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Phytosynthesized iron oxide nanoparticles andferrous iron on fermentative hydrogen productionusing Enterobacter cloacae: Evaluation andcomparison of the effects
Sundaresan Mohanraj a, Shanmugam Kodhaiyolii a,Mookan Rengasamy b, Velan Pugalenthi a,*
a Department of Biotechnology, Bharathidasan Institute of Technology, Anna University, Tiruchirappalli 620 024,
Tamil Nadu, Indiab Department of Petrochemical Technology, Bharathidasan Institute of Technology, Anna University,
Tiruchirappalli 620 024, Tamil Nadu, India
a r t i c l e i n f o
Article history:
Received 4 April 2014
Received in revised form
26 May 2014
Accepted 5 June 2014
Available online xxx
Keywords:
Iron oxide nanoparticles
Fermentative hydrogen
Enterobacter cloacae
Gompertz equation
Principal component analysis
* Corresponding author. Tel.: þ91 431 240799E-mail address: [email protected] (V. Pu
Please cite this article in press as: Mohantative hydrogen production using EnterHydrogen Energy (2014), http://dx.doi.org
http://dx.doi.org/10.1016/j.ijhydene.2014.06.00360-3199/Copyright © 2014, Hydrogen Ener
a b s t r a c t
The effects of FeSO4 and synthesized iron oxide nanoparticles (0e250 mg/L) on fermen-
tative hydrogen production from glucose and sucrose, using Enterobacter cloacae were
investigated, to find out the enhancement of efficiency. The maximum hydrogen yields of
1.7 ± 0.017 mol H2/mol glucose and 5.19 ± 0.12 mol H2/mol sucrose were obtained with
25 mg/L of ferrous iron supplementation. In comparison, the maximum hydrogen yields of
2.07 ± 0.07 mol H2/mol glucose and 5.44 ± 0.27 mol H2/mol sucrose were achieved with
125 mg/L and 200 mg/L of iron oxide nanoparticles, respectively. These results indicate that
the enhancement of hydrogen production on the supplementation of iron oxide nano-
particles was found to be considerably higher than that of ferrous iron supplementation.
The activity of E. cloacae in a glucose and sucrose fed systems was increased by the addition
of iron oxide nanoparticles, but the metabolic pathway was not changed. The results
revealed that the glucose and sucrose fed systems conformed to the acetate/butyrate
fermentation type.
Copyright © 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights
reserved.
Introduction
Hydrogen, a green energy source, is considered as an envi-
ronmentally safe, renewable, and suitable alternative for
fossil fuel energy in the future [1]. Hydrogen does not generate
3; fax: þ91 0431 2407999.galenthi).
raj S, et al., Phytosynthobacter cloacae: Evaluati/10.1016/j.ijhydene.2014
27gy Publications, LLC. Publ
pollutants, because its combustion results only in water
vapour and energy [2]. It has a high energy yield of 122 kJ/g,
which is 2.75 times higher than that of hydrocarbon fuels.
Among the hydrogen production processes, dark fermenta-
tion is a promising route to produce hydrogen from a diverse
range of substrates [3]. In this process, hydrogen is produced
esized iron oxide nanoparticles and ferrous iron on fermen-on and comparison of the effects, International Journal of.06.027
ished by Elsevier Ltd. All rights reserved.
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n en e r g y x x x ( 2 0 1 4 ) 1e1 02
as a co-product during the conversion of carbohydrate into
organic acid using anaerobic bacteria. Among the fermenta-
tive bacteria, Clostridium and Enterobacter have been exten-
sively used as inocula for fermentative hydrogen production
from glucose [4]. However, the yield and rate of hydrogen
production are yet to be improved in the fermentation pro-
cess. In the fermentative hydrogen production process, the
formation of propionate and end products (alcohol and
lactate), has been found to be the main reason for low
hydrogen yields [5]. Moreover, the formation of metabolites
from the fermentation process has beenmainly influenced by
the inocula, pH, temperature and nutritional requirements
[1,6]. Therefore, further efforts and new approaches are
required for regulating the bacterial metabolism towards the
formation of acetate and butyrate to produce high yield of H2
evolution.
Interestingly, iron plays an important role in the electron
transport, which produces more hydrogen by promoting hy-
drogenase activity [7]. The ironesulphur protein in ferredoxin
(Fd) acts as an electron carrier in pyruvate oxidation into
Acetyl CoA and CO2, as well as a proton reducer to molecular
H2 [8]. In addition, the ferredoxin reducing pathway is formed
by NADH ferredoxin oxidoreductase activity, from the acido-
genic metabolism of the fermentative hydrogen production
process [9,10]. However, in iron deficient conditions, fla-
vodoxin could replace ferredoxin as an electron transporter in
many redox reactions, including pyruvate ferredoxin oxido-
reductases, NADH oxidoreductase [11] and hydrogenases [12]
in bacterial metabolism. Therefore, the supplementation of
iron is required to improve the ferredoxin in the fermentative
hydrogen production process. Researchers noted that the lack
of iron lowered the enzyme activity for both FeeS (hydroge-
nase) and non FeeS (malic enzyme) proteins [13]. They also
reported that iron induced the metabolic changes in both the
FeeS and non FeeS proteins. A few studies investigated the
effect of iron supplementation on fermentative hydrogen
production by a mixed culture [14,15]. Recently, researchers
reported that the supplementation of nano-sized metal and
metal oxide particles extensively increased the microbial re-
action rates. Moreover, in microbial application, the inte-
grated nanoparticles withmicroorganisms exhibited a shorter
reaction time, when compared to the microorganism alone in
the reaction [16].
Han et al. [17], found the enhancement of hydrogen pro-
duction by the supplementation of haematite nanoparticles.
