Bioresource Technology 194 (2015) 354363

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Improvement of gaseous energy recovery from sugarcane bagasse

by dark fermentation followed by biomethanation process
Sinu Kumari a, Debabrata Das b,
a
Advanced Technology Development Center, Indian Institute of Technology Kharagpur, 721302, India
b
Department of Biotechnology, Indian Institute of Technology Kharagpur, 721302, India

h i g h l i g h t s g r a p h i c a l a b s t r a c t

 60% (w/w) lignin removal from the

biomass of using 0.25 N NaOH (50 C,
30 min).
 2.6-fold increase in enzymatic
conversion efciency of alkali treated
sugarcane bagasse than untreated
one.
 Gaseous energy recovery of two stage
process was signicantly higher than
dark fermentation process.

a r t i c l e i n f o a b s t r a c t

Article history: The aim of the present study was to enhance the gaseous energy recovery from sugarcane bagasse. The
Received 21 May 2015 two stage (biohydrogen and biomethanation) batch process was considered under mesophilic condition.
Received in revised form 9 July 2015 Alkali pretreatment (ALP) was used to remove lignin from sugarcane bagasse. This enhanced the enzy-
Accepted 10 July 2015
matic digestibility of bagasse to a great extent. The maximum lignin removal of 60% w/w was achieved
Available online 17 July 2015
at 0.25 N NaOH concentration (50 C, 30 min). The enzymatic hydrolysis efciency was increased to about
2.6-folds with alkali pretreated sugarcane bagasse as compared to untreated one. The maximum hydro-
Keywords:
gen and methane yields from the treated sugarcane bagasse by biohydrogen and biomethanation pro-
Sugarcane bagasse
Alkali pretreatment
cesses were 93.4 mL/g-VS and 221.8 mL/g-VS respectively. This process resulted in signicant increase
Hydrogen in energy conversion efciency (44.8%) as compared to single stage hydrogen production process (5.4%).
Methane 2015 Elsevier Ltd. All rights reserved.
Enzymatic hydrolysis

1. Introduction wide verities of feedstocks like lignocellulosic biomass and waste

Hydrogen is a clean fuel and it is one of the most important
alternative energy sources. The energy density of hydrogen is high-
est (142 kJ/g) as compared to other hydrocarbons and it can be pro-
duced from renewable sources (Perry and John, 1973) .Among the
various hydrogen production processes, biological hydrogen pro-
duction by dark fermentation has shown signicant advantages
due to high rate of hydrogen production and can be produced from
Corresponding author. Tel.: +91 9434717424.
E-mail address: ddas.iitkgp@gmail.com (D. Das).
materials. However, the total energy recovery of dark fermentative
hydrogen production is low (Pakarinen et al., 2011). In order to
enhance the overall gaseous energy recovery and complete utiliza-
tion of organic acids produced during dark fermentation, a
two-stage biohydrogen and subsequent biomethane production
process has been proposed recently and being investigated using
various feedstocks (Cheng and Liu, 2012; Chong et al., 2009; Guo
et al., 2010; Luo et al., 2011; Nasr et al., 2012; Pakarinen et al.,
2011). This process has several advantages as compared to the sin-
gle stage dark fermentative H2 production process. The process
involved the selection and enrichments of the stage specic

http://dx.doi.org/10.1016/j.biortech.2015.07.038
0960-8524/ 2015 Elsevier Ltd. All rights reserved.
 S. Kumari, D. Das / Bioresource Technology 194 (2015) 354363 355

