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 3 8 ( 2 0 1 3 ) 5 2 4 3 e5 2 5 0

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Electrochemically active biofilm mediated bio-hydrogen

production catalyzed by positively charged gold nanoparticles

Mohammad Mansoob Khan, Jintae Lee, Moo Hwan Cho*

School of Chemical Engineering, Yeungnam University, Gyeongsan-si, Gyeongbuk 712-749, South Korea

article info abstract

Article history: An electrochemically active biofilm (EAB) was used for the synthesis of positively charged
Received 26 November 2012 gold nanoparticles [(þ)AuNPs] and in-situ hydrogen production without any external en-
Received in revised form ergy input. The EAB generate electrons and protons by decomposing sodium acetate
11 February 2013 (carbon source) in water at 30  C. These electrons were used initially to reduce Au3þ to Au0,
Accepted 17 February 2013 and later in-situ, these generated electrons and protons were used for hydrogen produc-
Available online 16 March 2013 tion. The as-synthesized (þ)AuNPs acted as catalyst by providing a charged surface to
reduce the protons, leading to the formation of molecular hydrogen according to the
Keywords: Volmer-Heyrovsky mechanism. The hydrogen produced was confirmed and estimated by
Hydrogen production gas chromatography and a fuel cell test, respectively. The maximum rate of hydrogen
Electrochemically-active biofilm production reached w105  2 mL/L day. This suggests that hydrogen production is possible
Gold nanoparticles in a single chamber reactor using an EAB in the presence of sodium acetate as a substrate
Catalysis and (þ)AuNPs as a catalyst.
Volmer-Heyrovsky mechanism Copyright ª 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights
reserved.

1. Introduction nanoparticles is simple, the size and morphology of nano-

particles can be controlled, and the nanoparticles can be
Since the first report of gold colloids by Faraday [1], several separated easily without contamination. Therefore, the
studies of the synthesis, properties and applications of gold extracellular synthesis of nanoparticles might have a wide
clusters, colloids and nanoparticles have been reported [2e4]. range of applications compared to intracellular synthesis [13].
The interest in gold nanoparticles has increased rapidly over Electrochemically active bacteria found on electrochemically
the past years because of its intriguing properties, such as active biofilms (EABs) are well known for their high oxidation
strong surface plasmon [5], catalytic [6,7], redox behavior [7] power and ability to produce electrons and protons in solution
and applications in medical diagnostics [8], imaging [8], opti- by consuming sodium acetate or other organic matter [14,15].
cal [9] and forensic [10]. This group recently reported the extracellular synthesis of
The synthesis of gold nanoparticles (AuNPs) via a green metal nanoparticles and nanocomposites, such as gold
approach is encouraging [11]. In many of the microbial syn- nanoparticles (þ)AuNPs [16,17], cysteine capped silver nano-
thesis reported, the nanoparticles were produced within the particles (Cys-AgNPs) [18] and Au/TiO2 nanocomposites [17],
cell/organism. In general, nanoparticles that are synthesized using an EAB.
intracellularly are not monodispersed, become contaminated, Energy is one of the most important factors affecting
and are far inferior to those obtained from other methods global prosperity. Currently, the world is dependent on fossil
[12,13]. On the other hand, the extracellular synthesis of fuels for the primary energy needs. The consumption of

* Corresponding author. Tel.: þ82 53 810 2517; fax: þ82 53 810 4631.
E-mail address: [email protected] (M.H. Cho).
0360-3199/$ e see front matter Copyright ª 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.ijhydene.2013.02.080
5244 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 3 8 ( 2 0 1 3 ) 5 2 4 3 e5 2 5 0