Beckers et al. [18], investigated the effects of metal (Pd, Ag and
Cu) nanoparticles and metal oxide (FexOy) nanoparticles on
biohydrogen production, using Clostridium butyricum. Our
previous study explained the green synthesis of iron oxide
nanoparticles by Murraya koenigii leaf extract and the
enhancement effect of synthesized iron oxide nanoparticles
on fermentative hydrogen production, using Clostridium ace-
tobutylicum [19]. However, the study of nano-sized particles
effect on fermentative hydrogen production is very limited,
especially in the pure cultures. To the best of our knowledge,
there is no report available on the comparative study of the
supplementation of nano-sized iron oxide particles (FeNPs)
with iron (Fe2þ) for the enhancement of fermentative
hydrogen production from glucose and sucrose using Enter-
obacter cloacae. Therefore, there is a need to study the
Please cite this article in press as: Mohanraj S, et al., Phytosynthtative hydrogen production using Enterobacter cloacae: EvaluatiHydrogen Energy (2014), http://dx.doi.org/10.1016/j.ijhydene.2014
comparative analysis of the fermentative hydrogen produc-
tion process by the supplementation of iron (Fe2þ) and iron
oxide nanoparticles.
The objectives of this study were to compare the ferrous
sulphate (FeSO4) and synthesized iron oxide nanoparticles
effects on fermentative hydrogen production from glucose
and sucrose using E. cloacae, in order to find out the enhanced
efficiency. In addition, the kinetic parameters and principal
components analysis (PCA) were studied for the fermentative
hydrogen production process with the supplementation of
ferrous sulphate and iron oxide nanoparticles. Based on the
results, the metabolic pathway was proposed for the FeNPs
supplemented experiments.
Materials and methods
Microorganism and iron oxide nanoparticles
E. cloacae 811101 was obtained from the National Institute of
Agrobiological Science, Japan. The strain was grown in
nutrient broth (Himedia laboratories, India) under anaerobic
conditions for 24 h at 37 �C. The green synthesis of iron oxide
nanoparticles using Murraya koenigii leaf extract has been
recently reported [19], and green synthesized iron oxide
nanoparticles were used in this study.
Effect of iron oxide nanoparticles on fermentative hydrogenproduction
The batch experiment was conducted in a 250mL bottle with a
working volume of 200 mL. The fermentative hydrogen pro-
ducing medium contained the following compositions (g/L):
1.5 KH2PO4; 3.2 Na2HPO4; 0.5 NH4Cl; 0.8 MgCl2; 1.0 yeast
extract; 0.5 meat extract and 0.5 peptone. The concentrations
of glucose and sucrose were varied from 2.5 to 12.5 g/L. pHwas
varied from 4.0 to 10.0 in increments of 1.0, to determine the
optimumpH for the fermentation process, and the duration of
fermentationwas about 24 h. The concentrations of FeSO4 and
iron oxide nanoparticles were taken in the range of 0e250mg/
L to compare the enhancement effect of the fermentative
hydrogen production. The headspace air was displaced by
nitrogen gas to generate an anaerobic condition in the reactor.
The fermentative hydrogen was collected in a gas collector by
the water displacement method. The volume of the fermen-
tative hydrogen and glucose utilization was measured at
different time intervals.
Analytical methods
The head phase gas composition in each batch experiment
was analysed by the Shimadzu gas chromatograph (GC-2014)
(Shimadzu Co. Singapore), equipped with a thermal conduc-
tivity detector and a stainless steel column packed with Por-
apak Q (80/100 mesh) as described in our previous study [19].
Volatile fatty acids (VFAs) in the liquid phase were measured
by the Shimadzu gas chromatograph (GC-2014) (Shimadzu Co.
Singapore), equipped with a flame ionization detector (FID)
and stabilwax-DA capillary column [19]. The reducing sugars
were analysed, using the phenol-sulphuric acid method [20].
esized iron oxide nanoparticles and ferrous iron on fermen-on and comparison of the effects, International Journal of.06.027
0 5 10 15 20 250
100
200
300
400
Hyd
roge
n pr
oduc
tion
(mL
)
Time (h)
4 5 6 7 8 9 10
a
4 5 6 7 8 9 100.0
0.4
0.8
1.2
1.6
2.0
2.4 Hydrogen yield Final pH
Hyd
roge
n yi
eld
(mol
H2/m
ol g
luco
se)
b
0
1
2
3
4
5
6
7
8
9
10
Fina
l pH
i n t e r n a t i o n a l j o u r n a l o f h yd r o g e n e n e r g y x x x ( 2 0 1 4 ) 1e1 0 3
Kinetic and statistical analysis
The kinetic parameters of hydrogen production in the batch
experiments were carried out based on the followingmodified
Gompertz equation:
H ¼ P* exp
�� exp
�RmeP
ðl� tÞ þ 1
��
where H is the cumulative hydrogen production (mL), P is the
hydrogen production potential (mL), Rm is the maximum
hydrogen production rate (mL/h), e is 2.71828, l is the lag
phase time (h), and t is the incubation time (h). The kinetic
parameters (P, Rm and l) for fermentative hydrogen produc-
tion were estimated via Origin 7.5 [21].
The analysis of variance (ANOVA) followed by Dunnett'smultiple comparison and Tukey HSD was applied to test the
effects of FeSO4 and FeNPs on fermentative hydrogen pro-
duction using E. cloacae. The experimental data were sepa-
rately analysed for each supplementation. ANOVA analysis
was done with the XLSTAT program version 2014.1.04.
Principal component analysis
The principal component analysis (PCA) was carried out by
using PAST (Paleontological Statistics software) version 2.13
[22], to detect similarities and variances in the glucose and
sucrose fed experiment. PCA was used to identify the varia-
tion of metabolites including hydrogen, acetate, butyrate,
ethanol, and propionate in control, ferrous iron and iron oxide
nanoparticles supplemented experiments. The results of the
principal components (PC1 and PC2) are shown in the bi-plot.
pHFig. 1 e (a) Hydrogen production versus fermentation time
in glucose fed system at different initial pHs; (b) hydrogen
yield and final pH in glucose fed system at different initial
pHs.