microbial consortia, which enhanced the degradation efciencies evaluated. The changed in the morphological structure and chem-
of microbes and thereby, increased the overall process stability ical composition of biomass during the entire process was investi-
(Luo et al., 2011). The rst stage leads to the formation of hydrogen gated by scanning electron microscopy (SEM), X-ray diffraction
and volatile fatty acids. In the second stage, the volatile fatty acids (XRD) and Fourier transform infrared spectroscopy (FTIR), and
used by the specic microbial consortia to produce methane and the total energy recovery efciency for co-production of hydrogen
carbon dioxide (Monlau et al., 2015). Therefore, the methane pro- and methane from sugarcane bagasse was also evaluated.
duction in the 2nd stage can be improved by enhancing the hydrol-
ysis of biomass in the acidogenic phase, i.e., hydrogen production
stage (Nasr et al., 2012). The overall gaseous energy recovery is 2. Methods
high in two stage as compared to the single stage hydrogen pro-
duction. In the dark fermentative hydrogen production stage, 2.1. Feedstock
about 30% of the substrate energy can be recovered (Das and
Veziroglu, 2001). Subsequent utilization of the spent medium of The sugarcane bagasse used in the present study was collected
dark fermentation for methane production would represent a from the local market of IIT Kharagpur. Biomass were washed
promising alternative to improve the overall gaseous energy recov- properly under tap water, air dried, grinded and sieved through a
ery (Cooney et al., 2007). 3 mm screen before storing at room temperature prior to use.
The major bottleneck for using lignocellulosic materials as sub- The main composition of sugarcane bagasse was as follows (on
strate for the dark fermentation process is the structural complex- dry weight basis): volatile solid 97.19%, cellulose 42.3%, hemicellu-
ity of biomass (Monlau et al., 2015). Due to the heterogeneity and lose 21%, lignin 18.4%, carbon (C) 45.2%, nitrogen (N) 1.72%, hydro-
crystallinity of biomass, direct utilization of biomass by microbes is gen (H) 6.8% and sulfur (S) 0.194%. The carbon to nitrogen (C/N)
extremely slow. Therefore, pretreatment of the biomass is ratio of sugarcane bagasse was 26.
required. It disrupts the recalcitrant structure of the lignocellulosic
biomass by breaking the lignin seal and decreasing the crystallinity
2.2. Seed inoculum
of cellulose. As a consequence, the accessibility of cellulose to
enzymatic attack increases. This improves the enzymatic hydroly-
The acidogenic mixed consortia was developed from the cow
sis of carbohydrates, which can be easily fermented by microbes to
dung. The pH, total solid (TS) and volatile solid (VS) contents were
produce hydrogen and methane (Lim et al., 2013).
7.5, 28,100 and 25,000 mg/L respectively. Before being used as the
Over the past few years, many pretreatment techniques have
inoculum, the cow dung was subjected to heat shock pretreatment
been developed to decrease the crystallinity of cellulose, increase
(100 C, 20 min) to selectively enrich the hydrogen producing bac-
the biomass surface area, separation of lignin to increase the acces-
teria by inhibiting the growth of methanogenic bacteria (Ren et al.,
sibility of enzymes for maximum sugar recovery (Mosier et al.,
2008). The enrichments of pretreated inoculum were done in a
2005). Among the various pretreatment technologies, alkaline pre-
100 mL anaerobic serum bottles with working volume of 80 mL.
treatment has many advantages for effective delignication and
The mineral salt media was used for enrichments having the com-
swelling of biomass. Alkali pretreatment is basically a delignica-
position of (in g/L): glucose 10, yeast extract 3, NaHCO3 2.5,
tion process. The action of mechanism is based on the saponica-
NaH2PO42H2O 0.8, K2HPO43H2O 0.8, FeSO47H2O 0.1,
tion of intermolecular ester bonds cross-linking xylan
MgCl26H2O 0.1, NiCl26H2O 0.004, CuCl25H2O 0.005, CaCl2 0.05,
hemicelluloses and other components. Alkaline pretreatment
ZnCl2 0.005 (Wang and Wan, 2008). The pH of the media was
causes swelling of lignocellulosic biomass, leading to decreased
maintained to 6.5. The anaerobic condition was created by sparg-
degree of polymerization and crystallinity, increased internal sur-
ing with nitrogen gas for 5 min and sealed tightly by crimping
face area and separation of structural linkages between lignin
and incubated at 37 C and 180 rpm in shaker. The hydrogen pro-
and carbohydrates (Ganguly et al., 2013). It breaks the alkylaryl
ducing acidogenic mixed consortia was developed by repeated
linkages of lignin at higher pH concentration (Park and Kim,
sub-culturing in the above mentioned media after every 24 h.
2012). Additionally, the presence of a small amount of residual
The microbial prole of the hydrogen producing mixed consortia
alkali in the treated biomass may be helpful for preventing the
was analyzed by 16S-PCR-DGGE method and it was identied to
pH drop during dark fermentation process (Pavlostathis and
be dominated by Clostridium sp. (unpublished data).
Gossett, 1985). Among the various alkalis, sodium hydroxide
The anaerobic digester sludge was collected from the biogas
(NaOH) was found to be most effective for delignication and bio-
digester plant operating at mesophilic condition (35 C) near IIT
gas production (Zhu et al., 2010).
Kharagpur. The pH, total solid and volatile solid content of sludge
India is the world second largest producer of sugarcane
was 6.8, 7500 and 5600 mg/L respectively. Initially, the sludge
[341.2 million metric tons/year (MMT/Y)] and the residue gener-
was inoculated into the mineral salt media containing acetate as
