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 e n e r g y x x x ( 2 0 1 5 ) 1 e7

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The effects of Fe0 and Ni0 nanoparticles versus Fe2þ

and Ni2þ ions on dark hydrogen fermentation

Mohsen Taherdanak a,**, Hamid Zilouei a,*, Keikhosro Karimi a,b

a
Department of Chemical Engineering, Isfahan University of Technology, Isfahan 84156-83111, Iran
b
Industrial Biotechnology Group, Institute of Biotechnology and Bioengineering, Isfahan University of Technology,
Isfahan 84156-83111, Iran

article info abstract

Article history: The effects of Fe0 and Ni0 nanoparticles (NPs) versus Fe2þ and Ni2þ ions on the efficiency of
Received 28 June 2015 mesophilic dark hydrogen fermentation from glucose were investigated using the heat-
Received in revised form shock pretreated anaerobic sludge. The initial glucose concentration and the pH of the
20 November 2015 reactors were 5 g/L and 5.5, respectively, and the concentrations of the supplemented
Accepted 20 November 2015 metals were varied between 0 and 50 mg/L. The hydrogen and biogas yields of the control
Available online xxx test were 247 and 391 mL/g VS, respectively. The results showed that the effect of Ni2þ ions
on the hydrogen yield was highly significant (P < 0.01), improving the hydrogen yield by
Keywords: 55%. The effects of Fe0 NPs and Fe2þ ions were significant too (P < 0.05), enhancing the
Biohydrogen hydrogen yield by 37% and 15%, respectively, whereas the effect of Ni0 NPs was insignifi-
Dark fermentation cant (P > 0.05). The analysis of the soluble metabolites revealed that the hydrogen
Fe0 nanoparticles fermentative yield was mainly affected by ethanol and propionate production, while the
Ni0 nanoparticles production of butyrate favored the hydrogen fermentation.
Kinetics Copyright © 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights
Volatile fatty acids reserved.

Biohydrogen is produced either by photo-synthetic or

Introduction
Since the last century, the growing demand for energy has
challenged the societies lacking fossil fuel sources and facing
serious environmental problems [1,2]. By numerous attempts
over the last decades, bioenergy has been proposed as a po-
tential alternative which can efficiently mitigate the global
energy demand with the least adverse environmental effects.
Nowadays, the processes for the production of bioethanol,
biodiesel, biomethane, and biohydrogen have been mostly
focused to be more efficient and globally applicable, which
biohydrogen is the most clean alternative with the highest
energy content (142 KJ/g) [3e5].
fermentative processes. Fermentative hydrogen production is
more applicable since it has a higher production rate and
simpler operation [6]. Moreover, various organic feedstocks
can be used in this process. In comparison with photo-
fermentative, dark fermentative hydrogen production enjoys
more feasibility for commercial development [3,6]. Several
different parameters including the type of microorganisms,
temperature, pH, substrate complexity, availability of micro-
nutrients, and metal ions affect the dark hydrogen fermen-
tation [7]. Hydrogen production via dark fermentation process
is attributed to the reduction of protons to molecular
hydrogen, and this reaction is catalyzed by hydrogenases

* Corresponding author. Tel.: þ98 3133915632; fax: þ98 3133912677.