Results and discussion
Effect of initial pH on fermentative hydrogen production
Fig. 1(a) depicts the variation of hydrogen production from
glucose at different initial pHs (4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and
10.0). As seen in Fig. 1(a), the hydrogen production was
significantly increased from 128 ± 9 to 357 ± 12 mL, on varying
the initial pH from 5.0 to 7.0. Furthermore, the hydrogen
production was drastically decreased at the initial pH above
7.0. In Fig. 1(a), the lowest hydrogen production of 43 ± 8 and
28 ± 4 mL was found at the initial pH of 4.0 and 10.0, respec-
tively. Previously, Zhang et al. [23] reported that a high pH
inhibited the growth of microbes, and their abilities to pro-
duce hydrogen. According to our results, it was confirmed that
the E. cloacae activity was inhibited at both low and high pH.
The maximum hydrogen production (357 ± 12 mL) was ob-
tained at the initial pH of 7.0. Similarly, the hydrogen yieldwas
substantially increased from 0.2 ± 0.04 to 1.44 ± 0.05 mol H2/
mol glucose when the initial pH was varied from 4.0 to 7.0.
Also, the hydrogen yield was decreased from 1.3 ± 0.05 to
0.16 ± 0.02 mol H2/mol glucose on varying the initial pH from
8.0 to 10.0 (Fig. 1(b)). The hydrogen content in biogas was
significantly affected at the initial pH, and it was increased
from 8 to 41%, when the initial pH was varied from 4.0 to 7.0.
Further, the hydrogen content was decreased with the in-
crease of pH above 7, as presented in Table 1. The results of the
Please cite this article in press as: Mohanraj S, et al., Phytosynthtative hydrogen production using Enterobacter cloacae: EvaluatiHydrogen Energy (2014), http://dx.doi.org/10.1016/j.ijhydene.2014
study indicate that the optimal initial pH for efficient
hydrogen production using E. cloacae was observed to be 7.0.
The extent of hydrogen production using E. cloacae at
different time intervals for all the initial pHs is depicted in
Fig. 1(a). These experimental data were used to determine the
kinetic parameters (Table 1). The lag phase (l) time was
remarkably decreased from 10.1 to 7.8 h on varying the pH
from 4.0 to 7.0, and then continually increased to 10.3 h at pH
10.0. On the contrary, the hydrogen production potential (P)
and hydrogen production rate (Rm) were increased by varying
the initial pH up to 7.0. The hydrogen production potential and
hydrogen production rate were drastically decreased, on
varying the initial pH above 7.0. It is evident from the results,
that the lag phase time, hydrogen production potential and
hydrogen production rate were notably affected at both low
and high initial pH, thus being unfavourable for fermentative
hydrogen production using E. cloacae.
Table 1 shows the volatile fatty acids (VFA) and ethanol
concentration at various initial pH levels. The lowest con-
centration of soluble metabolites was at pH 4.0 and composed
of mainly acetate, butyrate and ethanol. The maximum
esized iron oxide nanoparticles and ferrous iron on fermen-on and comparison of the effects, International Journal of.06.027
Table 1 e Effect of pH on hydrogen content, kinetic parameters, glucose conversion efficiency and soluble metabolites.
pH H2a (%) Pb (mL) Rm
c (mL/h) ld (h) R2 Glucose conversionefficiency (%)
Soluble metabolites (mg/L)
EtOHe HAcf HBug HPrh
4 8 43 5.66 10.1 0.9967 87 123 ± 3 1123 ± 18 325 ± 12 e
5 33 128 12.33 8.4 0.9903 89 287 ± 12 983 ± 18 568 ± 15 e
6 37 262.6 24.66 7.9 0.9897 93 453 ± 16 872 ± 13 468 ± 14 254 ± 11
7 41 357 29.16 7.8 0.9932 99 468 ± 12 733 ± 20 657 ± 19 432 ± 13
8 34 282.7 27.16 8.4 0.9921 87 387 ± 14 783 ± 12 453 ± 11 387 ± 12
9 26 116.4 12.33 8.9 0.9953 74 290 ± 15 873 ± 14 388 ± 12 213 ± 14
10 6 28 3 10.3 0.9982 68 135 ± 17 982 ± 15 232 ± 18 123 ± 16
a Hydrogen content.b Hydrogen production potential.c Hydrogen production rate.d Lag phase.e Ethanol.f Acetate.g Butyrate.h Propionate.
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n en e r g y x x x ( 2 0 1 4 ) 1e1 04
concentrations of acetate (1123 ± 18 mg/L) and butyrate
(657 ± 19 mg/L) were obtained at pH 4.0 and 7.0, respectively.
Propionate concentration was not found at pH 4.0 and 5.0, and
it was noticeably produced (254 ± 11 mg/L) at pH 6.0. Further,
this concentration was increased (432 ± 13 mg/L) at pH 7.0.
Ethanol concentration was steadily increased from 123 ± 3 to
468 ± 12 mg/L as the initial pH varied from 4.0 to 7.0. The
amount of ethanol and propionate was evidently decreased at
the initial pH above 7.0 (Table 1). Also, it was clearly indicated
that the concentrations of major metabolites from fermenta-
tive hydrogen production process were in the order of
acetate > butyrate > ethanol > propionate at pH 7.0. The
maximum hydrogen was produced together with acetate and
butyrate at the initial pH of 7.0. Similar findings have been
reported by Khanna et al. [24], who found that the maximum
hydrogen production using E. cloacae was associated with ac-
etate and butyrate at the initial pH of 6.5, when comparedwith
the other pH values. However, the hydrogen production was
mainly affected, due to the formation of ethanol and propio-
nate. Likewise, it was observed that the final pH (3.8e9.1) at
the end of fermentation was significantly influenced by VFA
and ethanol accumulation, as illustrated in Fig. 1(b).