ated in the form of bagasse is around 101.3 MMT/Y. The sugarcane
the carbon source to enrich the methanogens and later it was accli-
bagasse is mostly consumed by the paper mill industries; however,
matized with spent medium from the dark fermentation to pro-
a signicant surplus amount (6.6 MMT/Y) is remain unused which
duce methane.
is almost always burnt in the eld itself (Sukumaran et al., 2010).
Use of this surplus amount of sugarcane bagasse for biofuel pro-
duction not only provides a renewable fuel but also help in 2.3. Alkali pretreatment
bioremediation.
The two stage process has been reported by few literature (as The effect of alkali concentration and incubation time on lignin
mentioned earlier), but this is the rst time when we are using removal of biomass was studied using sodium hydroxide (NaOH),
sugarcane bagasse for the two stage biohydrogen and subsequent SRL grade. Dry biomass of sugarcane bagasse was mixed with
biomethanation process. Therefore, the aim of the present study aqueous NaOH solution (solid loading of 5% w/v) at different con-
was to enhance the gaseous energy recovery from sugarcane centration of NaOH (0.12, 0.25 and 0.5 N) and kept at 50 C for 15,
bagasse. An efcient two stage process: dark fermentation fol- 30, 45 and 60 min. After pretreatment, solid biomass was sepa-
lowed by biomethanation process was developed. In addition, the rated by ltration using muslin cloth, and was repeatedly washed
effect of alkali pretreatment and enzymatic hydrolysis of biomass with tap water till neutral pH of wash water. The solid residue was
on two stage gaseous energy generation process was also then dried at 60 C in hot air oven till the constant weight and
356 S. Kumari, D. Das / Bioresource Technology 194 (2015) 354363
further used for enzymatic hydrolysis and chemical compositional
analysis.
2.4. Enzymatic hydrolysis
The dried untreated and pretreated biomass (solid loading 5%
w/v) in aqueous solution containing citrate buffer (0.05 M, pH 5)
was supplied with cellulase from Trichoderma reesei (0.8 units/mg
solid, SigmaAldrich) with different loading concentration (10,
15, 20, 25 and 30 U/g of dry biomass, DM) and incubated at 37 C
and 180 rpm for 48 h. The reactions were carried out in 250 mL
Erlenmeyer ask with total reaction volume of 100 mL. The hydrol-
ysis process was terminated by boiling in water bath for 10 min
(Yang et al., 2015). After cooling down to room temperature, liquid
and solid fractions were separated by centrifugation at 6000g for
10 min. Supernatant was collected as the enzymatic hydrolysate
and the released reducing sugars were determined by DNS method.
Optimal loading of cellulase was determined by enzymatic conver-
sion efciency (ECE) of biomass.
2.5. Two stage process: Biohydrogen followed by biomethanation
The two stage process was performed batch wise using double
jacketed reactor with working volume of 500 mL. Three different
processes were taken into consideration (Fig. 1).
Process 1: Raw sugarcane bagasse (RSCB) was directly used as a
substrate for hydrogen fermentation.
Process 2: RSCB was pretreated with NaOH (0.25 N, temp. 50 C,
incubation time 30 min). The dried alkali pretreated sugarcane
bagasse (ALPSCB) was used as substrate for hydrogen
fermentation.
Process 3: RSCB treated with alkali (conditions as mentioned in
Process 2) followed by enzymatic hydrolysis (Cellulase, 20 U/g
DB). The slurry of biomass after hydrolysis was used for hydro-
gen fermentation.
In all the processes, solid loading of 1% (w/v) in aqueous solu-
tion of mineral salt media (composition: refer Section 2.2) having
pH 6.5 was used for the 1st stage hydrogen production and the
spent medium (efuent) remained after dark fermentation was
used as inuent for the 2nd stage methane production by adjusting
the pH to 7.5. The acidogenic mixed consortia (10% v/v) was used
in hydrogen production process while the methanogenic mixed
consortia (25% v/v) was used in the methane production. Both
Fig. 1. Different two stage H2/CH4 production processes (initial substrate concentration was 1% (w/v); alkali treatment condition: substrate (5% w/v) in aqueous solution of
NaOH (0.25 N), temperature: 50 C, incubation time: 30 min; enzymatic hydrolysis: 20 U/g-DB, solid to liquid ratio (1:20), temperature: 37 C, 180 rpm).
the reactors were sparged with 100% nitrogen for 5 min prior to
inoculation to make the environment anaerobic. The temperature
and mixing speed of the stirrer were maintained at 37 C and
280 rpm respectively using M/s. Grand circulating water bath
and magnetic stirrer. Biogas production was monitored
periodically.
2.6. Analysis
The gas composition was analyzed by Gas Chromatography (GC
Agilent Technology 7890A, USA) using stainless steel packed with
Porapak Q (80/100) column connected with thermal conductivity
detector (TCD). The operational temperatures of the injection port,
the oven and the detector were 80, 110 and 100 C, respectively.
Nitrogen was used as the carrier gas at a ow rate of 20 mL/min.
Composition of soluble metabolites formed after dark fermentation
was analyzed by Gas Chromatography (GC Agilent Technology
7890A, USA) using capillary column coated with 10% PEG-20 M
and 2% H3PO4 (80/100 mesh) connected with ame ionization
detector (FID). The temperature of the injection port, detector
and programmed column were 220, 240 and 130175 C respec-
tively. Nitrogen was used as the carrier gas at a ow rate of
20 mL/min. The mixture of hydrogen and air at a ow rate of
30 mL/min was used for ame generation. The concentration of
sugars in the hydrolysate were analyzed by high performance liq-
uid chromatography (HPLC), equipped with a differential refrac-