** Corresponding author. Tel.: þ98 9361303035.
E-mail addresses: m.taherdanak@ce.iut.ac.ir (M. Taherdanak), hzilouei@cc.iut.ac.ir (H. Zilouei).
http://dx.doi.org/10.1016/j.ijhydene.2015.11.110
0360-3199/Copyright © 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Taherdanak M, et al., The effects of Fe0 and Ni0 nanoparticles versus Fe2þ and Ni2þ ions on dark
hydrogen fermentation, International Journal of Hydrogen Energy (2015), http://dx.doi.org/10.1016/j.ijhydene.2015.11.110
2 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 e n e r g y x x x ( 2 0 1 5 ) 1 e7
which have their optimum activity at certain temperatures
and pH dominants. Therefore, it is necessary to maintain the
process under optimum operational conditions [3].
Hydrogenases, which are interfered in dark fermentation,
are mainly classified in two clusters according to their metal
content: [FeeFe]-hydrogenase and [NieFe]-hydrogenase [8]. A
wide variety of bacteria can express [NieFe]-hydrogenase,
while a few are able to express [FeeFe]-hydrogenase [9,10].
Certain amounts of iron and nickel ions are needed to ex-
press and use the activity of these enzymes within dark
fermentation system. Several different studies have investi-
gated the effects of iron and nickel ions on dark fermentation
process, reporting the efficiency improvement with certain
concentrations of Fe and Ni ions [9]. Yang and Shen (2006)
investigated the effects of Fe2þ ions (0e4000 mg/L) on dark
hydrogen fermentation from starch using anaerobic sludge in
the batch reactor. They found that the addition of 150 mg
FeSO4/L increased the hydrogen yield by 163%, as compared
to the control test [11]. Wang and Wan (2008) investigated the
effect of Ni2þ (0e50 mg/L) on dark hydrogen fermentation
from glucose using the digested sludge in the batch reactor.
They found that the input of 0.1 mg Ni2þ/L increased the
hydrogen yield by nearly 65%, as compared to the control
test [9].
Because of the unique physical properties and chemical
activities of the nano-materials, such as high catalytic activity
and high specific surface area, a few studies investigated the
effect of metallic nanoparticles (NPs) on the efficiency of dark
hydrogen fermentation [8,12e16]. Mullai et al. (2013) reported
that 5.67 mg/L of Ni NPs increased the yield of dark hydrogen
fermentation from glucose by 22.7% [8]. Mohanraj et al. (2014)
investigated the effects of both iron oxide NPs and iron sulfate
on the hydrogen fermentation in a glucose fed system using
Enterobacter cloacae. They reported that iron oxide NPs sup-
plementation was superior to that of the iron sulfate and the
maximum hydrogen yield of 258 mL/g VS was obtained at iron
oxide NPs concentration of 125 mg/L [16]. Gadhe et al. (2015)
compared the effects of hematite and nickel oxide NPs on the
efficiency of dark hydrogen fermentation from dairy waste-
water in the batch reactor. They reported enhancement in the
hydrogen yield by 23.8% and 16%, as compared to the control
test, in the presence of 50 mg/L hematite NPs and 10 mg/L Ni-
oxide NPs, respectively. Based on Eq. (1), the supplementation
of zero-valent iron nanoparticles (Fe0 NPs) to dark fermenta-
tion can decrease the availability of the dissolved oxygen in
the system, leading to the better performance of the oxygen-
sensitive hydrogenases. Although this action is also per-
formed by facultative bacteria, the rate of oxygen consump-
tion by chemical reaction is much faster than its microbial
consumption. Therefore, chemical consumption of oxygen in
the presence of Fe0 NPs can be used as a complementary ef-
fect. Moreover, after consumption of the dissolved oxygen, the
Fe0 NPs is converted to Fe2þ ions accompanied by hydrogen
generation (Eq. (2)).
2Fe0 þ O2 þ 2H2O / 2Fe2þ þ 4OH, DE0 ¼ 1.67 V
Fe0 þ 2H2O / Fe2þ þ 2OH þ H2
Please cite this article in press as: Taherdanak M, et al., The effects of Fe0 and Ni0 nanoparticles versus Fe2þ and Ni2þ ions on dark
hydrogen fermentation, International Journal of Hydrogen Energy (2015), http://dx.doi.org/10.1016/j.ijhydene.2015.11.110
The main purpose of this study was to investigate the
effects of Fe0 and Ni0 NPs on the efficiency of mesophilic dark
hydrogen fermentation from glucose and compare them to
those of the Fe2þ/Ni2þ ions (FeSO4 and NiSO4). Furthermore,
their effects on the kinetic parameters of dark fermentation
process as well as the concentrations of the final soluble
metabolites were investigated.
Materials and methods
Inoculum and reagents
Anaerobic sludge was obtained from a 7000 m3 anaerobic
digester located at northern Isfahan wastewater treatment
plant, Isfahan, Iran. The sludge was sieved through a 1 mm
net to remove its large particles. Then, it was pretreated under
anaerobic conditions at 95  C for 15 min, and this heat-shock
pretreated sludge was used as an inoculum for biohydrogen
production. Total solids (TS) and volatile solids (VS) of the
pretreated inoculum were 53.8 and 28.2 g/L, respectively. The
composition of the nutrient solution was (mg/L in deionized
water): NaHCO3, 4000; K2HPO4.3H2O, 250; CuSO4.5H2O, 10;
MgCl2.6H2O, 200; MnSO4.4H2O, 30; CoCl2.6H2O, 0.25 [17].
Dark hydrogen fermentation
Dark hydrogen fermentations were performed in 118 mL dark
glass bottles as bioreactors containing 5 g/L of glucose, spec-
ified concentrations of iron or nickel (0e50 mg/L), 20 mL
inoculum, 40 mL nutrient solution, and a certain amount of
deionized water up to the total working volume of 80 mL.
Blank samples with only 20 mL inoculum, 40 mL nutrient so-
lution, and 20 mL deionized water were prepared to obtain the
hydrogen yield of inoculum individually. The initial pH of the
samples was adjusted to 5.5 by 1 N H2SO4. After filling, they
were capped with butyl rubber stoppers and aluminum caps
and purged with pure nitrogen for 3 min. All samples were
prepared in triplicate and incubated at 37  C. The bottles were
shaken manually before each gas sampling.
Analytical methods
Total solids (TS) and volatile solids (VS) of inoculum were
measured according to APHA standard method [18]. The
concentrations of glucose and the produced soluble metabo-
lites, including acetic acid (HAc), butyrate acid (HBu), propio-
nate acid (HPr) and ethanol (EtOH), were measured using high
performance liquid chromatography (HPLC, Jasco Interna-
tional Co., Tokyo, Japan) equipped with a Bio-Rad Aminex
HPX-87P column and a refractive index (RI) detector. The
temperatures of column and detector were set at 60 and 80  C,
respectively. Sulfuric acid solution (0.005 M) with a flow rate of
0.6 mL/min was used as the mobile phase.
The composition of the produced biogas was analyzed
using gas chromatograph (Sp-3420A, Beijing BeifenRuili
(1) Analytical Instrument CO) with a thermal conductivity de-
tector (TCD). The column was packed with Propack Q (3 m
length and 3 mm internal diameter, stainless steel, Chrom-
(2) pack, Germany) and the carrier was nitrogen at the flow rate of
 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 e n e r g y x x x ( 2 0 1 5 ) 1 e7 3

