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Fermentative hydrogen production using sorghum husk as a biomass

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Biotechnology and Bioprocess Engineering 20: 733-743 (2015)
DOI 10.1007/s12257-015-0172-3
RESEARCH PAPER
Fermentative Hydrogen Production Using Sorghum Husk as a
Biomass Feedstock and Process Optimization
Ganesh D. Saratale, Siddheshwar D. Kshirsagar, Rijuta G. Saratale, Sanjay P. Govindwar, and Min-Kyu Oh
Received: 14 March 2015 / Revised: 12 May 2015 / Accepted: 7 June 2015
© The Korean Society for Biotechnology and Bioengineering and Springer 2015
Abstract The potential of isolated actinomycetes and fungi
were evaluated for the cellulase and xylanase production
under solid state fermentation conditions. Maximal
secretion of enzymes was observed with Phanerochaete
chrysosporium using soybean straw. The potential of the
produced crude enzyme complex was demonstrated by
two-step enzymatic hydrolysis of untreated and mild acid-
pretreated sorghum husk (SH). A cellulase dose of 10 filter
paper units (FPU) released 563.21 mg of reducing sugar
(RS) per gram of SH with 84.45% hydrolysis and 53.64%
glucose yields, respectively. Finally, enzymatic hydrolysates
of SH were utilized for hydrogen production by Clostridium
beijerinckii. Effects of temperature, pH of media, and
substrate concentration on the biohydrogen production
from SH hydrolysates were investigated. The optimal
conditions for maximal hydrogen production using SH
hydrolysate were determined to be a loading of 5.0 g RS/L,
at 35°C, and controlled pH at 5.5. Under these optimal
conditions, the cumulative H2 production, H2 production
rate, and H2 yield were 1,117 mL/L, 46.54 mL/L/h, and
1.051 mol/mol RS, respectively. These results demonstrated
a cost-effective hydrogen production is possible with sorghum
Ganesh D. Saratale, Min-Kyu Oh*
Department of Chemical and Biological Engineering, Korea University,
Seoul 136-713, Korea
Tel: +82-2-3290-3308; Fax: +82-2-3290-3308
E-mail: mkoh@korea.ac.kr
Siddheshwar D. Kshirsagar
Department of Biotechnology, Shivaji University, Kolhapur 416-004,
India
Rijuta G. Saratale
Department of Environmental Science and Engineering, Ewha Womans
University, Seoul 120-750, Korea
Sanjay P. Govindwar
Department of Biochemistry, Shivaji University, Kolhapur 416-004, India
husk as a lignocellulosic feedstock.
Keywords: sorghum husk, lignocellulosic biomass, bio-
hydrogen, cellulolytic strain, enzyme saccharification, acid
pretreatment
1. Introduction
During the last few decades, lignocellulosic biomass has
been considered as an ideal feedstock for biofuel production
due to its enormous abundance and renewability [1].
Bioconversion of lignocellulosic biomass into fermentable
sugars can be further utilized for more environmentally
compatible and cleaner biofuel production [2]. Formation
of soluble sugars from cellulose of agricultural residues
relies on the sequential/coordinated action of cellulases,
primarily endoglucanase, exoglucanase, and cellobiase [3].
Moreover, xylanase plays a vital role in the efficient
conversion of xylan residues to readily available sugars for
biofuel production [4]. The comprehensive application of
cellulase and xylanase in lignocellulosic biorefineries is
hindered by its cost of synthesis and the demand for highly
stable enzymes at extreme conditions [5]. Several research
efforts have targeted the development of technologies for
enzyme production in an eco-efficient manner [6]. Cellulase
and xylanase can be produced by several microorganisms,
including bacteria, actinomycetes, and fungi, using submerged
and solid state fermentation processes [4,7]. Production
costs could be reduced by using efficient cellulolytic
microorganisms and by adopting suitable fermentation
processes.
Sweet sorghum is considered as an attractive nonfood
feedstock for biofuel production because it is easily
adaptable to diverse climate and soil conditions with high
734
photosynthetic efficiency and contains high fermentation
sugars in waste biomass. In India, sorghum is one of the
important industrial crops with an average of 15.6 million
metric tons of sorghum waste residues produced annually
[8]. Lignocellulosic biomass is typically resistant to
fermentation by microorganisms due to several factors
affecting hydrolysis, including the porosity (accessible
surface area) of the waste materials and crystallinity of
cellulose fibers [9,10]. In order to improve the enzymatic
hydrolysis of lignocellulosic biomass, it is necessary to
include a pretreatment step to degrade lignin layers in order
to increase the accessibility to the cellulose and hemicellulose
[11,12]. In the present study, conditions for obtaining a
high sugar yield from sorghum husk (SH) biomass using
dilute sulfuric acid as a pretreatment step and sequential
enzymatic saccharification were examined.