tometer using an Aminex HPX-87H column (Agilent
Technologies, USA) operating at 65 C with 5 mM H2SO4 as the
mobile phase at a ow rate of 0.5 mL/min.
The elemental composition of biomass were analyzed using
CHNS analyzer (Vario MACRO cube CHNS, Elementar, USA) and
the cellulose, hemicellulose and lignin content were analyzed
according to NREL protocol. Total solids (TS) and volatile solids
(VS) content were determined according to the APHA standard
protocol (Clesceri et al., 1999). The chemical oxygen demand
(COD) was also measured according to the APHA standard protocol
using a COD measurement instrument set (DRB200 and DR2800
Portable Spectrophotometer, HACH, USA). The caloric values of
samples were determined by using Automatic Oxygen Bomb
Calorimeter (6100, Parr Instruments, USA).
The changed in the morphology of sugarcane bagasse after
alkali pretreatment, enzymatic hydrolysis and hydrogen fermenta-
tion were analyzed by scanning electron microscopy (SEM). Prior
to imaging, the dried samples were spread on to the carbon tape
placed over the surface of SEM stub and were sputtered with
 S. Kumari, D. Das / Bioresource Technology 194 (2015) 354363
10 nm gold in a sputter coater. The image was observed using a
SEM (ZEISS EVO 60 scanning electron microscope).
The changed in the crystalline structure of biomass after pre-
treatment and enzymatic hydrolysis was investigated by X-ray
Diffractometer (D8 Advance, Bruker, Germany). The diffractometer
was adjusted to 40 kV, 40 mA; radiation was Cu Ka (k = 1.54 ).
Samples were scanned over the range of 2h = 530 with a step size
of 0.05. The crystallinity index (CrI) was calculated as per the
empirical formula proposed by Segal et al. (1959).
The chemical changes of biomass after pretreatment and enzy-
matic hydrolysis were analyzed using FTIR spectroscopy
(NEXUS-870, USA). The samples were prepared by mixing biomass
and KBr in a ratio of 1:100 w/w. The mixtures were grounded well
and the spectra were recorded in the range of 4004000 cm1
using 200 mg of biomass + KBr mixture in the form of pellets.
2.7. Statistical analysis
All reported data were the average value of the triplicates and
were expressed as mean value (SD). One way analysis of variance
(ANOVA) at the 95% condence level (p < 0.05) was performed
using statistical software package Minitab version 15.10 (Minitab
Inc., Pennsylvania, USA), to analyze the effect of pretreatment con-
ditions on lignin reduction, reducing sugars yield and biomass
composition.
3. Results and discussion
3.1. Effect of alkali pretreatment on lignin reduction of biomass
The reduction in the lignin content of sugarcane bagasse was
analyzed. Fig. 2 showed the effect of NaOH concentration and incu-
bation time on the percent reduction of lignin as compared to the
original biomass. The amount of lignin removal was varied from
11.3% to 74.6% of the original lignin content of sugarcane bagasse.
As the alkali concentration increased from 0.12 to 0.5 N, lignin
reduction increased. However, increasing the retention time from
15 to 60 min had insignicant effect on delignication. The maxi-
mum lignin reduction of 74.6% (w/w) was observed at 0.5 N
NaOH concentration having retention time of 60 min with hemi-
cellulose loss of 23.5% (w/w). This was due to the degradation of
Fig. 2. Effect of NaOH concentration and treatment time on lignin reduction of sugarcane bagasse.
357
hemicellulose at higher alkali concentration and longer treatment
time. Up to 60% (w/w) lignin removal of sugarcane bagasse was
observed by treating with 0.25 N NaOH with a residence time of
30 min. The loss of hemicellulose was low (Table 1). The composi-
tion of SCB hydrolysate obtained after alkali treatments were ana-
lyzed for sugar content. Glucose, xylose and arabinose were
obtained in low amount (Table 1). The concentrations of glucose,
xylose and arabinose were varied from 0.56% to 0.86%, 0.12
0.24% and 0.130.36% respectively. Hence, the total sugars (glu-
cose + xylose + arabinose) concentrations were observed in the
range of 0.821.44 g/L). Alkali generally breaks the ester bonds
crosslinking lignin and xylan, resulting the swelling of cellulose
bers and modication of cellulose crystallinity rather than direct
degradation (Yang et al., 2014). Therefore, alkali pretreatment was
used to remove the lignin from the biomass before enzymatic
hydrolysis and fermentation process. So, the most suitable pre-
treatment conditions were as follows: 0.25 N NaOH; reaction time
30 min; and temperature 50 C.
3.2. Effect of cellulase dosage on hydrolysis of treated sugarcane
bagasse
The effect of cellulase dosage on the hydrolysis of pretreated
sugarcane bagasse (0.25 N, 30 min, 50 C) with respect to percent
enzymatic conversion efciency (%ECE) is shown in Fig. 3.
Optimal loading of cellulase was determined in terms of reducing
sugars released of biomass. The pretreated sugarcane bagasse led
to signicant increase in the %ECE as compared to untreated one.
The highest %ECE of pretreated biomass was reached to 83.5% at
enzyme loading of 30 U/g-DB, which is approx. 2.6-fold higher than
that of the untreated one. It was observed that the %ECE of pre-
treated biomass was increased with increased in enzyme loading
and it reached around 77.5% at enzyme loading of 20 U/g-DB.
However, there was no signicant improvement of enzymatic
hydrolysis on further increase of enzyme loading to 30 U/g-DB.
Since, cellulase enzymes are one of the most expensive chemical,
therefore, the economical use of cellulases plays important role.
One can bypass the benet of slight increase in %ECE. Thus, the
optimum enzyme loading for hydrolysis was determined to be
20 U/g-DB.
358 S. Kumari, D. Das / Bioresource Technology 194 (2015) 354363