Fermentative hydrogen production from glucose and sucroseusing E. cloacae
The fermentative hydrogen production from glucose and su-
crose was investigated at different initial concentrations
(2.5e12.5 g/L). As shown in Fig. 2(a and b), the maximum
hydrogen production of 357 ± 12 mL and 417 ± 8 mL was ob-
tained from 10 g/L of glucose and 7.5 g/L of sucrose, respec-
tively. Further, the hydrogen production from glucose and
sucrose was found to be decreased on increasing the con-
centration above 10 g/L and 7.5 g/L, respectively. From Fig. 2(c),
the glucose conversion efficiency was found to be above 98%,
when the glucose concentration was changed from 2.5 to 10 g/
L. The glucose conversion efficiencywas observed to be 94% at
12.5 g/L. This result indicates that the conversion efficiency of
glucose during fermentative hydrogen productionwas slightly
decreased at 12.5 g/L of glucose. In contrast, the sucrose
Please cite this article in press as: Mohanraj S, et al., Phytosynthtative hydrogen production using Enterobacter cloacae: EvaluatiHydrogen Energy (2014), http://dx.doi.org/10.1016/j.ijhydene.2014
conversion efficiency was observed to be lower than that of
glucose conversion efficiency, as illustrated in Fig. 2(c). It was
noted that the sucrose conversion efficiency of 88, 87, 89, 75
and 71%was obtained for the experiment fed with 2.5, 5.0, 7.5,
10.0 and 12.5 g/L of sucrose, respectively. These results show
that the conversion efficiency was slightly increased up to
7.5 g/L of sucrose, and drastically decreased with the increase
of sucrose concentration above 7.5 g/L. The reason for the
decrease in conversion efficiency and hydrogen production
may be due to the substrate inhibition at high concentration
and accumulation of soluble metabolites at high levels. The
maximum hydrogen yields of 1.44 ± 0.05 mol H2/mol glucose
and 4.77 ± 0.09 mol H2/mol sucrose were obtained from 10 g/L
of glucose and 7.5 g/L of sucrose, respectively.
Tables 2 and 3 show the data for hydrogen content, kinetic
parameters and soluble metabolites from different glucose
and sucrose concentrations. From Table 2, it can be seen that
the lag phase time was increased from 5.85 to 8.55 h as the
glucose concentrations varied from 2.5 to 12.5 g/L. In addition,
the lag phase time was increased from 9.6 to 10.9 h as the
sucrose concentrations varied from 2.5 to 12.5 g/L (Table 3).
These findings demonstrate that the lag phase of the glucose
fed system was much shorter than that of the sucrose fed
system. As the glucose concentration was varied from 2.5 to
10.0 g/L, the maximum hydrogen production potential was
increased from 89 to 357 mL. Similarly, the maximum
hydrogen production potential was increased from 98 to
417 mL, when the initial sucrose concentration was varied
from 2.5 to 7.5 g/L. Further, the hydrogen production was
adversely affected with increasing sugar concentration above
10.0 g/L of glucose and 7.5 g/L of sucrose, due to the formation
of high levels of soluble metabolites. Themaximum hydrogen
content, hydrogen production potential and hydrogen pro-
duction rate were 41%, 357 mL and 29.16 mL/h for 10 g/L of
glucose, respectively (Table 2). In comparison, the maximum
hydrogen content, hydrogen production potential and
hydrogen production rate were 48%, 417 mL and 41.5 mL/h for
7.5 g/L of sucrose, respectively (Table 3). These results indicate
that the hydrogen production from the sucrose fed system
was significantly higher than that of the glucose fed system.
esized iron oxide nanoparticles and ferrous iron on fermen-on and comparison of the effects, International Journal of.06.027
0 5 10 15 20 250
100
200
300
400
Hyd
roge
n pr
oduc
tion
(mL
)
Time (h)
2.5 g/L 5.0 g/L 7.5 g/L 10.0 g/L 12.5 g/L
a
0 5 10 15 20 250
100
200
300
400
500
Hyd
roge
n pr
oduc
tion
(mL
)
Time (h)
2.5 g/L 5.0 g/L 7.5 g/L 10 g/L 12.5 g/L
b
0.0 2.5 5.0 7.5 10.0 12.5 15.00
2
4
6
8 Glucose Sucrose
Concentration (g/L)
Hyd
roge
n yi
eld
( mol
H2/
mol
glu
cose
/suc
rose
) c
0
25
50
75
100
125
Subs
trat
e co
nver
sion
eff
icie
ncy
(%)
Fig. 2 e Fermentative hydrogen production by E. cloacae: (a)
effect of glucose concentration; (b) effect of sucrose
concentration; (c) hydrogen yield and substrate conversion
efficiency at different initial concentrations of glucose and
sucrose.
i n t e r n a t i o n a l j o u r n a l o f h yd r o g e n e n e r g y x x x ( 2 0 1 4 ) 1e1 0 5
Please cite this article in press as: Mohanraj S, et al., Phytosynthtative hydrogen production using Enterobacter cloacae: EvaluatiHydrogen Energy (2014), http://dx.doi.org/10.1016/j.ijhydene.2014
As seen in Table 2, the concentrations of acetate from
453 ± 19 to 983 ± 23 mg/L and butyrate from 190 ± 19 to
765 ± 18 mg/L were steadily increased when the glucose
concentration was varied from 2.5 to 12.5 g/L. A similar trend
was observed in other metabolites, including ethanol
(128 ± 11 mg/L to 683 ± 23 mg/L) and propionate (162 ± 15 to
653 ± 20 mg/L), but they were considerably low concentra-
tions, at all the glucose concentration levels (Table 2). On the
contrary, the concentrations of acetate and butyrate were
increased as the sucrose concentration was changed from 2.5
to 10 g/L. The acetate and butyrate concentrations were
decreased above 10.0 g/L of sucrose. Propionate was not found
at sucrose concentration up to 5.0 g/L, but it was noticeably
formed at 7.5 g/L of sucrose. Further, this concentration was
increased with increasing sucrose concentration up to 12.5 g/
L. Ethanol concentration was steadily increased when the
sucrose concentration differed from 2.5 to 12.5 g/L. In the
present investigation, it was clearly demonstrated that the
concentrations of major metabolites from fermentative
hydrogen production process were in the order of
acetate > butyrate > ethanol > propionate. Therefore, the
fermentation process for hydrogen production from glucose
and sucrose using E. cloacae, was considered as the acetate/
butyrate fermentation type. Similar observations have been
reported by Khanna et al. [24], who investigated the hydrogen
production from glucose using E. cloacae IIT-BT 08.