Cellulase and xylanase production by solid state fer-
mentation (SSF) was considered as an alternative production
route for enzymatic saccharification to reduce the production
costs. SSF is generally characterized by the growth of
microorganisms on moist solid substrates in which the
inter-particle spaces are filled with a continuous gas phase
with low moisture content [13]. SSF resembles the natural
habitat for fungi and holds potential in the production of
hydrolytic enzymes [7,13]. For example, Phanerochaete
chrysosporium, a white rot fungus, has capacities for
interparticle penetration and secretion of cellulases and
lignin degrading enzymes [14]. Agro-industrial wastes are
regarded as promising substrates for culturing microbial
cells under SSF, by which it is possible to reduce the cost
of enzyme production and boost its ecofriendly management
[3,5,6].
Biohydrogen production has been established as a pro-
spective alternative and integral component of sustainable
energy because of its clean, renewable and eco-friendly
nature [15]. Compared to photo fermentative H2 production,
dark fermentative H2 production offers several advantages,
including process simplicity, low energy requirements, and
ability to generate H2 from a variety of organic substrates
frequently obtained as refuse or waste products [16,17].
Clostridium spp. and Enterobacter spp. have been found to
be very efficient dark-fermentative microorganisms [17,18].
Recent studies for dark fermentative biohydrogen production
using cellulosic biomass suggested that conditions for
cellulose degradation and dark H2 fermentation are quite
different, and these microorganisms are inefficient utilizers
of cellulose in producing H2 in one stage H2 production
process [19,20]. Therefore, a two-step separate hydrolysis
and fermentation (SHF) process has been applied in which
cellulose hydrolysis is carried out in the first stage and the
hydrolysate is efficiently converted into H2 in the second
stage [9,17]. Biohydrogen production is a complex process
Biotechnology and Bioprocess Engineering 20: 733-743 (2015)
and is greatly influenced by many factors, including micro-
organism seeding, pH, temperature, organic loading rate,
metal ions, and oxidation-reduction potential. Optimization
of these factors and controlling suitable environmental
conditions could enhance dark fermentative H2 production
[21,22].
In the present study, we evaluated the capacity of isolated
actinomycetes and fungi grown on agricultural wastes under
SSF for cellulase and xylanase production. Furthermore,
the ability of crude enzyme complexes to hydrolyze
untreated and pretreated SH was evaluated. Finally, the
resulting SH hydrolysates were utilized for dark fer-
mentative H2 production by Clostridium beijerinckii. In
addition, to optimize the H2 production from SH hydrolysates,
different operational conditions were investigated. The aim
of this work was to evaluate the feasibility of prepared
lignocellulosic feedstock for dark fermentative H2 production.
2. Materials and Methods
2.1. Microorganisms and culture conditions
Pure cultures of Phanerochaete chrysosporium MTCC
787, and isolated actinomycetes Streptomyces sp. MDS [2]
and Nocardiopsis sp. KNU [4] were used in this study.
Pure cultures were maintained on Dubos salt medium (g/L):
NaNO3, 0.5; K2HPO4, 1.0; MgSO4·7H2O, 0.5; KCl, 0.5;
FeSO4·7H2O, 0.01; agar powder, 20; CMC, 10 at pH 6.5,
stored at 4°C, and subcultured monthly.
2.2. Lignocellulosic substrates and their pretreatment
Lignocellulosic substrates such as SH, rice husk, Vigna
mungo (Urad) harvesting waste, soybean straw, and waste
tea powder were selected in this study. The raw substrates
were collected from local markets and air-dried, milled,
and consecutively sieved through 0.5 and 0.2 mm screens
before storing at room temperature. The commercial cellulosic
materials carboxymethyl cellulose (CMC), avicel, 4-
nitrophenyl β-D-glucopyranoside (PNPG), and Whatman
filter paper No.1 were obtained from Sigma-Aldrich (St.
Louis, MO, USA). All other chemicals were of highest
purity available and of analytical grade. The pretreatment
of SH was performed separately with mild acid: 0.1 ~ 0.4%
(w/v) H2SO4 at 121°C for 30 min. The pretreated residues
were further washed at neutral pH and dried at 70°C
until constant weight. The pretreated biomass was used
immediately for the hydrolysis experiments or stored in
airtight containers at 4°C until use.
2.3. Cellulase and xylanase production under SSF
SSF was carried out in 250 mL Erlenmeyer conical flasks
containing 5 g of dry lignocellulosic substrate moistened
Fermentative Hydrogen Production Using Sorghum Husk as a Biomass Feedstock and Process Optimization
with Dubos salt medium to attain the final substrate-to-
moisture ratio for cellulase and xylanase production. The
media were sterilized by autoclaving at 121°C for 15 min
and thereafter cooled at room temperature. For inoculation,
1 mL of spore suspension (absorbance of 1.0 at 600 nm) of
the fungi and actinomycetes, prepared in sterile saline from
7-day-old cultures, was used. All contents of the flasks
were mixed well with a sterile glass rod to distribute the
inoculum throughout the substrate and incubated in an
environmental chamber at 30°C. After every 24 h, the
fermented substrate was aseptically removed from the flask
and suspended in 50 mL of McIlvaine’s buffer (0.1 M citric
acid, 0.2 M Na2HPO4, pH 5) and incubated with orbital
shaking at 120 rpm for three hours. To maximize the enzyme
yield, the cultures were extruded through muslin cloth and
then centrifuged at 10,000 × g at 4°C for 10 min. The
enzyme solution thus obtained was assayed for various
enzyme activities and to study their time course of
production. The effects of incubation time (1 ~ 8 days) and
moisture content (1:1 ~ 1:3), while maintaining the initial
pH (5.0) and temperature (30oC) on cellulase and xylanase
production by P. chrysosporium with soybean straw under
SSF were investigated.
