International Journal of Engineering Trends and Technology (IJETT) – Volume3 Issue 6 Number1–Nov 2012
Biological Hydrogen Production by Bacillus sp. Using Hydrolysed
Sugarcane Bagasse
S.SUGANYA
DEPARTMENT OF BIOTECHNOLOGY
BHARATH UINVERSITY, CHENNAI 73
ABSTRACT
In the present investigation, sugarcane bagasse was used to produce Biohydrogen. The
microorganism used was Bacillus sp. Bagasse was first hydrolyzed by using 1 % NaOH and
for 60 min at 121◦C and 1.5 kg/cm2 in an autoclave. The highest hydrogen production rate
(HPR) and hydrogen yield of 1.89 ml/hr and 28.6 ml /g-COD wastewater were obtained
respectively, at an optimal initial pH and temperature of 7 and 37◦C respectively. The Initial
total sugar in sugarcane bagasse hydrolysate was 3.45 g/l. The hydrogen production
performance was comparable with those reported in the literature ensuring that sugarcane
bagasse hydrolysate could be used as a source for hydrogen production by Bacillus sp.
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1. INTRODUCTION
Hydrogen is considered as a one of the optional fuel and “energy carrier” for the coming
days. No emission of CO2 occurs in hydrogen fuel and can easily be used in fuel cells for
electricity production. According to the recent reviews on hydrogen it shows that the demand
of hydrogen is increasing day by day.
o
Some methods are energy intensive processes and it requires high temperatures (> 850 C).
Conventional hydrogen production is one the example. The cleanest technology for hydrogen
gas production of water is Electrolysis. This process should be used in areas where electricity
is less expensive, since electricity costs account for 85% of the operating cost for H2
production. Water is the main product in combustion of hydrogen and thus hydrogen is
considered as a clean non polluting fuel. Fuels like water gas, hydrogen is harmless to
humans and the environment as compared to other gases.
Hydrogen is used as a reactant in hydrogenation process, as a fuel in rocket engines, as a O2
scavenger and also as a coolant in electrical generation. The hydrogen demand is increasing
day by day and because of this increase in demand of hydrogen, development of cost effective
and efficient hydrogen production technologies has earned the notable attention. In accordance with
sustainable development and waste minimization issues, bio-hydrogen gas production from renewable
sources, also known as “green technology” has received considerable attention in recent years.
Production of clean energy source and effective utilization of waste materials make biological
hydrogen production a novel and promising approach to meet the increasing energy needs as a
substitute for fossil fuels. Hydrogen is produced biologically through bio-photolysis of water
using algae and cyanobacteria, photodecomposition of organic compounds by photosynthetic
bacteria, fermentative hydrogen production from organic compounds, and hybrid systems
using photosynthetic and fermentative bacteria.
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2. MATERIALS AND METHODS
2.1 Sugarcane Bagasse
The sugarcane bagasse used in this study was obtained from the local sugar industry. Bagasse
was dried, milled and sieved through a 0.5 mm screen and stored at room temperature. The
composition of the sugarcane bagasse is 35% cellulose, 26% hemicellulose, 3% lignin,
1% ash and remaining 25% other components.
2.2 Isolation and Screening of Hydrogen Producing Microorganism
Hydrogen producing bacterial samples was isolated from soil using serial dilution technique.
The screening for best strains to obtain maximum hydrogen production was conducted by
using Durham tubes filled with the fermentation broth in an inverted position in a test tube.
Ten strains were selected on the basis of gas collected in the Durham tubes; finally three
bacteria were screened for further studies for maximum hydrogen production using a
selective media of (NH4)2SO4 -3g/l, MgSO4-0.5 g/l, K2HPO4-0.5 g/l, KH2PO4-0.5 g/l, yeast
extract-1 g/l, glucose-5g/l, (pH-7) under asceptic conditions. These were biochemically tested
and identified according to Bergeys manual of systematic bacteriology (Garrity et al., 2005)
and was found to be Bacillus sp. as shown in Table 1, bacterial samples and seed sludge were
enriched in a synthetic medium consisting of sucrose-10g/l, MgSO4.7 H2O-0.2 g/l, FeSO4.7
H2O-0.015 g/l, CaSO4-0.2 g/l, Na2MoO4.2H2O-0.005 g/l, casamino acids-0.2 g/l, K2HPO40.2 g/l (pH-7) under anaerobic conditions.
2.3 Alkaline hydrolysis pretreatment
Alkali pre-treatment refers to the use of NaOH to remove lignin and a part of the
hemicellulose, and to efficiently increase the accessibility of enzyme to the cellulose. The
alkali pretreatment can result in a sharp increase in saccharification, with manifold increase in
yield. Pretreatment can be performed at low temperatures but with a relatively long time and
at high concentration of base. Alkali hydrolysis was conducted at 121◦ C for 60 min in an
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autoclave with a mass ratio of solid (g of dry weight) to liquid (ml) 1:20 using 1% NaOH.