30 mL/min. The temperatures of the column, the injection
port, and the detector were 40, 100 and 150  C, respectively.
Gas samples were withdrawn at regular intervals with a
pressure-lock syringe (VICI, Precision Sampling, Inc., USA) and
analyzed by gas chromatography. Gas volumes were calcu-
lated based on the ideal gas law by taking into consideration
the pressure in the bottle, the volume of gas head and the
hydrogen and carbon dioxide proportion. The results of the
gas analysis were reported on the basis of cumulative yield at
the standard temperature (273 K) and pressure (101.3 kPa) and
normalized in terms of the total initial organic matter added
to each sample, as mL/g VS [5].
In order to determine the effect of Fe0 and Ni0 nano-
particles (NPs) on the kinetic parameters of dark hydrogen
fermentation from glucose, experimental methane pro-
ductions (mL) were modeled using modified Gompertz model
(Eq. (3)) and the kinetic parameters were obtained:
  
Rmax e
HðtÞ ¼ Hmax exp  exp ðl  tÞ þ 1
Hmax
where H(t), Hmax, Rmax, l, e, and t represent the cumulative
hydrogen production (mL), ultimate hydrogen production
potential (mL), maximum hydrogen production rate (mL/h),
lag-phase (h), Euler's number (e z 2.71828) and incubation
time (h), respectively [19].
The significance of the effects of each supplementation
was determined using the analysis of variance (ANOVA) with
the confidence interval of 95%. Principal component analysis
(PCA) was used to determine the similarities and the variances
of the produced hydrogen and soluble metabolites in the
control and metal supplemented experiments. Both ANOVA
and PCA were performed using Minitab software (version 17).
Results and discussion
The effects of different concentrations of Fe0 NPs, Ni0 NPs,
Fe2þ ions and Ni2þ ions on dark hydrogen fermentation from
glucose as well as the fermentation byproducts, including
acetate (HAc), butyrate (HBu), propionate (HPr) and ethanol
(EtOH), were investigated. The results are depicted in Figs. 1
and 2. The gas production yields were calculated based on
the consumed glucose. The glucose degradation efficiency (%)
and the final pH of the samples are presented in Table 1. The
hydrogen and biogas yields of the control test were 247 and
391 mL/g VS, respectively. The produced HAc, HBu, HPr and
EtOH from the control test were measured to be 0.54, 0.86,
1.04, and 0.86 g/L, respectively.
(3)
The effect of Fe0 nanoparticles on hydrogen production
The effect of Fe0 NPs supplementation on the biohydrogen
production was significant based on the ANOVA (p < 0.05). The
low Fe0 NPs input of 0.5 mg/L enhanced the hydrogen yield by
8% in comparison with the control test, probably due to the
promoted activity of hydrogenases as well as the ferredoxin
electron transfer after the Fe0 NPs supplementation, as re-
ported by Mohanraj et al. [15]. This supplementation
increased the HAc production by 57%, while HBu and HPr
productions were reduced by 80% and 24%, respectively, and

