Phytosynthesized iron oxide nanoparticles and ferrous iron on fermentative hydrogen production using...

ww.sciencedirect.com

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

Available online at w

ScienceDirect

journal homepage: www.elsevier .com/locate/he

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.

mailto:[email protected]

www.sciencedirect.com/science/journal/03603199

www.elsevier.com/locate/he

http://dx.doi.org/10.1016/j.ijhydene.2014.06.027



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 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].




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.
pH
Fig. 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


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




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.


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


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.




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.



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




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)


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



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



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)


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




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.



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].




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.


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.


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




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).


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.)


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





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.

r e f e r e n c e s

[1] Sinha P, Pandey A. An evaluative report and challenges forfermentative biohydrogen production. Int Hydrogen Energy2011;36:7460e78.

[2] Nath MK, Das D. Improvement of fermentative hydrogenproduction: various approaches. Appl Microbiol Biotechnol2004;65:520e9.

[3] Peintner C, Zeidan AA, Schnitzhofer W. Bioreactor systemsfor thermophilic fermentative hydrogen production:evaluation and comparison of appropriate systems. J CleanProd 2010;18:S15e22.

[4] Wang J, Wan W. Factors influencing fermentative hydrogenproduction: a review. Int J Hydrogen Energy 2009;34:799e811.

[5] Hawkes FR, Dindale R, Hawkes DL, Hussy I. Sustainablefermentative biohydrogen: challenges for processoptimization. Int J Hydrogen Energy 2002;27:1339e47.

[6] Levin DB, Pitt L, Love M. Biohydrogen production: prospectsand limitations to practical application. Int J HydrogenEnergy 2004;29:173e85.

[7] Liu BF, Ren NQ, Ding J, Xie GJ, Guo WQ. The effect of Ni2þ,Fe2þ and Mg2þ concentration on photo-hydrogen productionby Rhodopseudomonas faecalis RLD-53. Int J Hydrogen Energy2009;34:721e6.

[8] Schonheit P, Brandis A, Thauer RK. Ferredoxin degradationin growing Clostridium pasteurianum during periods of irondeprivation. Arch Microbiol 1979;120:73e6.

[9] Vasconcelos I, Girbal L, Soucaille P. Regulation of carbon andelectron flow in Clostridium acetobutylicum grown inchemostat culture at neutral pH on mixtures of glucose andglycerol. J Bacteriol 1994;176:1443e50.

[10] Demuez M, Cournac L, Guerrini O, Soucaille P, Girbal L.Complete activity profile of Clostridium acetobutylicum[FeFe]-hydrogenase and kinetic parameters for endogenousredox patners. FEMS Microbiol Lett 2007;275:113e21.

[11] Petitdemange H, Marczak R, Gay R. NADH-flavodoxinoxidoreductase activity in Clostridium tyrobutyricum. FEMSMicrobiol Lett 1979;5:291e4.


[12] Fitzgerald MP, Rogers LJ, Rao KK, Hall DO. Efficiency offerredoxins and flavodoxins as mediators in systems forhydrogen evolution. Biochem J 1980;192:665e72.

[13] Vanacova S, Rasoloson D, Razga J, Hrdy I, Kulda J,Tachezy J. Iron-induced changes in pyruvate metabolismof Tritrichomonas foetus and involvement of iron inexpression of hydrogenosomal proteins. Microbiology2001;147:53e62.

[14] Lee YJ, Miyahara T, Noike T. Effect of iron concentrationon hydrogen fermentation. Bioresour Technol2001;80:227e31.

[15] Karadag D, Puhakka JA. Enhancement of anaerobic hydrogenproduction by iron and nickel. Int J Hydrogen Energy2010;35:8554e60.

[16] Zhang X, Yan S, Tyagi RD, Surampalli RY. Synthesis ofnanoparticles by microorganisms and their application inenhancing microbiological reaction rates. Chemosphere2011;82:489e94.

[17] Han H, Cui M, Wei L, Yang H, Shen J. Enhancement effect ofhematite nanoparticles on fermentative hydrogenproduction. Bioresour Technol 2011;102:7903e9.

[18] Beckers L, Hiligsmann S, Lambert SD, Heinrichs B, Thonart P.Improving effect of metal and oxide nanoparticlesencapsulated in porous silica on fermentative biohydrogenproduction by Clostridium butyricum. Bioresour Technol2013;133:109e17.

[19] Mohanraj S, Kodhaiyolii S, Rengasamy M, Pugalenthi V.Green synthesis of iron oxide nanoparticles effect onfermentative hydrogen production by Clostridiumacetobutylicum. Appl Biochem Biotechnol 2014;173:318e31.

[20] Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F.Colorimetric method for determination of sugars and relatedsubstances. Anal Chem 1956;28:350e6.

[21] Cappelletti BM, Reginatto V, Amante ER, Antonio RV.Fermentative production of hydrogen from cassavaprocessing wastewater by Clostridium acetobutylicum. RenewEnerg 2011;36:3367e72.

[22] Hammer Ø, Harper DAT, Ryan PD. PAST: paleontologicalstatistics software package for education and data analysis.Palaeontol Electron 2001;4(1):9.

[23] Zhang ML, Fan YT, Xing Y, Pan CM, Zhang GS, Lay JJ.Enhanced biohydrogen production from cornstalk wasteswith acidification pretreatment by mixed anaerobic cultures.Biomass Bioenerg 2007;31:250e4.

[24] Khanna N, Kotay SM, Gilbert JJ, Das D. Improvement ofbiohydrogen production by Enterobacter cloacae IIT-BT 08under regulated pH. J Biotechnol 2011;152:9e15.

[25] Lima DMF, Moreira WK, Zaiat M. Comparison of the use ofsucrose and glucose as a substrate for hydrogen productionin an upflow anaerobic fixed bed reactor. Int J HydrogenEnergy 2013;38:15074e83.

[26] Gorrel TE. Effect of culture medium iron content on thebiochemical composition and metabolism of Trichomonasvaginalis. J Bacteriol 1985;163:1228.

[27] Shanmugam SR, Chaganti SR, Lalman JA, Heath DD. Effect ofinhibitors on hydrogen consumption and microbialpopulation dynamics in mixed anaerobic cultures. Int JHydrogen Energy 2014;39:249e57.


http://refhub.elsevier.com/S0360-3199(14)01667-X/sref1




























































































































Phytosynthesized iron oxide nanoparticles and ferrous iron on fermentative hydrogen production using...

Documents

Transcript of Phytosynthesized iron oxide nanoparticles and ferrous iron on fermentative hydrogen production using...