2.4. Enzyme assays
Endoglucanase, exoglucanase, and xylanase activities were
determined according to a method reported previously
[2,4]. Total cellulase activity (filter paper units, FPU) was
assayed according to IUPAC recommendations using
Whatman filter paper as the substrate [3]. Glucose and
xylose standard curves were used to calculate cellulase and
xylanase activities. One unit of enzyme activity was defined
as the amount of enzyme required to release 1 µmol of
reducing sugars (RS) per minute. Cellobiase activity was
determined by assaying the release of p-nitrophenol (pNP)
using a previously reported procedure [23]. One unit of
cellobiase activity was defined as the amount of enzyme
required to release 1 µmol of pNP per minute under the
assay conditions. All enzyme assays were conducted in
triplicate and the average rates were calculated to represent
the enzyme activity.
2.5. Application of crude enzyme complex to biomass
hydrolysis
Enzymatic hydrolysis of untreated and mild acid-pretreated
SH was performed at a biomass loading of 2.0% (w/v) in
20 mL of 50 mM McIlvaine’s buffer (pH 5.0) containing
0.005% (w/v) sodium azide. Crude enzyme solution
equivalent to FPU activity of 10 IU/g of untreated and
pretreated substrate was added to the flasks and incubated
at 50°C and 150 rpm for 24 h (first hydrolysis step). Two-
step enzymatic hydrolysis was performed at the designated
735
time (24 h) and the sample was vacuum-filtered to separate
the liquid and solid fractions. The solid fraction was then
re-inoculated aseptically into 20 mL of 50 mM McIlvaine’s
buffer and incubated with orbital shaking (150 rpm) at
50°C (second hydrolysis step). After each hydrolysis step,
the samples were immediately heated to 100°C for 15 min
to denature the enzymes, cooled, and then centrifuged for
10 min at 10,000 rpm. The supernatants were stored at
–20°C and used for the RS and glucose analyses. The
reducing sugars released during two steps hydrolysis was
combined together to calculate the overall hydrolysis yield
and glucose yields according to the following equation
[24]:
Hydrolysis yield (%) = RS (mg) × 0.9 × 100/cellulose
content in the substrate.
Glucose yield (%) = Glucose (mg) × 0.9 × 100/cellulose
content in the substrate.
Hydrolysis of polysaccharides involves water. For each
mole of reducing sugar released, 1 mol of H2O is required.
A correction factor of 0.9 was therefore included in the
calculation of the amount of polysaccharides hydrolyzed.
2.6. Biohydrogen production using SH hydrolysates by
C. beijerinckii and optimization of environmental conditions
C. beijerinckii KCTC 1785 was evaluated for dark hydrogen
fermentation using lignocellulosic biomass hydrolysate. The
medium for dark H2 fermentation containing SH hydrolysate
was (g/L): yeast extract, 4.0; NH4HCO3, 6.72; NaHCO3,
5.24; K2HPO4, 0.125; MgCl2·6H2O, 0.1; MnSO4·6H2O,
0.015; FeSO4·7H2O, 0.025; CuSO4·5H2O, 0.005; CoCl2·5H2O,
0.00012. The initial conditions for biohydrogen production
were pH 6.5, 35°C, and an SH hydrolysate containing
5.0 g/L RS. To optimize the biohydrogen production, initial
pH values of 6.0 ~ 7.5, incubation temperatures of 30 ~
45°C, and 0.2% acid-pretreated SH hydrolysates with RS
concentrations of 2.5 ~ 10.0 g/L were used. The inoculum
of a 24 h preculture (OD 600 nm = 2.0) was diluted to a
final concentration of 10% (v/v) in 40 mL of fresh cultivation
medium and incubated for 24 h. As a control experiment,
biohydrogen fermentation was also conducted using
autoclaved SH biomass without any chemical treatment.
During the course of fermentation, cell concentration, pH,
residual RS, production of biogas, and soluble metabolites
were monitored with respect to culture time.