600 600 (c)

Carbon dioxide (a) Carbon dioxide
Hydrogen Hydrogen
500 500
Biogas (mL/g VS)
Biogas (mL/g VS)

400 400

300 300

200 200

100 100

0 0
Control 0.25 0.5 2.5 5 10 25 50 Control 0.25 0.5 2.5 5 10 25 50
Ni0 concentration (mg/L) Fe2+ concentration (mg/L)

700 800 (d)

Carbon dioxide (b) Carbon dioxide
600 Hydrogen 700 Hydrogen

600
500
Biogas (mL/g VS)
Biogas (mL/g VS)

500
400
400
300
300
200 200
100 100

0 0
Control 0.25 0.5 2.5 5 10 25 50 Control 0.25 0.5 2.5 5 10 25 50
Fe0 concentration (mg/L) Ni2+ concentration (mg/L)

Fig. 1 e The effects of Fe0 NPs, Ni0 NPs, Fe2þ ions, and Ni2þ ions on dark hydrogen fermentation from glucose, as compared
to the control test.

Please cite this article in press as: Taherdanak M, et al., The effects of Fe0 and Ni0 nanoparticles versus Fe2þ and Ni2þ ions on dark
hydrogen fermentation, International Journal of Hydrogen Energy (2015), http://dx.doi.org/10.1016/j.ijhydene.2015.11.110
4 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 e n e r g y x x x ( 2 0 1 5 ) 1 e7

1.8 (a)
EtOH HAc 1.8 (c)
1.6 EtOH HAc
HPr HBu 1.6
Soluble metabolite (mg/L)

1.4 HPr HBu

Soluble metabolite (mg/L)

1.4
1.2
1.2
1
1
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0
0
Control 0.25 0.5 2.5 5 10 25 50
Control 0.25 0.5 2.5 5 10 25 50
Fe0 concentration (mg/L)
Fe2+ concentration (mg/L)

1.8 (b)
EtOH HAc 1.6 (d)
1.6 EtOH HAc
HPr HBu 1.4
Soluble metabolite (mg/L)

1.4 HPr HBu

Soluble metabolite (mg/L)

1.2
1.2
1 1

0.8 0.8
0.6 0.6
0.4 0.4
0.2
0.2
0
Control 0.25 0.5 2.5 5 10 25 50 0
Control 0.25 0.5 2.5 5 10 25 50
Ni0 concentration (mg/L)
Ni2+ concentration (mg/L)

Fig. 2 e The effects of Fe0 NPs, Ni0 NPs, Fe2þ ions, and Ni2þ ions on the fermentation soluble metabolites including acetate
(HAc), butyrate (HBu), propionate (HPr) and ethanol (EtOH).

no EtOH was produced. Since the production of EtOH and HPr production was enhanced by 74%; the HPr production was
reduced the yield of hydrogen production, the enhancement reduced by 75%. The maximum hydrogen yield enhancement
in the hydrogen yield could be attributed to the inhibition of of 37% was achieved after Fe0 NPs input of 25 mg/L, as
HPr and EtOH producing bacteria. However, the small compared with the control test. While no EtOH was produced,
hydrogen yield enhancement could indicate the low in- the HAc production was nearly constant; the HPr production
teractions between the supplemented Fe0 NPs and the mi- was decreased by 54%, and the HBu production was increased
croorganisms, highlighting the need for higher Fe0 NPs input, by 88%. It could be understood that the supplementation of Fe0
as demonstrated by Choi et al. [20]. The higher Fe0 NPs input NPs enhances the HBu production in addition to the inhibition
(5 mg/L) led to the higher enhancement in the hydrogen yield of EtOH and HPr productions. Further increase in Fe0 NPs
(11%), as compared with the control test. No EtOH was pro- concentration (up to 50 mg/L) reduced the hydrogen yield by
duced; the HAc production remained constant; the HBu 65%. While the HAc production was enhanced by 65%, the HBu