Effect of iron oxide nanoparticles on fermentative hydrogenproduction using E. cloacae
The effects of the synthesized iron oxide nanoparticles on
fermentative hydrogen production from optimum glucose
and sucrose were investigated using E. cloacae, to find out the
enhancement of efficiency. In Fig. 3(a and b), the maximum
hydrogen production of 423 ± 16 and 497 ± 25mL from glucose
and sucrose was noticeably observed at 125 and 200 mg/L of
iron oxide nanoparticles, respectively. It was evident that the
hydrogen production from glucosewas significantly increased
with increasing the iron oxide nanoparticles concentration
from 25 to 125 mg/L. The hydrogen production from sucrose
was gradually increased on increasing the iron oxide nano-
particles concentration from 25 to 200mg/L. The results of the
present study demonstrate that the hydrogen production
from glucose and sucrose was found to be decreased above
125 and 200 mg/L of iron oxide nanoparticles concentration,
respectively. This is in good agreement with the report of Han
et al. [17], who found that the hydrogen production from
glucose increased on increasing the haematite nanoparticles
concentration from 0 to 200 mg/L, and decreased on
increasing the haematite nanoparticles concentration from
400 to 1600 mg/L.
The parameters including hydrogen production potential,
hydrogen production rate and lag phase time, were deter-
mined for glucose and sucrose fed experiments, at different
concentrations of iron oxide nanoparticles (0e250 mg/L), by
using themodifiedGompertz equation (Table 4). In the glucose
fed system, the lag phase time was decreased from 7.65 to
7.2 h, when the addition of iron oxide nanoparticles was var-
ied from 25 to 125 mg/L. This finding indicates that the lag
phase time of iron oxide nanoparticles supplemented
esized iron oxide nanoparticles and ferrous iron on fermen-on and comparison of the effects, International Journal of.06.027
Table 2 e Effect of glucose concentration on hydrogen content, kinetic parameters, and soluble metabolites.
Glucose concentration (g/L) H2a (%) Pb (mL) Rm
c (mL/h) ld (h) R2 Soluble metabolites (mg/L)
EtOHe HAcf HBug HPrh
2.5 33 89 11 5.85 0.9928 128 ± 11 453 ± 19 190 ± 19 162 ± 15
5 36 165 15.66 6.45 0.9965 187 ± 14 562 ± 20 268 ± 22 268 ± 14
7.5 37 258 21.22 6.85 0.9919 299 ± 13 678 ± 27 439 ± 20 324 ± 16
10 41 357 29.16 7.8 0.9932 468 ± 12 733 ± 20 657 ± 19 432 ± 13
12.5 38 343 28.83 8.55 0.993 683 ± 23 983 ± 23 765 ± 18 653 ± 20
a Hydrogen content.b Hydrogen production potential.c Hydrogen production rate.d Lag phase.e Ethanol.f Acetate.g Butyrate.h Propionate.
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n en e r g y x x x ( 2 0 1 4 ) 1e1 06
experiments (25e125mg/L) wasmuch shorter than that of the
control experiment (7.8 h). It was also noted that the lag phase
time was gradually increased from 8.1 to 11.6 h as the iron
oxide nanoparticles concentration was altered from 150 to
250mg/L. On the other hand, the lag phase time of the sucrose
fed system was decreased from 10.0 to 9.26 h, when the iron
oxide nanoparticles concentration was increased from 25 to
200 mg/L. Moreover, the lag phase time was increased with
increasing the iron oxide nanoparticles above 200 mg/L, and
further reached 10.8 h at 250 mg/L. The results showed that
the hydrogen production using E. cloacae was inhibited by the
addition of excess iron oxide nanoparticles. A similar trend
was observed byHan et al. [17] who reported that the lag phase
time significantly decreased from 35.4 to 24.4 h on increasing
the haematite nanoparticles concentration from 25 to
1600 mg/L. They also found that the high concentration of
haematite nanoparticles (200e1600mg/L) supported the start-
up of the hydrogen production rate, but inhibited the growth
of mixed microorganisms, and hence, its effect led to a
decrease in the hydrogen production. Similarly, the present
study shows that the lowest hydrogen production rate for
glucose and sucrose was 23.5 and 36.66mL/h respectively, at a
high concentration of iron oxide nanoparticles (250 mg/L).
Table 3 e Effect of sucrose concentration on hydrogen content
Sucrose concentration (g/L) H2a (%) Pb (mL) Rm
c (mL/h)
2.5 37 98 5.66
5 39 212 28
7.5 48 417 41.5
10 43 409 42.83
12.5 38 387 41.3
a Hydrogen content.b Hydrogen production potential.c Hydrogen production rate.d Lag phase.e Ethanol.f Acetate.g Butyrate.h Propionate.
Please cite this article in press as: Mohanraj S, et al., Phytosynthtative hydrogen production using Enterobacter cloacae: EvaluatiHydrogen Energy (2014), http://dx.doi.org/10.1016/j.ijhydene.2014
However, the maximum hydrogen production rates of 37.33
and 45.66 mL/h for glucose and sucrose fed systems were
obtained at 50 and 100 mg/L of iron oxide nanoparticles,
respectively (Table 4). It was clearly indicated that the
hydrogen production potential and hydrogen production rate
for glucose and sucrose fed experiments were remarkably
improved by the supplementation of iron oxide nanoparticles.
These results demonstrate that the supplementation of iron
oxide nanoparticles in glucose and sucrose fed systems had a
higher influence on the kinetic parameters of H2 production,
than on the total volumetric production.
