactor, FBR: Fluidized Bed Reactor, UASB: Up-flow Anaerobic Sludge Blanket Reactor, AF: Anaerobic Filter.

Please cite this article in press as: Kongjan P, et al., Hydrogen production from xylose by moderate thermophilic mixed cultures using
granules and biofilm up-flow anaerobic reactors, International Journal of Hydrogen Energy (2018), https://doi.org/10.1016/
j.ijhydene.2018.09.066
 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y x x x ( 2 0 1 8 ) 1 e8
that are rich in decaying plant materials and therefore have the
enzymatic machinery to hydrolyze polymers like cellulose,
xylan, pectin, chitin, and starch. Thermoanaerobacterium species
have optimal growth at a moderate thermophilic temperature
(60
C) and can convert xylose to H2 via butyrate and acetate
type fermentation [16]. Thermoanaerobacteriumspecies are well
known as good H2 producing bacteria [5,6]. Thermoanaer-
obacterium and Clostridium species are particularly important in
converting lignocellulosic biomass to hydrogen. Hydrogen for-
mation from NADH requires cytoplasmic monomeric Fe only
hydrogenases or multisubunit membrane-bound [NiFe] hy-
drogenases, which found in Thermoanaerobacterium and Clos-
tridium species [24]. According to the experimental results
obtained from this investigation and other previous reports as
shown in Table 2, a continuous mode of operation hydrogen
production from xylose could be potentially possible by using
various reactors. Both mixed-cultures and pure-culture are
capable to be enriched and/or cultivated in both suspended and
attached growth systems.
Conclusions
Hydrogen can be successfully produced with rather a high
yield and rate from the granular reactor with an H2 yield of
251 mL H2/g-xylose and H2 production rate of 15.1 L H2/L,d at
the organic loading rate (OLR) of 60 g-xylose/L,d and hydraulic
retention time (HRT) of 4 h. Meanwhile, the H2 production rate
of 13.3 L H2/L,d, corresponding to an H2 yield of 221 mL H2/
gexylose was achieved from biofilm reactors at the same
operating condition. Both reactors were dominated by Ther-
moanaerobacterium species with acetate and butyrate main
fermentation products. The granular reactor was more mi-
crobial diversity and more balance between economic effi-
ciency in term of the hydrogen production rate and technical
efficiency in term of hydrogen yield leading to more robust for
operation.
Acknowledgments
The authors would like to thank Thailand Research Fund for
Mid-Career Research Grant (Grant No.RSA6180048), Senior
Research Scholar (Grant No.RTA5980004), and Research Group
for Development of Microbial Hydrogen Production Process,
Khon Kaen University for the financial support.
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