After hydrolysis, a solid residue was filtered and pH of the hydrolysate was adjusted to 7.
2.4 Acid Hydrolysis Pretreatment
Treatment of lignocellulosic materials with acid at a high temperature can efficiently improve
the enzymatic hydrolysis. Sulfuric acid is the most applied acid, while other acids such as
HCl and nitric acid were also reported (Taherzadeh et al., 2007). Acid hydrolysis was
conducted at 121◦ C for 60 min in autoclave with a mass ratio of solid (g of dry weight) to
liquid (ml) 1:20 using 1% H2SO4. After hydrolysis, a solid residue was filtered through a
thin cloth. The pH of hydrolysate was adjusted to 7.
2.5 Experimental Setup
The hydrogen production experiments were carried out in a 1500 ml glass bioreactor with a
1000ml working volume and with 10% inoculum. The schematic diagram of the experimental
setup is shown in Fig.1. The glass reactors were purged with N2 gas to maintain anaerobic
conditions. There is an inlet for the medium and outlet for the hydrogen and other gases.
Hydrogen gas was collected in a gas collector by water displacement method using a
graduated cylinder. Biogas samples were collected from the headspace of the reactor
regularly and analyzed for hydrogen and volatile acids (Cruwys et al., 2002). The samples
were drawn at regular time intervals and subjected to Chemical Oxygen Demand (COD),
biomass concentration, pH, volatile suspended solids (VSS) and residual sugar analysis.
2.6 Gas Analysis
The biogas production was measured regularly by gas displacement method. The hydrogen
and other gases were analyzed by a gas chromatography equipped with a thermal
conductivity detector (Chemito GC 2865). The biogas hydrogen content was measured using
2.4mm × 6mm stainless column packed with porapak Q (80/100 mesh). Nitrogen was used as
a carrier gas at a flow rate of 30ml/min. The temperatures for the injection port, the column
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International Journal of Engineering Trends and Technology (IJETT) – Volume3 Issue 6 Number1–Nov 2012
and the detector were set to 50, 50 and 100C respectively. The concentrations of volatile
fatty acids including acetate, propionate, butyrate and ethanol in the solution were determined
with gas chromatography equipped with a flame ionization detector and 30mm×0.25mm
fused silica capillary column (DB-FFAP). The sugar content was determined by using phenol
sulphuric method. COD, volatile fatty acids (VFA), biomass concentration, pH, volatile
suspended solids and residual sugars were measured according to the standard methods. Cell
concentration was determined by centrifuging 1 mL of culture broth at 12,000 rpm for 15
min. Cell pellets were washed twice with distilled water, dried at 105 C and cell weight was
determined.
2.7 Kinetic Modeling
Modified Gompertz Equation was used to determine the hydrogen production potential (P),
hydrogen production rate (R) and lag phase (λ) as given in Eqn. (1).
H(t) = P exp {- exp [ Rm . e ( λ – t ) +1] }
(1)
P
Where H (t) = Cumulative Hydrogen Production (ml) at time (t), λ is time of lag phase (h), PHydrogen Production Potential (ml), R-Hydrogen Production Rate (ml/h), e is the exp (12)
i.e. 2.71828 ( Van Ginkel et al., 2001)
The analysis of variance (ANOVA) was used to test the significance of estimated parameters
at a confidence interval (CI) of 95%. The maximum specific hydrogen production rate
(mlg-1VSS.hr) was calculated by dividing Rm by initial sludge VSS. The hydrogen yield
(mlg-1 COD wastewater) was calculated by dividing P by the g COD wastewater and the
specific hydrogen production potential (ml H2 g-1substrate) was calculated by dividing P by
substrate concentration (Lay et al.,2001)
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3. RESULTS AND DISCUSSION
3.1 Composition of Bagasse
The composition of sugar present in bagasse after hydrolysis using HCl and NaOH were 2.2
g/l and 3.46 g/l respectively. Glucose and xylose are the important components present in
bagasse extract and glucose is the main carbon source. It can be noticed that the maximal
glucose production was obtained with alkali treatment.
3.2 Gas Production
The cumulative gas production with respect to incubation time during the fermentation of
bagasse extract by Bacillus sp. is shown in Fig.2. The Gompertz equation (Eqn.3) was used to
fit the experimental data and the relevant parameters determined were presented in Table 2.
The cumulative hydrogen production was well correlated with the modified Gompertz
equation (R2 > 0.99). The parameters estimated (P, R and λ) for Bacillus sp. for the bagasse
extract was statistically significant at a confidence interval (CI) of 95%.
Hydrogen
production potential (P) for acid hydrolysed bagasse was 48 ml at a lag phase time of 11 hrs.