Table 1
Composition of sugarcane bagasse and hydrolysate after alkali pretreatment.

Treatment Residence time (min) Pretreated SCB Hydrolysate of pretreated SCB

Glucan (% w/w) Xylan (% w/w) Glucose (g/L) Xylose (g/L) Arabinose (g/L) Total sugar (g/L)
Untreated 42.3 0.76 21.00 0.4
0.12 N NaOH 15 60.3 0.13 20.86 0.4 0.56 0.05 0.13 0.01 0.13 0.01 0.82 0.09
30 56.16 0.87 18.45 0.52 0.65 0.08 0.12 0.01 0.14 0.01 0.91 0.07
45 55.46 0.79 20.53 0.72 0.66 0.05 0.13 0.02 0.15 0.01 0.94 0.05
60 54.85 0.82 17.53 0.56 0.71 0.08 0.18 0.01 0.13 0.01 1.02 0.08
0.25 N NaOH 15 66.16 0.13 19.86 0.40 0.66 0.07 0.13 0.01 0.15 0.02 0.94 0.08
30 62.16 0.88 19.62 1.65 0.73 0.07 0.15 0.02 0.18 0.01 1.06 0.07
45 61.46 0.75 18.60 0.53 0.71 0.05 0.18 0.03 0.24 0.01 1.13 0.08
60 60.85 0.92 17.65 0.57 0.76 0.05 0.20 0.01 0.26 0.01 1.22 0.05
0.5 N NaOH 15 55.12 0.73 17.85 0.89 0.81 0.06 0.19 0.03 0.23 0.03 1.23 0.08
30 52.85 0.92 18.60 0.69 0.76 0.08 0.23 0.01 0.28 0.01 1.27 0.07
45 51. 78 0.95 19.10 0.4 0.86 0.7 0.22 0.01 0.34 0.01 1.42 0.09
60 50.33 0.79 16.06 0.40 0.84 0.07 0.24 0.02 0.36 0.01 1.44 0.07

Fig. 3. Effect of enzyme loading on enzymatic conversion efciencies (ECE) of biomass. (A) Untreated sugarcane bagasse, (B) alkali pretreated sugarcane bagasse using 0.25 N
NaOH at 50 C for 30 min.

3.3. Comparison of different hydrogen production in batch processes
The comparisons of hydrogen yields from the three different
processes are depicted in Fig. 4a. Raw sugarcane bagasse was used
directly for microbial conversion in Process 1 to evaluate the feasi-
bility of acidogenic mixed consortia for hydrogen production. A,
total hydrogen yield in mesophilic dark fermentation of sugarcane
bagasse was reached to 55 mL/g-VS. When lignocellulosic biomass
was used as carbon source in mesophilic dark fermentation by
using mixed culture, signicant decrease of hydrogen yield was
also reported by Cheng and Liu (2012). This low hydrogen yield
was mainly due to the crystalline structure and presence of lignin
barrier of biomass, which prevents the direct contact of microbes
with substrate. This reduces the substrate conversion efciencies
(Cheng and Liu, 2012).
The hydrogen yield increased from 55 to 77.2 mL/g-VS (Fig. 4a)
when sugarcane bagasse was pretreated with NaOH before hydro-
gen fermentation (Process 2). This result indicated that, NaOH pre-
treatment removed the lignin barrier and opened up the
recalcitrant structure of biomass, which improved the degradation
efciencies of microbes resulted in increased in hydrogen yield.
The increased in hydrogen yield after NaOH pretreatment was also
reported by Cheng and Liu (2012). They observed that the
increased in hydrogen yield from 37.6 to 45.7 mL/g-cornstalk,
when the cornstalk was pretreated with 0.83% (w/v) NaOH at
120 C for 20 min. The hydrogen yield was further improved by
integrating the alkali pretreated sugarcane bagasse with enzyme
hydrolysis (Process 3). The total hydrogen yield of 93.4 mL/g-VS
was achieved. This nding was ascribed the capability of enzymes
to breakdown the complex sugars of alkali pretreated sugarcane
bagasse. Therefore, increased yield of monomeric sugars, which
easily utilized by the microbes for fermentative hydrogen produc-
tion. Lin et al. (2015) reported the increased hydrogen yield from
51.7 mL/g-TVS (NaOH treated water hyacinth) to 63.9 mL/g-TVS
(NaOH treatment + enzymatic hydrolysis of water hyacinth).
3.4. Methane production from the spent medium of dark fermentative
hydrogen production in batch process
Signicant amount of organic acids was remained in the spent
medium after the hydrogen production, which can reutilized by
the methanogen mixed consortia for methane production to max-
imize the gaseous energy recovery. The organic acid content of
spent medium was determined (Table 2). In the hydrogen
 S. Kumari, D. Das / Bioresource Technology 194 (2015) 354363 359

Fig. 4a. Hydrogen production proles from sugarcane bagasse.