Table 1 e The effects of Fe0 and Ni0 nanoparticles versus Fe2þ and Ni2þ ions on the glucose degradation efficiency (%), final
pH, and acetate/butyrate ratio (%).
Conc. (mg/L) Glucose degradation efficiency Final pH HAc/HBu ratio (%)
(%)
Fe0 Fe2þ Ni0 Ni2þ Fe0 Fe2þ Ni0 Ni2þ Fe0 Fe2þ Ni0 Ni2þ
a
0.00 64 64 64 64 5.2 5.2 5.2 5.2 160 62 62 62
0.25 63 63 65 66 5.2 5.1 5.1 5.4 25 267 143 248
0.50 65 66 64 70 5.4 5 5.1 5.3 20 87 378 77
2.50 64 63 65 64 5.3 4.9 4.9 5.3 183 208 155 140
5.00 62 63 66 68 4.5 5 5.3 5.6 60 129 34 168
10.00 63 62 64 66 4.9 5 5 5 21 90 567 258
25.00 65 64 67 65 5.5 5.2 5.3 5.2 310 44 107 59
50.00 65 62 68 66 5.5 5.3 5.2 5 41 58 132 68
a
Control sample.

Please cite this article in press as: Taherdanak M, et al., The effects of Fe0 and Ni0 nanoparticles versus Fe2þ and Ni2þ ions on dark
hydrogen fermentation, International Journal of Hydrogen Energy (2015), http://dx.doi.org/10.1016/j.ijhydene.2015.11.110
 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 e n e r g y x x x ( 2 0 1 5 ) 1 e7
production was decreased by 57%. Therefore, it could be un-
derstood that the hydrogen was produced through HBu type
fermentation reaction. The higher Fe0 NPs input (50 mg/L)
disturbed the HBu production bacteria and therefore, the
hydrogen production was decreased. This phenomenon has
also been reported by Mohanraj et al. (2014), who investigated
the effect of hematite NPs on the efficiency of dark hydrogen
fermentation. They reported that the hydrogen yield was
enhanced with increasing the hematite concentration and
then decreased at higher concentrations [16]. Almost at all
concentrations, the Fe0 NPs supplementation led to the higher
biogas production, in comparison to the control test, and the
highest biogas yield of 551 mL/g VS was obtained at Fe0 NPs
concentration of 25 mg/L.
The effect of Ni0 nanoparticles on hydrogen production
In contrast to Fe0 NPs, the effect of Ni0 NPs on the biohydrogen
production was insignificant (p > 0.05). The only enhancement
in hydrogen yield was achieved from the Ni0 NPs input of
2.5 mg/L, which enhanced the hydrogen yield by 0.9%, in
comparison with the control test. This result was not consis-
tent with the obtained results by Mullai et al. (2013), who
investigated the effect of Ni NPs on the efficacy of dark
hydrogen fermentation from glucose. They reported that the
input Ni NPs of 5.37 mg/L could increase the hydrogen yield by
22.7%, as compared to the control test [8]. No straight effect on
the EtOH, HAc, HBu or HPr production was observed. At some
concentrations, the EtOH production was increased in com-
parison with the control test. The highest EtOH production
was observed at Ni0 NPs concentration of 5 mg/L, leading to
the lowest hydrogen yield of 99 mL/g VS, which was 60% lower
than that of the control test. When the concentration of Ni0
NPs was increased to 50 mg/L, the process was drastically
inhibited; hydrogen, HAc, HBu, HPr and EtOH were reduced by
41%, 9%, 57%, 82%, and 93%, respectively, as compared with
control test. The increasing concentration of Ni0 NPs reduced
the hydrogen content. The lowest hydrogen content of 32%
was obtained at the Ni0 NPs concentration of 50 mg/L.
Generally, unlike the Fe0 NPs supplementation, the Ni0 NPs
supplementation was not favorable for dark hydrogen
fermentation using anaerobic sludge.
The effect of FeSO4 on hydrogen production
The effect of FeSO4 or Fe2þ ions on the hydrogen yield was
significant based on the ANOVA (p < 0.05). The Fe2þ input of
0.5 mg/L enhanced the hydrogen yield by 7%, as compared
with the control test. EtOH and HPr productions were reduced
by 37% and 28%, respectively; HAc production remained
constant and HBu was reduced by 27%. Further increase in the
concentration of Fe2þ ions up to 10 mg/L enhanced the
hydrogen yield by 15%. The EtOH and HPr productions were
decreased by 43% and 35%, respectively; HAc production was
increased by 8%; HBu was decreased by 25%. Therefore, these
enhancements in hydrogen yield could be attributed to the
inhibition of EtOH and HPr productions, which consumed the
hydrogen; therefore, the process was shifted to more
hydrogen production than hydrogen consumption. At higher
concentrations of Fe2þ ions (50 mg/L), the hydrogen yield was
Please cite this article in press as: Taherdanak M, et al., The effects of Fe0 and Ni0 nanoparticles versus Fe2þ and Ni2þ ions on dark
hydrogen fermentation, International Journal of Hydrogen Energy (2015), http://dx.doi.org/10.1016/j.ijhydene.2015.11.110
5
reduced by 32%, as compared to the control test. In this test,
EtOH was increased by 15%; HPr was decreased by 26%; HAc
and HBu were increased by 28% and 38%, respectively. No
significant disturbance in the HAc or HBu production was
observed. It could be understood that the hydrogen yield was
more affected by the EtOH production than the production of
other byproducts. As in the Fe0 NPs, the supplementation of
Fe2þ ions at all concentrations enhanced the biogas yield and
the highest biogas yield of 493 mL/g VS was obtained at Fe2þ
ions concentration of 5 mg/L.
The effect of NiSO4 on hydrogen production
The effect of NiSO4 on the biohydrogen production was highly
significant based on the ANOVA (P < 0.01). The initial Ni2þ ions
concentration of 2.5 mg/L enhanced the hydrogen yield by
28%, in comparison with the control test. As the concentration
of Ni2þ ions was increased to 10 mg/L and 25 mg/L, the
hydrogen yield was enhanced by 46% and 55%, respectively, as
compared to the control test. These enhancements might be
due to the interaction of the Ni2þ ions and the [NieFe]-hy-
drogenases [21]. The higher concentration of Ni2þ ions led to
the higher positive interaction. However, increasing the con-
centration of Ni2þ ions from 25 to 50 mg/L reduced the
hydrogen yield by 5%, probably due to the inhibitory effect of
Ni2þ at higher concentrations [9,22,23]. Supplementation of
Ni2þ ions at all concentrations drastically enhanced the biogas
yield, and the highest biogas yield of 680 mL/g VS was ob-
tained at Ni2þ ions concentration of 50 mg/L, which was 74%
higher than that of the control test. The presence of Ni2þ ions
significantly reduced the EtOH production, and it could be
seen that the higher hydrogen yields were achieved at lower
EtOH productions.
Kinetic study
The experimental hydrogen production data were modeled
using modified Gompertz equation. The results are presented
in Table 2. For all samples, the fine coefficients of de-
terminations (Adjusted R2 z 1) indicated the good correlation
between the experimental data and the used model. The lag-
phase, maximum hydrogen production rate, and the
hydrogen potential of the control test were 25.5 h, 4.5 mL/h,
and 63 mL, respectively. Generally, the supplementation of
Fe2þ/Ni2þ ions and Fe0/Ni0 NPs increased the lag-phase times,
probably due to the disturbance effect of additives on the
microbial metabolism [3]. This phenomenon has also been
reported by Mohanraj et al. (2014), who observed that the
supplementation of palladium NPs at all concentrations
enhanced the lag-phase time of hydrogen fermentation [12].
Supplementation of Ni0 NPs had a more negative effect on the
lag-phase than the other additives. Except Ni0 NPs, the other
additives at certain concentrations increased the maximum
hydrogen production rate. Supplementation of Fe0 NPs (25 mg/
L), Fe2þ (0.5 mg/L) and Ni2þ (10 mg/L) increased the maximum
hydrogen production rate by 44%, 13%, and 62%, respectively.
Moreover, positive effects on the maximum hydrogen poten-
tial after Fe0 NPs, Fe2þ, and Ni2þ supplementations and the
highest achievement were observed after Ni2þ input of 25 mg/
L, which was 57% higher than the hydrogen potential of the
6 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 e n e r g y x x x ( 2 0 1 5 ) 1 e7