The effect of iron oxide nanoparticles on the VFA and
ethanol concentrations was investigated during fermentative
hydrogen production from glucose and sucrose (Table 5). As
shown in Table 5, the maximum concentrations of acetate
(980 ± 11 mg/L) and butyrate (687 ± 14 mg/L) were obtained in
the glucose fed experiment at 125 mg/L of iron oxide nano-
particles. The acetate concentration was increased in the
glucose fed experiment, with the addition of iron oxide
nanoparticles up to 125 mg/L, when compared with the con-
trol experiment. The ethanol and propionate concentrations
were found to be low with the supplementation of iron oxide
nanoparticles. On the other hand, in the sucrose fed
, kinetic parameters, and soluble metabolites.
ld (h) R2 Soluble metabolites (mg/L)
EtOHe HAcf HBug HPrh
9.6 0.9415 69 ± 10 982 ± 22 783 ± 8 0
11.5 0.989 124 ± 13 1242 ± 12 948 ± 12 0
10.2 0.9955 199 ± 16 1257 ± 12 987 ± 12 124 ± 10
10.54 0.995 243 ± 14 1349 ± 12 1119 ± 8 156 ± 21
10.9 0.996 267 ± 15 1124 ± 16 1008 ± 10 167 ± 15
esized iron oxide nanoparticles and ferrous iron on fermen-on and comparison of the effects, International Journal of.06.027
0
100
200
300
400
Hyd
roge
n pr
oduc
tion
(mL
)
Time (h)
Control 25 mg/L 50 mg/L 75 mg/L 100 mg/L 125 mg/L 150 mg/L 175 mg/L 200 mg/L 225 mg/L 250 mg/L
a
0
100
200
300
400
500
600
Hyd
roge
n pr
oduc
tion
(mL
)
Time (h)
0 mg/L 25 mg/L 50 mg/L 75 mg/L 100 mg/L 125 mg/L 150 mg/L 175 mg/L 200 mg/L 225 mg/L 250 mg/L
b
FeSO4; FeNPs; FeSO4; FeNPs
0 5 10 15 20 25
0 5 10 15 20 25
0 50 100 150 200 2501
2
3
4
5
6
**
**
****
**
**************
******
**
******
**
****
****
**
****
**
Iron concentration (mg/L)
Hyd
roge
n yi
eld
( mol
H2/
mol
sucr
ose)
**
c
0
1
2
3
4
Hyd
roge
n yi
eld
(mol
H2/
mol
glu
cose
)
Fig. 3 e Hydrogen production versus fermentation time at
different concentrations of iron oxide nanoparticles
(FeNPs): (a) 10 g/L of glucose; (b) 7.5 g/L of sucrose; (c)
comparison of hydrogen yield at different concentrations
of iron oxide nanoparticles (FeNPs) and FeSO4. ** Highly
significant to control at P < 0.01 level.
i n t e r n a t i o n a l j o u r n a l o f h yd r o g e n e n e r g y x x x ( 2 0 1 4 ) 1e1 0 7
Please cite this article in press as: Mohanraj S, et al., Phytosynthtative hydrogen production using Enterobacter cloacae: EvaluatiHydrogen Energy (2014), http://dx.doi.org/10.1016/j.ijhydene.2014
experiment, the maximum concentrations of acetate
(1513 ± 14 mg/L) and butyrate (1199 ± 11 mg/L) were obtained
with 200 mg/L of iron oxide nanoparticles. The highest con-
centrations of ethanol (287 ± 14 mg/L) and propionate
(155 ± 13 mg/L) were obtained with 225 and 50 mg/L of iron
oxide nanoparticles, respectively. The acetate and butyrate
concentrations were increased, when the addition of iron
oxide nanoparticles was varied from 25 to 200 mg/L (Table 5).
These findings reveal that the supplementation of iron oxide
nanoparticles increased the activity of E. cloacae, and
improved the soluble metabolites concentration in the
glucose and sucrose fed experiments, when compared with
the control experiment. But, the supplementation of FeNPs
did not change the metabolic pathway. Further, the soluble
metabolites from the glucose and sucrose fed systems were
decreased on increasing the iron oxide nanoparticles above
125 and 200 mg/L, respectively.
Comparative analysis of ferrous iron and iron oxidenanoparticles effects on fermentative hydrogen production
The enhancement effect of the iron oxide nanoparticles was
compared with different concentrations of FeSO4 on
fermentative hydrogen production from glucose and sucrose
using E. cloacae, as depicted in Fig. 3(c). In the glucose fed
system, the maximum hydrogen yield of 1.44 ± 0.05 mol H2/
mol glucose was obtained in the control experiment, whereas
the maximum hydrogen yield of 1.7 ± 0.017 mol H2/mol
glucose was achieved at 25 mg/L of FeSO4 supplementation.
Previously, Karadag and Puhakka [15] reported that the
maximum hydrogen yield of 1.13 mol H2/mol glucose was
achieved with optimum concentration of Fe2þ at 50 mg/L
from glucose using a mixed culture. According to our results,
the low concentration of FeSO4 (25 mg/L) supplementation
improved the hydrogen yield in the glucose fed system. In
FeNPs supplemented experiments, the maximum hydrogen
yield of 2.07 ± 0.07 mol H2/mol glucose was achieved with
125 mg/L of iron oxide nanoparticles. The obtained
maximum hydrogen yields from iron (FeSO4 and FeNPs)
supplemented experiments was significantly (P < 0.01) higher
than that of the control experiment. Moreover, the hydrogen
yield from the iron oxide nanoparticles supplemented
fermentation was significantly higher than that of the FeSO4
(P < 0.01). In the sucrose fed experiments, the hydrogen
yields of 4.77 ± 0.09 mol H2/mol sucrose and 5.19 ± 0.12 mol
H2/mol sucrose (P < 0.01) were obtained from the control and
FeSO4 supplemented experiments (25 mg/L of FeSO4),
respectively. The observed result was noticed to be higher
than that of the reported values, by Lee et al. [14]. They re-
ported that the maximum hydrogen yield was 131.9 mL/g of
sucrose with the iron concentration of FeCl3 at 800 mg/L. In
the present study, the maximum hydrogen yield of
5.44 ± 0.27 mol H2/mol sucrose was achieved with 200 mg/L
of iron oxide nanoparticles supplementation. These results
indicate that the hydrogen yield in the iron oxide nano-
particles supplemented experiment was significantly higher
than that of the control experiment (P < 0.01). On supple-
mentation of the iron oxide nanoparticles, the hydrogen
yields from glucose and sucrose were found to be relatively
higher than the reported values [25].