Whereas with alkali hydrolysis, the H2 production potential was 97 ml for a lag phase time of
10 hrs. Hydrogen production rate (R) for acid and alkali hydrolysis was 1.89 and 3.38 ml/hr
respectively. As shown in Table 2, the maximum hydrogen production yield was observed
with alkali hydrolysed bagasse extract using Bacillus sp. (28.6 ml/g COD wastewater) and
maximum hydrogen production rate was 0.99 ml/g VSS.hr at a substrate (glucose)
concentration of 3.45 g/l.
3.3 Variation of pH
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In the present investigation, experiments were performed at a constant pH of 7. Cell growth
rate and hydrogen yield decreased with decrease in initial pH of the medium. The variation of
pH of the acid and alkali hydrolysed bagasse extract during hydrogen production is shown in
Table 2. The pH was found to decrease with an increase in time and it is mainly due to the
formation of organic acids.
3.4 Effect of Initial Sugar Concentration and Biomass Concentration
The growth of hydrogen producing microorganisms was closely associated with the substrate
consumption. Net cell growth concentration increased to 2.6 g/l with alkali hydrolysed
bagasse extract and the substrate concentration decreased to 0.8 g/l as shown in Fig 3. The
cell concentration was 2 g/l with a substrate consumption of 0.3 g/l in acid hydrolysed
bagasse extract (Fig. 4).
The results indicated that substrate concentration apparently
influenced the hydrogen production and increase in substrate concentration could increase the
hydrogen production up to a certain level. However, an excessive substrate concentration will
cause a build up of VFAs in the system leading to a decline of pH in the reactor and could
inhibit the growth of hydrogen producing organisms (Fan et al., 2006). Moreover, an increase
in substrate concentration could lead to a partial pressure in the fermentation system. When
partial pressure increases in the headspace of reactor to some level, the hydrogen production
will be switched to solvent production, thus inhibiting the hydrogen production
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Fig. 2 Cumulative gas production from bagasse extract using Bacillus sp.
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Fig 3. Effect of initial biomass concentration and substrate degradation on alkali hydrolysed
bagasse extract.
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Fig 4. Effect of initial biomass concentration and substrate degradation on acid hydrolysed
bagasse extract.
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Table 1. Biochemical characteristics of the culture
Biochemical
characteristics
Gram stain
Gram positive
Shape and arrangement
Oxygen requirements
Motility
Rods in Single and in
Chains
Facultative
(+)
Temperature
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Bacillus sp.
37 - 44 C
Oxidase
(+)
Indole production
(+)
Methyl red
(+)
Voges-Proskauer
(+)
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International Journal of Engineering Trends and Technology (IJETT) – Volume3 Issue 6 Number1–Nov 2012
Simmon –Citrate
(+)
Hydrogen-sulphide
production
(+)
Urea hydrolysis
(+)
Gelatin Hydrolysis
(+)
Growth in KCN
(+)
D-Glucose-gas
production
(+)
Acid with Gas
(+) - Positive; (−) - Negative
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Table 2. Modified Gompertz equation parameters for hydrogen production from bagasse
extract using Bacilllus sp.
λ - Time of lag phase (h) P - Hydrogen Production Potential (ml) R - Hydrogen
Sample
Initial
pH
Final
pH
P
(ml)
λ
(h)
R
(ml/h)
SHPP
(ml /g
substrate)
SHPR
(ml /g
VSS.hr
)
Acid
Hydrogen
yield(ml
R2
/g-COD
wastewate
r)
7.0
5.8
49
11
1.89
35
0.55
12.4
0.9998
7.0
5.0
97
10
3.38
78
0.994
28.6
0.9999
Hydrolysed
Alkali
hydrolysed
Production Rate (ml/h) e - Exponential constant i.e. 2.71828, R2- Regression coefficient,
SHPR- Specific hydrogen production rate (mlg-1-VSS.hr) SHPP- Specific hydrogen
production potential (ml H2 g-1substrate)
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CONCLUSIONS
The present investigation demonstrated that acid hydrolysate of sugarcane bagasse was
suitable for hydrogen by Bacillus sp., due to its high sugar concentration (glucose, xylose,
arabinose) and low growth inhibitors concentration.The use of 1% NaOH under 121◦C and
1.5 kg/cm2 in an autoclave for 60 min has been found to be the optimal condition for the
hydrolysis of sugar cane bagasse (SCB). The highest HPR and hydrogen yield were 1.89
ml/hr and 12.86 ml /g-COD wastewater respectively, obtained at an optimal fermentation
condition of initial pH 7 and 37◦C at an initial total sugar of 3.45 g/l. The hydrogen
production performance was compared favourably to those reported in the literature ensuring
that sugarcane bagasse hydrolysate could be used as a fermentation media for the hydrogen
production by Bacillus sp.
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International Journal of Engineering Trends and Technology (IJETT) – Volume3 Issue 6 Number1–Nov 2012
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