Table 2 e The effects of Fe0 and Ni0 nanoparticles versus Fe2þ and Ni2þ ions on the kinetic parameters of hydrogen
fermentation from glucose.
Conc. (mg/L) l (h) Rmax (mL/h) Hmax (mL) Adj. R2
Fe0 Fe2þ Ni0 Ni2þ Fe0 Fe2þ Ni0 Ni2þ Fe0 Fe2þ Ni0 Ni2þ Fe0 Fe2þ Ni0 Ni2þ
a
0.00 25.5 25.5 25.5 25.5 4.5 4.5 4.5 4.5 63 63 623 63 0.998 0.998 0.998 0.998
0.25 29.3 27.8 37.1 24.0 3.7 2.8 2.2 2.8 56 56 59 63 1.000 0.999 1.000 0.999
0.50 28.0 26.5 28.4 27.5 6.0 5.1 3.2 3.7 74 70 60 68 1.000 1.000 0.999 1.000
2.50 27.4 26.5 27.8 28.1 2.1 4.8 3.5 4.7 57 65 68 79 0.999 1.000 0.995 0.999
5.00 29.0 28.0 42.7 27.4 4.0 2.1 1.2 4.2 73 55 26 56 1.000 1.000 1.000 0.999
10.00 28.8 25.3 32.3 28.6 4.6 4.0 1.7 7.3 70 69 51 90 1.000 0.999 0.998 1.000
25.00 28.5 26.8 40.7 24.4 6.5 5.0 1.6 6.8 87 70 35 98 1.000 1.000 0.990 1.000
50.00 26.4 22.9 NDb 28.2 1.0 2.4 ND 4.8 27 42 ND 90 0.994 1.000 ND 0.994
a
Control.
b
ND: not determined.