esized iron oxide nanoparticles and ferrous iron on fermen-on and comparison of the effects, International Journal of.06.027
Table 4 e Kinetic parameters for fermentative hydrogen production at various concentrations of iron oxide nanoparticles.
FeNP's concentration (mg/L) Glucose fed system Sucrose fed system
Pa (mL) Rmb (mL/h) lc (h) R2 Pa (mL) Rm
b (mL/h) lc (h) R2
0 357 29.16 7.8 0.9932 417 41.5 10.2 0.9955
25 342.4 30.66 7.65 0.997 424 43.83 10 0.997
50 347.6 37.33 7.54 0.996 429 44.5 9.93 0.998
75 361.5 27.11 7.5 0.987 421 45.16 9.92 0.993
100 382.4 30 7.35 0.995 431 45.66 9.9 0.995
125 423 32.66 7.2 0.992 436 43.83 9.87 0.998
150 411.2 32.66 8.1 0.984 426 43.5 9.55 0.995
175 314 28.33 9.45 0.989 453 41.83 9.47 0.997
200 286 31.5 9.6 0.985 497 45 9.26 0.998
225 269 26.16 10.2 0.983 412 40.83 10.4 0.996
250 215 23.5 11.6 0.983 329 36.66 10.8 0.994
a Hydrogen production potential.b Hydrogen production rate.c Lag phase.
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n en e r g y x x x ( 2 0 1 4 ) 1e1 08
A low concentration (25 mg/L) of FeSO4 addition was
favourable for hydrogen production, and further increasing
the concentration above 25 mg/L drastically affected the
hydrogen production. On the contrary, the hydrogen produc-
tion from glucose was gradually increased with an increase in
the supplementation of iron oxide nanoparticles from 25 to
125 mg/L. A similar trend was observed for the sucrose fed
system, with an increase in the supplementation of iron oxide
nanoparticles from 25 to 200 mg/L. Further, the hydrogen
production was slightly affected with increasing the concen-
tration above 125 mg/L of iron oxide nanoparticles for the
glucose fed system, and 200 mg/L of iron oxide nanoparticles
for the sucrose fed system, due to the excess soluble iron
oxide nanoparticles concentration. The synthesized iron
oxide nanoparticles used in the current study without any
encapsulation method produced the maximum hydrogen
yield of 2.07 ± 0.07 mol H2/mol glucose and the obtained value
was relatively higher than that of the literature value as re-
ported, by Beckers et al. [18]. They reported the maximum
hydrogen yield of 1.08 ± 0.06 mol H2/mol glucose with the
Table 5 e Effect of iron oxide nanoparticles on soluble metabo
FeNP's concentration (mg/L) Soluble metabolites in glucose(mg/L)
EtOHa HAcb HBuc
0 468 ± 12 733 ± 20 657 ± 19
25 421 ± 4 943 ± 10 546 ± 7
50 369 ± 12 880 ± 4 621 ± 12
75 342 ± 12 816 ± 9 519 ± 11
100 429 ± 10 932 ± 13 567 ± 13
125 347 ± 13 980 ± 11 687 ± 14
150 328 ± 15 878 ± 19 348 ± 16
175 287 ± 14 782 ± 12 479 ± 14
200 194 ± 17 918 ± 8 412 ± 18
225 128 ± 11 892 ± 14 487 ± 10
250 124 ± 14 883 ± 14 458 ± 11
a Ethanol.b Acetate.c Butyrate.d Propionate.
Please cite this article in press as: Mohanraj S, et al., Phytosynthtative hydrogen production using Enterobacter cloacae: EvaluatiHydrogen Energy (2014), http://dx.doi.org/10.1016/j.ijhydene.2014
addition of iron oxide nanoparticles encapsulatedwith porous
silica catalysts (10�6 mol L�1). In comparison with the control
experiment, the enhancement of fermentative hydrogen
production from glucose and sucrose, by the supplementation
of iron oxide nanoparticles was found to be 18% and 19%,
respectively. The improvement in hydrogen production from
glucose and sucrose was observed to be 11% and 12% with the
addition of ferrous iron, respectively. These results prove that
the nano-sized iron oxide particles were considerably
favourable to produce high hydrogen production from the
glucose and sucrose fed systems,when compared to the FeSO4
supplementation. It was indicated that both forms of iron
(FeSO4 and iron oxide nanoparticles) were considered to in-
crease the activity of ferredoxin in hydrogenase, which led to
enhance the fermentative hydrogen production. Previously,
Gorrell [26] reported that a high concentration of iron was
required in the medium, to keep up the maximum level of
pyruvate ferredoxin oxidoreductase and ferredoxin activity.