2.7. Improvement of biohydrogen production using SH
hydrolysates by a pH-control strategy
A pH-control strategy was employed to improve the
performance of biohydrogen production from 0.2% acid-
pretreated SH hydrolysates (RS, 5.0 g/L). The pH of the
medium was adjusted to 6.5 prior to cultivation. After the
736
start of fermentation, the culture pH gradually decreased
due to formation of acidic metabolites. The pH was
allowed to decrease to 5.0, 5.5, or 6.0, and the pH was then
kept at that value by automatic titration with 1 N NaOH.
The incubation time was 24 h and the working volume was
150 mL.
2.8. Analytical methods
The presence of sugars (glucose, xylose, and arabinose)
and soluble products (volatile fatty acids and ethanol) in
the filtered (with 0.2 µm porosity) supernatants of culture
broth during bioH2 production were detected by high-
performance liquid chromatography (HPLC) with refraction
index detection (RID-10A, Shimadzu; Kyoto, Japan). The
column used in HPLC analysis was an ICSep ICE-
COREGEL 87H3 column (Transgenomic; Omaha, NE,
USA) with 0.008 N H2SO4 as eluent at a flow rate of
0.4 mL/min and a column oven temperature of 55°C. The
gas products (mainly H2 and CO2) were analyzed by gas
chromatography (GC-17A, Shimadzu) using a thermal
conductivity detector. The detailed procedures for GC
analyses were described in our previous work [10]. Cellulose,
hemicellulose, and lignin contents of SH biomass were
estimated by the method of Goering and Van Soest [25].
RS content was determined by the dinitrosalicylic acid
(DNS) method [26]. Fourier-transform infrared (FTIR)
spectroscopic (Agilent, Cary 630; USA) analysis of native
and dilute sulfuric acid-pretreated SH was performed to
detect the changes in functional groups. The dried samples
of cellulosic materials (approximately 1%) were embedded
in KBr pellets and fixed in the sample holder for analyses.
FTIR analysis was conducted in the mid-IR region by
averaging 32 scans in the range of 400 ~ 4000/cm with a
resolution of 4/cm. Scanning electron microscope (SEM)
images of the particles coated with platinum were recorded
using a JEOL JSM-6360A microscope (Tokyo, Japan) with
operating voltage 20 kV as previously described [2].
2.9. Statistical analysis
Data were analyzed by one-way analysis of variance
(ANOVA) with a Tukey-Kramer multiple comparisons test.
Differences were considered significant when P was ≤ 0.05.
3. Results and Discussion
3.1. Selection of cellulolytic enzymes produced under
SSF
For selecting lignocellulose-degrading enzymes, production
of endoglucanase, exoglucanase, cellobiase, FPU, and
xylanase activity was evaluated using three cellulolytic
Biotechnology and Bioprocess Engineering 20: 733-743 (2015)
strains: isolated Streptomyces sp. MDS, Nocradiopsis sp.
KNU, and P. chrysosporium. Several lignocellulosic
substrates (tea powder, SH, Urad waste, rice husk, and
soybean straw) were used for enzyme production using
SSF. SSF quality is governed by various factors such as
temperature, pH, water activity, and concentration of
nutrients. Each factor is generic and holds a significant
impact for the technical and economic feasibility of the
process development [6,7,13] The maximal production of
cellulase and xylanase by P. chrysosporium were previously
obtained at 30°C with initial pH set to 5.0 in SSF [27]. The
optimal moisture content in the solid-state substrate (soybean
straw) by P. chrysosporium for cellulase and xylanase
production was 1:1.5 (w/v) (60% water level) (Fig. S1).
With the condition, all three strains grew and showed the
ability to metabolize these cellulosic substrates by expressing
cellulase and hemicellulase. The maximum production of the
cellulase and xylanase by all three strains was observed in
the presence of soybean straw (Figs. 1A and 1B). Moderate
production of all enzymes was observed in the presence of
urad waste, SH, and rice husk, whereas lower enzyme
activities were observed in the case of waste tea powder
(Figs. 1A and 1B). Under optimal condition, P. chrysosporium
displayed highest production of endoglucanase (212.78 units
per gram of dry solid [U/gds]), exoglucanase (22.34 U/gds),
cellobiase (176.50 U/gds), FPU (26.20 U/gds), and xylanase
(371 U/gds) in the presence of soybean straw after seven
days of incubation (Fig. S2), which were much higher than
those produced by the other strains (except exoglucanase)
(Fig. 1C). This variability in enzyme production was likely
due to compositional variation, physical properties, and
nutrient availability of the substrate [5].
The endoglucanase (212.78 U/gds) and cellobiase activities
(176.50 U/gds) exhibited by P. chrysosporium in this study
were significantly higher than those reported with Trichoderma
reesei RUT-C30 (90.5 and 61.6 U/gds, respectively) [1],
Fomitopsis sp. RCK 2010 (71.52 and 69.08 U/gds,
respectively) [3], and with a consortium of Aspergillus
niger and T. reesei (131.3 and 33.7 U/gds, respectively) [6].