Table 2 fermentation of 15 d signicant enhancement of methane pro-

Performance of the three processes in terms of hydrogen yield, methane yield, COD duction was obtained in Process 3, and the total methane reached
mass balance and total gaseous energy recovered (H2 + CH4).
221.8 mL CH4/g-VS. The VFAs and ethanol were hardly detected
Feedstock parameters Process 1 Process 2 Process 3 at the end of methane fermentation. The analytical results of liq-
TS (mg/L) 10,000 10,000 10,000 uid phase after methane production showed the utilization ratio
VS (mg/L) 9790 9790 9790 of both acetate and butyrate were more than 97%. This conrms
pH feeding 6.5 6.5 6.5 the effectiveness of using VFAs produced by the biological hydro-
H2 batch assay gen production process as a substrate to produce methane via
H2 yield (mL/g-VS added) 55 1.4 77.2 0.93 93.4 1.7 anaerobic process. Table 3 summarizes the literature data on
Acetate (mg/L) 280 10 380 15 600 10
Butyrate (mg/L) 510 15 630 10 1200 12
two stage hydrogen and methane production using lignocellulosic
pH outlet 4.8 4.5 4.21 wastes.
Energy recovery (%) 3.2 4.5 5.4
CH4 batch assay 3.5. Analysis of raw and treated sugarcane bagasse
pH feeding 7.5 7.5 7.5
VS (mg/L) 6853 6266 5834
3.5.1. Scanning electron microscopy (SEM)
CH4 yield (mL/g-VS added) 122.3 2.3 173.3 4.6 221.8 5.3
pH outlet 6.1 6.0 6.0 The changed in the morphology of biomass during the entire
Acetate (mg/L) 5.2 12.8 11.0 process was observed using scanning electron microscopy. Fig. S1
Butyrate (mg/L) 10.9 10.8 12.5 illustrated that signicant morphological changes were observed
Total energy yield (H2 + CH4) 4.6 6.6 8.4 during the pretreatment, hydrolysis and fermentation process.
(MJ/kg-VS)
The surface of the untreated biomass was smooth, at, highly rigid
Total gaseous energy recovery 24.5 35.2 44.8
(H2 + CH4) (%) and undamaged (Fig. S1A), whereas after alkali pretreatment, the
biomass appeared to be disordered. The micro brils were also sep-
COD mass balance
VSS out (mg/L) 783 25 886 34 851 28 arated from each other and fully exposed thus increasing the
SCOD out (mg/L) 6935 82.5 7658 74 8576 83 porosity and the enzyme accessible surface area (Fig. S1B). This
TVFA (mgCOD/L) 25.25 1.5 33.12 1.2 34.29 1.5 in turn led to enhance the enzymatic hydrolysis of biomass. After
H2 (mgCOD/L) 334 18 480 15 584 13
the enzymatic hydrolysis, the biomass seemed more degraded
CH4 (mgCOD/L) 3160 76 4480 45 5720 55
COD balance (%) 102 4 110 6 108 5 and the big pores occurred, which conrm the degradation of long
micro brils upon hydrolysis (Fig. S1C). The structure of biomass
residue left over after fermentation was appeared more damaged
which revealed the degradation of biomass by acidogenic mixed
production processes, the acetate and butyrate were the major consortia to some extent (Fig. S1D).
end products. A negligible amount of ethanol production was
observed in all cases, with the concentrations values below 3.5.2. X-ray diffraction
40 mg/L (data not shown). Acetate and butyrate production was The crystallinity of sugarcane bagasse is a major factor which
increased during hydrogen fermentation of alkali + enzyme trea- inuences the hydrolysis as well as utilization of biomass for
ted sugarcane bagasse (ALP + ETSCB) as compared to that of bioenergy production. Fig. S2 shows the XRD pattern of the raw,
RSCB and ALPSCB. The spent of three different processes were pretreated and enzymatically hydrolyzed sugarcane bagasse. A
collected for batch methane production. Fig. 4b illustrates the major diffraction peak of cellulose crystallographic plane at 2h of
methane production prole of three different processes after 2223 indicated the presence of highly organized crystalline
360 S. Kumari, D. Das / Bioresource Technology 194 (2015) 354363

Fig. 4b. Methane production proles from spent medium of dark fermentation.

Table 3
Comparison of experimental results with respect to the reported two stage H2/CH4 production processes.