control test. These positive achievements could be attributed
to the promoted activity of hydrogenases as well as the
enhanced electron transfer and microbial growth in the sys-
tem [3,14].
Principal component analysis
In order to come to a better understating of the effect of the
supplemented metals on the fermentation process, the
experimental data (hydrogen yields and the produced soluble
metabolites) of the control test were accompanied by those of
the metal supplemented experiments and used for the anal-
ysis of the principal components. Fig. 3 depicts the biplot of
the first principal component (PC1) and the second principal
component (PC2), demonstrating nearly 85% variance in the
data set. It could be seen that the control experiment was
closely associated with the HPr, HAc and EtOH productions.
Moreover, both the HBu production and the Control test were
positively correlated with PC2 and therefore, they were asso-
ciated to each other, but in a lower intensity. The HAc, HPr and
EtOH productions were positively correlated with PC1 while
HBu and hydrogen productions were negatively correlated
with PC2, indicating the hydrogen production through the HBu
fermentation pathway. Both Fe0 NPs and Ni2þ were linked
with hydrogen and HBu productions. The enhancement in
hydrogen yield by Fe0 NPs could be attributed to the activation
of HBu production as well as the inhibition of EtOH produc-
tion; also, the enhancement effect of the Ni2þ could be mainly
attributed to the inhibition of HPr production.
To summarize, the supplementation of Fe0 NPs, Fe2þ ions
and Ni2þ ions had positive effects on dark hydrogen fermen-
tation from glucose using anaerobic sludge, while no signifi-
cant effect was observed after the supplementation of Ni0 NPs.
According to the ANOVA results, the significance of the sup-
plemented metals on the hydrogen yield was as follows: Ni2þ
ions > Fe0 NPs > Fe2þ ions > Ni0 NPs. The hydrogen was pro-
duced through the butyrate fermentation pathway, where the
major effects of Fe0 NPs supplementation were the inhibition
of HPr and EtOH production and the increase in HBu
production.
Conclusion
The yield of the dark hydrogen fermentation could be inter-
estingly enhanced by the supplementation of iron and nickel
at the optimum concentrations. The results revealed that the
effect of the supplemented iron and nickel was as follows:
Ni2þ ions > Fe0 nanoparticles > Fe2þ > Ni0 nanoparticles.
Moreover, it was found that the yield of hydrogen was dras-
tically affected by ethanol and propionate production.

references

[1] Ghimire A, Frunzo L, Pirozzi F, Trably E, Escudie R, Lens PNL,

et al. A review on dark fermentative biohydrogen production
from organic biomass: process parameters and use of by-
products. Appl Energy 2015;144:73e95.
[2] Chu C-Y, Sen B, Lay C-H, Lin Y-C, Lin C-Y. Direct
fermentation of sweet potato to produce maximal hydrogen
and ethanol. Appl Energy 2012;100:10e8.
Fig. 3 e The obtained biplot from principal component
[3] Zilouei H, Taherdanak M. Biohydrogen from lignocellulosic
analysis (Fe0, Ni0, Fe2þ, and Ni2þ refer to the experiments wastes. In: Karimi K, editor. Lignocellulose-based
containing the mentioned metals; control: experiments bioproducts. Switzerland: Springer International Publishing;
without metal supplementation). 2015. p. 253e88.