Moreover, the exoglucanase activity (22.34 U/gds) was
found to be higher than that in Thermoascus aurantiacus
(4.0 U/gds) [5]. The FPU activity (26.20 U/gds) was also
found to be several-fold higher than that reported in other
studies [3,5,6] and comparable to that of T. reesei RUT-
C30 (15.0 U/gds) [1]. In this study, the xylanase production
(371 U/gds) was insignificant and lower than that reported
in other studies [5,14]. Overall, P. chrysosporium exhibited
greater potential than the other two strains with respect to
cellulolytic enzymes production (Figs. 1A, 1B, and1C);
thus, further experiments were conducted with enzymes
produced by P. chrysosporium using soybean straw.
Fermentative Hydrogen Production Using Sorghum Husk as a Biomass Feedstock and Process Optimization 737

3.2. Effect of acid pretreatment of SH

Several physical and chemical pretreatment methods have
been reported to overcome the recalcitrance of lignocellulose,
such as sorghum husk, and to increase enzyme accessibility
for higher yield of fermentable sugars [12]. Dilute acid
pretreatment, which solubilizes hemicellulose and lignin
and thereby disrupts the lignocellulosic composite material,
is one of the most commonly used commercial pretreatment
processes [11]. Therefore, sorghum husk was treated with
sulfuric acid before enzymatic saccharification, and its
effects on structure were studied. Cellulose-related bands
in the FTIR spectra were seen at 2850, 2918, 2927, and
3340/cm [12,31]. The FTIR spectrum of the region from
3800 to 3000/cm increased in both width and asymmetry of
the curves after acid pretreatment, indicative of perturbations
in the crystalline structure of cellulose (Fig. 2). Hemicellulose
is associated with the region from 1700 to 1740/cm, and
the band at 1733/cm was missing after treatment, indicating
that the hemicellulose fraction, which is inherently
susceptible to hydrolytic degradation, was hydrolyzed by
the acid pretreatment. Lignin-related bands in the FTIR
spectra were seen at 1273, 1518, and 1715/cm [12]. The
region from 1500 to 1610/ is associated with the vibration
of aromatic rings and indicated stretching of the phenyl
ester side chain C=O bonds of lignin in the pretreated
sample (Fig. 2). Bands in the 1200 ~ 1000/cm spectral
regions are associated with the structural features of
cellulose and hemicellulose, with overlap of primary and
secondary alcohol C–O–H stretching at 1064/cm. The peak
at 900/cm corresponds to removal of amorphous cellulose
[12]. The FTIR results supported that dilute acid pretreatment
removed most of the hemicellulose and lignin by cleaving
the acetyl linkages of cellulose from the SH biomass. SEM

Fig. 1. Cellulase and xylanase production in the presence of different

lignocellulosic waste materials by (A) Nocardiopsis sp. KNU, (B)
Streptomyces sp. MDS, (C) Phanerochaete chrysosporium MTCC
787 after seven days of incubation (at 30°C, initial pH 5.0) under
solid state fermentation (SW- soybean straw; RH- rice husk; Udid- Fig. 2. FTIR spectroscopic analysis of sorghum husk: black line,
urad harvesting waste; SH- sorghum husk; TP- waste tea powder). untreated and grey line, steam + acid-pretreated sorghum husk.
738
observations of native SH and acid-pretreated SH confirmed
these physical changes in the biomass. The native SH had
a smooth and continuous surface, whereas that of the acid-
pretreated SH was rough in appearance (Fig. S3). These
results indicate that the steam and acid pretreatment removed
the external fibers and increased the surface area such that
cellulose and hemicellulose became more accessible to the
enzymes. Similar structural changes were observed in
sugarcane bagasse after microwave-assisted pretreatment
and in rice straw pretreated with aqueous ammonia [31,32].
3.3. Enzymatic saccharification of lignocellulosic substrate
(SH)
The efficiency of crude enzymes produced by P. chrysosporium
in hydrolyzing the untreated and mild sulfuric acid-pretreated
SH was evaluated. Most fungal cellulases possess catalytic
and cellulose-binding domains; the capability of cellulase
to adsorb to cellulose is an important factor that affects the
rate and enzymatic hydrolysis of the biomass [24]. To
overcome this potential rate-limiting step, a two-step
hydrolytic process that couples enzymatic hydrolysis with
fiber separation was proposed. The differences in hydrolytic
and glucose yields between the two processes are depicted
in Fig. 3. Compared to one-step hydrolysis, the two-step
process exhibited a significant improvement in the hydrolysis
and glucose yields. The maximum hydrolysis and glucose
yields after one-step enzymatic hydrolysis were 84.45 and
49.45%, respectively, which increased to 89.28 and 53.64%,
respectively, after two-step enzymatic hydrolysis. The
saccharification yield was improved by 6.6% in the two-step
enzymatic hydrolysis process. The cellulose, hemicellulose,
and lignin concentrations of untreated and acid-treated SH
and total RS obtained after two-step enzymatic hydrolysis
are presented in Table S1. Two-step enzymatic hydrolysis
Fig. 3. Changes in hydrolysis yield and glucose yield after one-
step and two-step enzymatic hydrolysis of untreated and mild
acid-pretreated sorghum husk.