Substrate Pretreatment Mode of Temp. H2 yield Mode of Temp. CH4 yield References
method operation (C) (L/kg-VS) operation (C) (L/kg-VS)
Sorghum stalk Water extract CSTR 35 10.4 CSTR 35 29 Antonopoulou et al. (2008)
Grass silage NaOH treatment Batch 55 6.46 Batch 35 473 Pakarinen et al. (2009)
Water hyacinth NaOH treatment Batch 35 51.7 Batch 35 143.4 Cheng et al. (2010)
Defatted algal biomass NaOH treatment Batch 37 46 Batch 37 394 Yang et al. (2011)
Maize Raw Batch 55 5.6 Batch 35 342 Pakarinen et al. (2011)
Maize Water extract Batch 55 1.9 Batch 35 358 Pakarinen et al. (2011)
Maize HCl treatment Batch 55 Batch 35 397 Pakarinen et al. (2011)
* *
Cornstalk Raw Batch 55 37.6 Batch 34 111.6 Cheng and Liu (2012)
* *
Cornstalk NaOH treatment Batch 55 45.7 Batch 34 103.1 Cheng and Liu (2012)
Food waste Raw Batch 37 55.2 Batch 37 94.8 Nathao et al. (2013)
**
Durum wheat Raw Batch 35 55 Batch 35 274 Giordano et al. (2014)
**
Common wheat Raw Batch 35 33 Batch 35 251 Giordano et al. (2014)
Sunower stalk Raw Batch 37 7.1 Batch 35 150 Monlau et al. (2015)
Sunower stalk NaOH treatment Batch 37 6.3 CSTR 35 196 Monlau et al. (2015)
Water hyacinth Microwave-NaOH + enzymatic Batch 35 63.9 Batch 35 172.5 Lin et al. (2015)
hydrolysis
Sugarcane bagasse Raw Batch 37 55 Batch 37 122.3 This study
Sugarcane bagasse NaOH treatment Batch 37 77.2 Batch 37 173.3 This study
Sugarcane bagasse NaOH treatment + enzymatic Batch 37 93.4 Batch 37 221.8 This study
hydrolysis
*
Unit in L/kg-TS.
**
Unit in L/kg-COD.

region whereas the peak at 2h of 18 represents the less organized
amorphous region (Chen et al., 2012). The crystallinity index (CrI)
is the amount of crystalline cellulose present in the lignocellulosic
biomass and it is inuenced by the chemical composition of bio-
mass. The CrI (Spectrum-B) of ALPSCB (at optimum condition)
was approximately 64%, which was higher than the RSCB
(Spectrum-A). Upon alkali pretreatment, a signicant portion of
the lignin layer was removed or, the crystalline structure was bro-
ken by swelling, resulted in more expose of crystalline structure as
compared to RSCB. The high CrI indicates the high enzymatic
accessibility and digestibility (Kim et al., 2013). After further enzy-
matic hydrolysis, the CrI rapidly dropped from 64% to 46.5%
(Spectrum-C), which indicated the crystalline cellulose was
degraded by cellulase enzyme.
3.5.3. FTIR
The changed in the chemical composition of sugarcane bagasse
after alkali pretreatment (at optimum condition) and enzymatic
hydrolysis (at optimum condition) was analyzed and it is pre-
sented in Fig. S3. The spectrum 13 corresponds to RSCB, ALPSCB
and the ALP + ETSCB. Signicant changes in the biomass were
observed in the bands associated with lignin, cellulose and hemi-
cellulose. The band intensities provided the following information:
3383, OH stretching vibration; 2928, CH stretching vibration;
1253, ester absorbance; 1054, C@O stretching due to carbohy-
dratelignin linkage; 896, glycosidic linkage; 667, COH bending
deformation; 558, glycosidic linkage for hemicellulose. The absor-
bance peak at 3383 and 1054 is very sharp in spectrum 1 but it is
blunt in spectrum 2 and 3. The peak at 2928 and 896 is becoming
 S. Kumari, D. Das / Bioresource Technology 194 (2015) 354363 361

sharper that indicate the purity of biomass, i.e., the biomass
becoming more pure after alkali pretreatment and enzyme hydrol-
ysis. Which also support the data of X-ray diffraction. The reduc-
tion in intensities of vibration of the aromatic rings and
carbohydratelignin linkage (1253, 1054 cm1) in spectrum 2
and 3 prove that alkali pretreatment could effectively dissolve
the lignin. Other changes were associated with hemicellulose
dissolution.
3.6. Overall mass balance and gaseous energy recovered for the three
different congurations
toxicity and a very high C/N ratio might lead to slowing down of
fermentation due to lack of growth of the microbial cells. In the
present study, the C/N ratio of the digested biomass was found
to increase as compared to raw one after the biogas generation
process. This indicates the consumption of nitrogen is directly pro-
portional to the overall gaseous energy production.
The COD mass balances for the three processes 13, computed
considering the inuent and the efuent CODs and the equitant
CODs for both gases and biomass are shown in Table 2. The closure
of COD mass balances at 102110% proves the reliability of the
data.