The author explained that the hydrogen production with ac-
etate was dependent on the concentration of iron in the
lites in glucose and sucrose fed systems.
fed system Soluble metabolites in sucrose fed system(mg/L)
HPrd EtOHa HAcb HBuc HPrd
432 ± 13 199 ± 16 1257 ± 12 987 ± 12 124 ± 10
345 ± 15 213 ± 9 1126 ± 12 979 ± 13 132 ± 13
467 ± 12 189 ± 11 1290 ± 14 1024 ± 15 155 ± 13
432 ± 14 198 ± 14 1297 ± 16 1043 ± 14 123 ± 14
454 ± 11 213 ± 16 1378 ± 11 1110 ± 11 112 ± 13
335 ± 13 234 ± 12 1398 ± 10 1114 ± 14 110 ± 12
311 ± 13 245 ± 16 1412 ± 14 1135 ± 18 109 ± 11
274 ± 10 265 ± 14 1432 ± 14 1166 ± 14 113 ± 19
189 ± 13 278 ± 14 1513 ± 14 1199 ± 11 104 ± 11
198 ± 11 287 ± 14 1329 ± 9 1093 ± 10 124 ± 14
127 ± 14 213 ± 11 1239 ± 7 967 ± 14 122 ± 14
esized iron oxide nanoparticles and ferrous iron on fermen-on and comparison of the effects, International Journal of.06.027
H2
EtOH
HAc
HBu
HPr
Control
FeSO4
FeNP's
-2.4 -1.6 -0.8 0.0 0.8 1.6 2.4PC 1 (97 %)
-0.3
-0.2
-0.1
0.0
0.1
0.2
0.3
0.4
PC 2
(2 %
)
a
H2
EtOH
HAcHBu
HPr
Control
FeSO4
FeNP's
-1.8 -1.2 -0.6 0.0 0.6 1.2 1.8 2.4PC 1 (96 %)
-0.36
-0.24
-0.12
0.00
0.12
0.24
0.36
PC 2
(3 %
)
b
Fig. 5 e Principal component analysis: (a) glucose fed
system; (b) sucrose fed system (note: control- without
addition of iron oxide nanoparticles and ferrous iron;
FeSO4- ferrous iron; FeNPs- iron oxide nanoparticles; HAc-
acetate; Hbr- butyrate; HPr- propionate, EtOH- ethanol).
i n t e r n a t i o n a l j o u r n a l o f h yd r o g e n e n e r g y x x x ( 2 0 1 4 ) 1e1 0 9
fermentationmedium. A similar result was observed with our
present investigation. The results showed that the high
hydrogenwith acetate and butyrate wasmainly dependent on
the optimal concentration of iron oxide nanoparticles sup-
plementation. Also, it was noted that the ethanol and propi-
onate concentrations were decreased on the supplementation
of the iron oxide nanoparticles. Based on the obtained results,
the possible pathway of fermentative hydrogen production
from glucose by the supplementation of iron oxide nano-
particles, is given in Fig. 4. The proposed possible metabolic
route suggested that the optimum level of iron oxide nano-
particles supplementation may increase the ferredoxin
oxidoreductase and ferredoxin activity, which led to high
hydrogen production.
Principal component analysis (PCA)
The hydrogen yield and soluble metabolites data were used
for the analysis of the principal components. The PCA exhibits
the similarities and differences between the control and iron
(FeSO4 and FeNPs) supplemented experiments for fermenta-
tive hydrogen production from glucose and sucrose. The ob-
tained resultswere represented as a bi-plot of PC1 against PC2,
and showed 99% variances in the data set for both glucose and
sucrose systems (Fig. 5(a and b)). In the glucose fed system, the
butyrate, propionate and ethanol were positively correlated
with PC1, whereas acetate, propionate and ethanol were
positively correlated with PC2. The butyrate was associated
with the control experiment. As shown in the bi-plot,
hydrogen was negatively correlated with propionate and
ethanol (Fig. 5(a)). This result indicates that the propionate
and ethanol affected the hydrogen production. In the sucrose
fed system, the hydrogen and all the soluble metabolites were
positively correlated with PC1, whereas hydrogen was posi-
tively correlated with PC2. The high variance of propionate
Fig. 4 e Proposed possible metabolic pathway for FeNPs
supplemented fermentative hydrogen production process.
(The dotted colour represents the enhancement of
hydrogen production with the supplementation of FeNPs).
(For interpretation of the references to colour in this figure
legend, the reader is referred to the web version of this
article.)
Please cite this article in press as: Mohanraj S, et al., Phytosynthtative hydrogen production using Enterobacter cloacae: EvaluatiHydrogen Energy (2014), http://dx.doi.org/10.1016/j.ijhydene.2014
and ethanol affected the FeNPs supplemented fermentation
process. Acetate and butyrate did not contribute significantly
to the FeNPs supplemented experiments due to the low vari-
ances (Fig. 5(b)). In bi-plots of glucose and sucrose fed systems,
the responses in the control, FeSO4 and FeNPs supplemented
experiments were located in different places due to the weak
relationship. A detailed explanation of the PCA has been given
by Shanmugam et al. [27].
Conclusions
The maximum hydrogen yields of 2.07 ± 0.07 mol H2/mol
glucose and 5.44 ± 0.27 mol H2/mol sucrose were achieved
with 125 and 200 mg/L iron oxide nanoparticles, respectively.
The results of the comparative study proved that, the
enhancement effect of the iron oxide nanoparticles on
fermentative hydrogen production was found to be higher
than that of FeSO4. The hydrogen production from the glucose
and sucrose fed systems with the supplementation of iron
oxide nanoparticles using E. cloacae, conformed to the acetate/
butyrate fermentation type. In conclusion, the supplementa-
tion of the synthesized iron oxide nanoparticles was believed
esized iron oxide nanoparticles and ferrous iron on fermen-on and comparison of the effects, International Journal of.06.027
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n en e r g y x x x ( 2 0 1 4 ) 1e1 010
to enhance the ferredoxin activity in fermentative hydrogen
production. Further studies are required to understand the
role of iron oxide nanoparticles in hydrogenase activity.
Acknowledgements
This researchwas funded by the Department of Biotechnology
(Ref. No. BT/PR12051/PBD/26/213/2009, dated 19th November
2010), New Delhi, India. The author S. Mohanraj gratefully
thanks theMinistry of New and Renewable Energy, NewDelhi,
India, for the Senior Research Fellowship (NREFeSRF). The
authors are thankful to theNational Institute of Agrobiological
Science, Japan, for providing the bacterial culture of Enter-
obacter cloacae 811101.
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