Biotechnology and Bioprocess Engineering 20: 733-743 (2015)
after 0.4% acid pretreatment of SH resulted in the maximum
RS released (approximately 563 mg/g of substrate), which
is significantly higher than that of reported for pine biomass
using enzymes from Fomitopsis pinicola (70.9 mg/g) and
Laetiporus sulphureus (370 mg/g) [28]. In addition, the
present value is higher than that reported for wheat straw
and sunflower hull hydrolysis using crude enzyme extract
from Fomitopsis sp. RCK2010 (214.044 mg/g) and T. reesei
RUT-C30 (288 mg/g) [3,29]. The cellulolytic enzymes produced
by P. chrysosporium showed higher saccharification efficiency
(89.28%) compared to cellulase produced in previous
studies by combinations of Agaricus arvensis and Sistotrema
brinkmannii (RS, 507 mg/g; 76.7% saccharification yield)
and commercial enzymes (Celluclast 1.5 L and Novozyme
188; RS, 429 mg/g; 60% saccharification yield) [23,30].
3.4. Dark fermentative H2 production from SH hydro-
lysates
Hydrogen is a promising alternative to fossil fuels with
many social, economic, and environmental benefits to its
credit. Acidic pretreatment followed by enzymatic hydrolysis
of SH may result in different types of reducing sugars and
Fig. 4. Performance of dark fermentative H2 fermentation from
untreated and acid-pretreated sorghum husk hydrolysates using
Clostridium beijerinckii KCTC 1785 (A) cumulative H2 production,
H2 yield, and H2 content; (B) soluble metabolites formation.
Fermentative Hydrogen Production Using Sorghum Husk as a Biomass Feedstock and Process Optimization 739

generates solubilized furfural derivatives, which creates an
unfavorable environment for microbial growth and fer-
mentation (biofuels) processes [11,12]. Considering this
potential drawback, it is important to determine that the
obtained SH hydrolysates can be efficiently converted to
H2 by C. beijerinckii. Much lower H2 production (186 mL/L)
observed with untreated SH hydrolysates (only autoclaved)
suggested the need for acid pretreatment for maximum H2
production. With 0.2% acid-pretreated SH hydrolysates,
C. beijerinckii exhibited a maximum cumulative H2 production
of 952 mL/L, a maximum production rate of 39.70 mL/L/h,
and an H2 yield of 0.955 mmol H2/mol of RS (or 0.859 mmol
H2/g cellulose) (Fig. 4A). The 0.4% acid-pretreated SH
hydrolysates had higher sugar content but produced less H2
(677 mL/L), with a maximum production rate of 25.66 mL/L/h
and H2 yield of 0.945 mmol H2/mol of RS (Fig. 4A). The
soluble metabolites formed along with H2 production from
SH hydrolysates consisted mainly of acetate and lactate,
followed by butyrate and formate (Fig. 4B). The presence of
acidogenic soluble metabolites decreased the pH (6.5 ~ 2.0)
of the fermentation media. At the end of the fermentation,
dominant ethanol production was observed, which is
unfavorable to biohydrogen production [20,33]. These
results suggest that biohydrogen production efficiency is
influenced not only by the initial sugar concentration but
also by the soluble metabolites generated during fermentation.
increase in the concentration of SH hydrolysates for the
concentration range of 2.5 ~ 7.5 g RS/L. The highest
cumulative H2 production (952 and 1,025 mL/L), H2
production rate (39.66 and 42.70 mL/L/h), and H2 yield
(0.95 and 0.92 mol H2/mol of RS) occurred at concentrations
of 5.0 and 7.5 g RS/L, respectively (Fig. 5B). When the
concentration was increased to 10.0 g RS/L, a significant
decrease in the H2 production and H2 yield was observed
(Fig. 5B). At higher concentration (10 RS/L) enhancement
of soluble metabolites accumulation was observed. When
the concentration of acid metabolites was over a certain
threshold, initiated the solventogenesis process which leads
further inhibition of H2 production.
In SH hydrolysates, the RS composition consists of
glucose (55%), xylose (24%), and arabinose (5%), with the
remainder being unidentified sugars. During this increase
in the substrate concentration, glucose can be utilized
efficiently; however, the utilization of xylose and arabinose
remains poor. Nearly 100% utilization of glucose was
observed at up to 7.5 g/L RS; however, at 10.0 g/L RS, a
glucose utilization of approximately 90% was observed
(data not shown). In contrast, the utilization of xylose and
arabinose at 5.0 g/L RS sharply decreased, and at 10.0 g/L

3.5. Effects of temperature and substrate concentration

on biohydrogen production
Temperature is an important environmental factor that
influences the growth rate and metabolic activities of H2-
producing bacteria and fermentative H2 production [15,34].