3.6.1. Energy efciency

The performance of the two-stage hydrogen and subsequent
The traditional methane and hydrogen fermentation reaction
methane production in terms of their hydrogen yields, methane
for glucose is shown in Eqs. (1) and (2) respectively. The heating
yields, COD mass balance and total energy recoveries of the three
value of glucose is 2826.6 kJ/mol and the heating value of hydro-
different processes are shown in Table 2. Sugarcane bagasse in
gen and methane is 241 and 801 kJ/mol respectively. The theoret-
terms of volatile solid content was considered as the input vari-
ical hydrogen and methane production efciency from one mole of
ables and the hydrogen, methane, carbon dioxide and the undi-
glucose is calculated as: 4  242/2826.6 = 34.1% and 3  801/282
gested biomass as output variables. The undigested biomass
6.6 = 85% respectively. As the heating value of hydrogen is
(digestate) was analyzed with respect to cellulose, hemicellulose,
242 kJ/mol, the theoretical hydrogen production efciency is very
lignin content as well as C, H, N, S content (Fig. 5).
low as compared to that of methane. The residue of hydrogen pro-
Total solid removal in case of ALPSCB and ALP + ETSCB were
duction can be utilized by methanogens to produce methane (Eq.
52.8% and 58% respectively which is higher than that of the
(3)). Thus, the two stage hydrogen and methane production can
untreated biomass (41.85%) (Fig. 5). Increased in TS removal sug-
be expressed by Eq. (4). The theoretical combined (H2 + CH4) pro-
gest that, the alkali pretreatment enhanced the accessibility of fer-
duction efciency is calculated as: (2  801 + 4  242)/
mentative bacteria for better conversion of biomass to biogas.
2826.6 = 91%.
Cellulose, hemicellulose, lignin and CHNS analysis of digestate pro-
vide the information about which components were preferably C6 H12 O6 ! 3CH4 3CO2 1
degraded during fermentation process. In all the processes, hemi-
cellulose were almost completely degraded whereas, the degrada- C6 H12 O6 2H2 O ! 2CH3 COOH 2CO2 4H2 2
tion of cellulosic part was comparatively less as compared to
hemicellulose especially with untreated biomass. The less degrada- 2CH3 COOH 2H2 O ! 2CH4 4CO2 2H2 O 3
tion of cellulose might be due to its recalcitrant structure. The
decreased in lignin content in Processes 2 and 3 was mainly due
C6 H12 O6 2H2 O ! 4CO2 2CH4 4H2 4
to its partial solubilization during the alkali pretreatment. In addi-
tion to compositional analysis, the elemental (CHNS) analyses also Hence, the overall energy values and maximum gaseous energy
provide interesting information. During biogas production, proper recovered from each processes was calculated based on the lower
C/N ratio (2030) is desired. Low C/N ratio might lead to ammonia heating value of the gases and the caloric value of sugarcane

Fig. 5. Elemental and compositional analysis of digestate after two stage H2/CH4 fermentation processes (Percent values are calculated on the raw solid and on the digestate
solid remained after two stage fermentation process. All the values are the mean of triplicate).
362 S. Kumari, D. Das / Bioresource Technology 194 (2015) 354363
bagasse. Using raw sugarcane bagasse, 55 mL hydrogen and
122.3 mL methane per g-VS sugarcane bagasse were obtained as
described above, reaching 24.5% of energy recovery with the
total energy value of 4600 kJ/kg-VS sugarcane bagasse. Process 2 with
alkaline pretreatment enhanced microbial degradation of
biomass during hydrogen fermentation, and total energy recovery
rate of 35.2% was obtained with the total energy value of
6600 kJ/kg-VS sugarcane bagasse. In Process 3, 93.4 mL/g-VS of hydro-
gen and 221.8 mL/g-VS of methane were produced in hydrogen
fermentation and following methane fermentation of the efuent,
corresponding to total energy value and recovery rate of
8400 kJ/kg VS sugarcane bagasse and 44.8% respectively, i.e., the overall
energy recovery in the two stage processes was varied from 24.5%
to 44.8% which was signicantly high as compared to 1st stage
biohydrogen production by dark fermentation processes (from
3.2% to 5.4%). By performing the two stage sequential hydrogen
and methane production, Xie et al., obtained the gaseous energy
recovery (GER) of 82% by using the glucose as the substrate
(Xie et al., 2008). Cheng et al., obtained GER of 21.3% with
from untreated water hyacinth (Chuang et al., 2011). The GER
increased to 33.2% when water hyacinth was pretreated with
NaOH (Cheng et al., 2010). When water hyacinth was treated by
microwave heated alkali pretreatment (24 h NaOH soaking) and
enzymatic hydrolysis, the GER enhanced to 40% (Lin et al., 2015).
The GER from cornstalk varied from 37.7% (raw corn stalk) to
70% (alkali treated) (Cheng and Liu, 2012). These results suggest
that, incorporation of integrated dark fermentationanaerobic
biomethanation can prove to be an attractive strategy to improve
the gaseous energy recovery from lignocellulosic wastes.
4. Conclusion
ALP effectively removed the lignin, increased the porosity and
also enhanced the enzymatic digestibility of lignocellulosic bio-
mass. This improved hydrogen and methane production signi-
cantly. SEM, XRD and FTIR analysis revealed the removal of
lignin layer. Maximum gaseous energy recovery of 44.8% was
observed by integrating the biohydrogen production and
biomethanation processes. The total energy recovery is still not
attractive as compared to the theoretically yield of 91%.
Therefore, the degradation mechanism of the complex structure
of lignocellulosic biomass is to be investigated further to enhance
the overall gaseous energy recovery and process energy require-
ment is to be determined.
Acknowledgements
The authors gratefully acknowledge Ministry of New and
Renewable Energy (MNRE), Govt. of India, and IIT Kharagpur for
the funding and facilities provided to conduct this work.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.biortech.2015.07.
038.
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