Please cite this article in press as: Taherdanak M, et al., The effects of Fe0 and Ni0 nanoparticles versus Fe2þ and Ni2þ ions on dark
hydrogen fermentation, International Journal of Hydrogen Energy (2015), http://dx.doi.org/10.1016/j.ijhydene.2015.11.110
 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 e n e r g y x x x ( 2 0 1 5 ) 1 e7
[4] Dhar BR, Elbeshbishy E, Nakhla G. Influence of iron on sulfide
inhibition in dark biohydrogen fermentation. Bioresour
Technol 2012;126:123e30.
[5] Taherdanak M, Zilouei H. Improving biogas production from
wheat plant using alkaline pretreatment. Fuel
2014;115:714e9.
[6] Wieczorek N, Kucuker MA, Kuchta K. Fermentative hydrogen
and methane production from microalgal biomass (Chlorella
vulgaris) in a two-stage combined process. Appl Energy
2014;132:108e17.
[7] Wang J, Wan W. Factors influencing fermentative hydrogen
production: a review. Int J Hydrogen Energy 2009;34:799e811.
[8] Mullai P, Yogeswari MK, Sridevi K. Optimisation and
enhancement of biohydrogen production using nickel
nanoparticles e a novel approach. Bioresour Technol
2013;141:212e9.
[9] Wang J, Wan W. Influence of Ni2þ concentration on
biohydrogen production. Bioresour Technol 2008;99:8864e8.
[10] Bao MD, Su HJ, Tan TW. Dark fermentative bio-hydrogen
production: effects of substrate pre-treatment and addition
of metal ions or L-cysteine. Fuel 2013;112:38e44.
[11] Yang H, Shen J. Effect of ferrous iron concentration on
anaerobic bio-hydrogen production from soluble starch. Int J
Hydrogen Energy 2006;31:2137e46.
[12] Mohanraj S, Anbalagan K, Kodhaiyolii S, Pugalenthi V.
Comparative evaluation of fermentative hydrogen
production using Enterobacter cloacae and mixed culture:
effect of Pd (II) ion and phytogenic palladium nanoparticles. J
Biotechnol 2014;192. Part A:87-95.
[13] Han H, Cui M, Wei L, Yang H, Shen J. Enhancement effect of
hematite nanoparticles on fermentative hydrogen
production. Bioresour Technol 2011;102:7903e9.
[14] Mohanraj S, Kodhaiyolii S, Rengasamy M, Pugalenthi V.
Green synthesized iron oxide nanoparticles effect on
Please cite this article in press as: Taherdanak M, et al., The effects of Fe0 and Ni0 nanoparticles versus Fe2þ and Ni2þ ions on dark
hydrogen fermentation, International Journal of Hydrogen Energy (2015), http://dx.doi.org/10.1016/j.ijhydene.2015.11.110
7
fermentative hydrogen production by Clostridium
acetobutylicum. Appl Biochem Biotechnol 2014;173:318e31.
[15] Gadhe A, Sonawane SS, Varma MN. Enhancement effect of
hematite and nickel nanoparticles on biohydrogen
production from dairy wastewater. Int J Hydrogen Energy
2015;40:4502e11.
[16] Mohanraj S, Kodhaiyolii S, Rengasamy M, Pugalenthi V.
Phytosynthesized iron oxide nanoparticles and ferrous iron
on fermentative hydrogen production using Enterobacter
cloacae: evaluation and comparison of the effects. Int J
Hydrogen Energy 2014;39:11920e9.
[17] Zhao W, Zhang Y, Du B, Wei D, Wei Q, Zhao Y. Enhancement
effect of silver nanoparticles on fermentative biohydrogen
production using mixed bacteria. Bioresour Technol
2013;142:240e5.
[18] APHA. Standard methods for the examination of waste and
wastewater. 16th ed. New York: American Public Health
Associations; 1995.
[19] Nath K, Das D. Modeling and optimization of fermentative
hydrogen production. Bioresour Technol 2011;102:8569e81.
[20] Choi O, Yu C-P, Esteban Ferna  ndez G, Hu Z. Interactions of
nanosilver with Escherichia coli cells in planktonic and biofilm
cultures. Water Res 2010;44:6095e103.
[21] Karadag D, Puhakka JA. Enhancement of anaerobic hydrogen
production by iron and nickel. Int J Hydrogen Energy
2010;35:8554e60.
[22] Gou C, Guo J, Lian J, Guo Y, Jiang Z, Yue L, et al.
Characteristics and kinetics of biohydrogen production with
Ni2þ using hydrogen-producing bacteria. Int J Hydrogen
Energy 2015;40:161e7.
[23] Liu B-F, Ren N-Q, Ding J, Xie G-J, Guo W-Q. The effect of Ni2þ,
Fe2þ and Mg2þ concentration on photo-hydrogen production
by Rhodopseudomonas faecalis RLD-53. Int J Hydrogen Energy
2009;34:721e6.