The H2 production rate by C. beijerinckii varied with
temperature ranging from 30 to 45°C. The maximum H2
production level and rate were attained at 35°C and from
40 to 50°C there was a sharp decrease in the H2 production
(Fig. 5A). The H2 production pattern was very similar with
soluble metabolites production. At the end of fermentation,
solventogenesis was observed between 35 and 40°C where
the ethanol (55 mg/L) production was also dominant. The
results were consistent with C. beijerinckii RZF-1108 and
C. beijerinckii Fanp3, where maximum H2 production was
observed at 35°C [35,36]. Therefore, 35°C (Fig. 5A) was
selected as the optimal temperature condition for H2
production in the following experiments.
The composition and concentration of substrate is also
an important factor affecting the kinetics of biohydrogen
production [20,34,36]. In the present investigation, the
effect of the concentration of acid-pretreated (0.2%) SH
Fig. 5. Effect of (A) temperature using 0.2% acid-pretreated sorghum
hydrolysate (2.5 ~ 10.0 g RS/L) on biohydrogen production husk hydrolysates (5.0 g RS/L), (B) concentration of 0.2% acid-
was determined (Fig. 5B). The cumulative H2 production pretreated sorghum husk hydrolysates (RS 2.5 to 10.0 g/L) on
and maximum H2 production rate increased along with the biohydrogen production by Clostridium beijerinckii KCTC 1785.
740 Biotechnology and Bioprocess Engineering 20: 733-743 (2015)

the utilization was only approximately 10 and 5%, incomplete degradation of organic substrates and the
respectively (data not shown). Thus, an increase in sugar production of H2 gas is accompanied by the formation of
concentration beyond a certain level does not lead to an acetate and/or butyrate with a stoichiometric ratio of 2 mol
increase in utilization, and H2 yield consequently of H2 per mole of acetate or butyrate [15,18,34]. The
decreases. In this study the optimal sugar concentration of results suggest that the soluble metabolites, mainly acetate,
SH hydrolysate was found lower than many studies that may contribute important role and act as an inducer for H2
used pure carbon source using Clostridium strains for the production in the set of controlled pH at 5.5.
hydrogen production [15]. Based on these results, 5.0 g Utilization of xylose and arabinose by C. beijerinckii in
RS/L of SH hydrolysate was used for further experiments. without pH control was approximately 22 and 14%,
The low yield of H2 at higher SH hydrolysate concentration respectively, while in case of pH control, it increased the
could also be due to the generation of chemical inhibitors efficiencies around 54 and 45%, respectively, at pH 5.5
including terpenes and furfural derivatives which may be
present in small quantity [31,32]. Similar observations
were obtained in various H2 production studies including
Clostridium butyricum [34], Clostridium beijerinckii RZF-
1108 [35], Clostridium saccharoperbutylacetonicum N1-4
[38], and Clostridium butyricum CGS5 [20], where
increasing the substrate concentration beyond a certain
level inhibited the fermentation process. We supposed that
for efficient conversion of SH hydrolysate into H2 is
possible by using molecular biology tools to modify the
strain for the conversion of xylose and arabinose into H2
and also need detoxification of hydrolysates to remove the
toxic compounds may present in small quantity.

3.6. Using a pH-control strategy to enhance biohydrogen

production from SH hydrolysate
It has been reported that low pH inhibits H2 production
because it affects hydrogenase activity and many aspects of
microbial metabolism, including utilization of substrate,
biogas content, and types of organic acids [15,18,33]. Low pH
of fermentation media usually results in lower intracellular
ATP levels and causes inhibition of bacterial growth and
change in their metabolic pathways (acidogenesis to
solventogenesis) resulting in low H2 production [21,33].
Considering these, a pH-control strategy was employed
to improve the performance of biohydrogen production.
During dark fermentation, the culture pH was initially 6.5,
and when the pH decreased to a desired level (i.e., 5.0, 5.5,
and 6.0), the pH was maintained at that value. Incubation
at pH 5.5 showed the highest H2 yield (1.05 mol H2/mol of
RS) with higher cumulative H2 production (1,117 mL/L)
and production rate (46.54 mL/L/h) (Fig. 6A). In without
pH control set, high level of acids and ethanol production
during fermentation was observed and these metabolites
could act as inhibitor for the H2 production. Whereas in the
set of controlled pH at 5.5, higher production of acetate
compared to lactate, formate and butyrate and lower ethanol
production in contrast to the set of controlled pH at 5.0 was
Fig. 6. Effect of pH-control strategy on (A) hydrogen production,
observed (Fig. 6B). Moreover less production of metabolites (B) soluble metabolites production, and (C) sugar utilization by
and H2 was observed in the set of controlled pH at 6.0 Clostridium beijerinckii KCTC 1785 using 0.2% acid-pretreated
(Fig. 6B). It was reported that during dark fermentation sorghum husk hydrolysates (RS 5.0 g/L; at 35°C).
Fermentative Hydrogen Production Using Sorghum Husk as a Biomass Feedstock and Process Optimization 741

Table 1. Comparison of biohydrogen production performance using pure cellulose or waste biomass hydrolysates under dark fermentation
Maximum
Operation Temp Maximum
H2 producer Substrate (g/L) pH H2 production Reference
mode (°C) H2 yield
(mL H2/L)
a
Clostridium butyricum Sugarcane bagasse Batch 37 5.5 1,611 1.73 mol H2/mol [34]
(20 g COD/L) hexose
Clostridium Rice bran (100 g/L) Batch 30 6.0 1,132 3.37 mol H2/mol [38]
saccharoperbutylacetonicum hexose
N1-4
Clostridium pasteurianum Hydrolyzed carboxymethyl Batch 35 7.0 23.8 1.21 mol H2/mol [39]
cellulose (10 g/L) hexose
Clostridium beijerinckii Glucose (10 g/L) Batch 36 6.5 2,450 2.0 mol H2/mol [40]
AM21B hexose
Starch (10 g/L) 2,255 1.8 mol H2/mol
hexose
Clostridium thermocellum Delignified wood fibers Batch 60 7.0 2,528 1.6 mol H2/mol [41]
27405 (0.1 g/L) hexose
Clostridium butyricum CGS5 Sugarcane bagasse Batch 37 7.5 72.6 1.91 mol H2/g [10]
hydrolysates (RS 1.545 g/L) sugarcane bagasse
Clostridium acetobutylicum Microcrystalline cellulose Batch 37 5.0 755 0.188 mol H2/mol [42]
X9 (10 g/L) hexose
Thermotoga neapolitana Cellulose (5 g/L) Batch 75 7.0 39-49 1.07 mol H2/ mol [43]
(DSM 4359) hexose
Clostridium beijerinckii Food waste (50 g COD/L) Batch 40 5.5 1,368.5 128 mL H2/g COD [21]
KCTC1785 degraded
Clostridium butyricum CGS5 Hydrolysate of NaOH Batch 37 7.5 1,006 0.76 mol H2/mol [17]
pretreated rice straw (100 g/L) xylose
Clostridium butyricum CGS5 Chlorella vulgaris ESP6 Batch 37 5.5 246 1.15 mol/mol RS [20]
(RS 9 g/L)
Clostridium beijerinckii Sorghum husk hydrolysates Batch 35 6.5 1,117 1.051 mol H2 /mol This study
KCTC 1785 (RS 5.0 g/L) RS
a
mL H2/L/day.
COD: chemical oxygen demand.
RS: reducing sugar.

(Fig. 6C). Thus, higher utilization of all sugars present in
SH hydrolysates and lower soluble metabolites production
in the pH-control approach (initial pH 6.5 followed by pH-
control at 5.5) may contribute to the increase in H2
production and production rate. Our results are consistent
with those reported with C. butyricum CGS5 using
carbohydrate-rich Chlorella vulgaris hydrolysates [20] and
C. beijerinckii KCTC 1785 using food waste [21], where
maintaining pH at 5.5 exhibited maximum H2 production
and production rate. In the present study, the maximum H2
production and H2 yield obtained under the optimal
conditions is found to be better or comparable to most of
the reported results from relevant studies suggesting that
SH hydrolysates can be used as fermentation media for H2
production (Table 1).
The foregoing results suggested that pH controlled
strategy (initial pH 6.5 followed by pH-control at 5.5) was
better option for the efficient utilization of hydrolysates and
their conversion into hydrogen. Future investigations will
be devoted to optimizing the bioreactor design and operation
conditions to favor H2-producing metabolic processes, and
to reduce the cost of biohydrogen production via more
efficient hydrolysis-fermentation approaches.
4. Conclusion
In this study, we have investigated the capacity of
actinomycetes and fungi for cellulase and xylanase production
under SSF conditions. The crude enzymes could effectively
hydrolyze SH biomass and release maximum RS with
higher hydrolysis and glucose yield. Mild acidic pretreatment
(0.2% H2SO4) was the preferred method for increasing the
accessibility of SH for enzymatic hydrolysis. The resulting
SH hydrolysates used for H2 production by C. beijerinckii
exhibited maximum H2 yield using dark fermentation. The
optimal conditions for H2 production were 35°C, an SH
hydrolysate loading of 5.0 g RS/L, and pH maintained at
5.5. These results demonstrate that mild acid pretreatment
followed by enzymatic hydrolysis is a promising technology
742
for the enhancement of biohydrogen production from
lignocellulosic biomass. This work may provide a promise
of large-scale biohydrogen production using different
abundant lignocellulosic feedstock (rice waste biomass)
which is abundant in South Korea.
Acknowledgements
This research was supported by a grant from the New &
Renewable Energy Program of the Korea Institute of Energy
Technology Evaluation and Planning (no. 20133030000300)
and a grant from the National Research Foundation of Korea
funded by the Korean Government (2012M1A2A2026560).
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