fossil fuels leads to global climate change, environmental

degradation and human health problems [19]. Hydrogen is 2. Experimental
considered the fuel of the future and hydrogen fuel-based
industries will have less pollution than fossil fuel-based in- 2.1. Materials
dustries. Hydrogen is a clean renewable energy source
because the only oxidation product is H2O. In addition, it has Chloroauric acid (HAuCl4$nH2O; n ¼ 3.6) (Kojima Chemicals,
the highest gravimetric energy density (122 kJ g1), which is Japan), sodium citrate (Na3C6H5O7$2H2O), sodium acetate and
w2.75 times higher than that of hydrocarbon fuels. extra pure NaOH (Duksan Pure Chemicals Co. Ltd. Korea) were
Furthermore, hydrogen is compatible with combustion and used as received. Carbon paper (without wet proof, Fuel Cell
electrochemical processes for energy production without Earth LLC, USA) and stainless steel mesh of type SUS 304
producing any harmful gases (CO2, CO, CH4 etc) that (Ildong Wire Cloth, Korea), which is a commercial alloy con-
contribute to environmental pollution and climate change taining 68e72% Fe, 18e20% Cr, 8e10% Ni and very small
[19]. On the other hand, most of the hydrogen gas produced amounts of C, Mn and Si were used. De-ionized (DI) water was
in the world today is prepared from fossil fuels, which re- prepared by using a water purification system (PURE ROUP 30,
sults in the release of greenhouse gases. Renewable Hanascience, Korea).
hydrogen production by water electrolysis using renewable
sources, such as wind, solar or biomass, is possible but the 2.2. Methods
energy requirements are high (5.6 kW h/m3 H2) and typical
electrolyzer energy efficiencies are only 56e73% [20]. Photo- Glass syringes (HAMILTON Co. USA) of 100 mL and 25 mL were
synthetic bacteria and algae can use sunlight to produce H2 used to collect the hydrogen gas produced for analysis. The
autotrophically from water, but the efficiencies are currently hydrogen gas produced was analyzed by gas chromatography
low, and most researchers believe that the process will never (GC, Hewlett Packard, GC 5890, USA) equipped with a thermal
be feasible because of the large surface area required for the conductivity detector (TCD) and a stainless steel column
process [20]. Polysaccharides, such as starch and cellulose, (RESTEK, 800-356-1688, USA), 2 m long with a 1 mm ID and
and carbohydrates, such as glucose, can be fermented by packed with Shin Carbon ST. The operating temperatures of
certain bacteria to H2 gas at average rates of 2.5  0.3 m3/ the oven, injection port and detector were set to 40, 100 and
m3 d. Fermentation results in a range of soluble organic 200  C, respectively. Nitrogen was used as the carrier gas at a
byproducts. The conversion of these byproducts to useful flow rate of 10 mL min1. A fuel cell kit (Carl’s Electronics,
amounts of hydrogen requires endothermic reactions. USA) connected to a digital multimeter (Agilent 34405A, USA)
Therefore, these molecules cannot be converted to hydrogen to measure the generated voltage was used to reconfirm the
without external energy input. A voltage must be applied hydrogen gas produced. Size of fuel cell electrode was
because hydrogen formation using acetate or other substrate 3 cm  3 cm. Membrane electrode assembly (MEA) was
is not spontaneous under standard conditions [21]. “platinum on carbon paper/proton exchange membrane/
This paper reports a novel, efficient and extracellular platinum on carbon paper” (Pt/PEM/Pt). At the anodic end of
synthesis of positively charged gold nanoparticles and in- fuel cell, produced hydrogen, collected in syringe, was injec-
situ hydrogen production. Hydrogen production was ach- ted whereas cathodic end of fuel cell was connected to air
ieved using an electrochemically active biofilm (EAB) on a pump. The generated voltage was recorded by multimeter.
stainless steel mesh, sodium acetate as the carbon source Fuel cell was operated at room temperature.
and as-synthesized (þ)AuNPs as a catalyst in water at 30  C
without any external energy input (except for magnetic 2.3. Preparation of the electrochemically active biofilm
stirring and temperature control). The electrochemically (EAB)
active bacteria used were mixed cultures. In general, an EAB
formed on carbon paper [14,16e18] was used for the syn- An EAB was prepared on a stainless steel mesh and carbon
thesis of Cys-AgNPs, (þ)AuNPs, and Au@TiO2 nano- paper, as reported earlier [16e18]. Briefly, a stainless steel
composites. On the other hand, in the present study, an EAB mesh and carbon paper (2.5 cm  4.5 cm) were dipped into an
was developed on a stainless steel mesh as a support aqueous mineral salt medium containing sodium acetate as a
[22e24]. The combined effects of the EAB and the stainless substrate [25]. 10 mL of an anaerobic sludge (from a biogas
steel mesh enhanced the availability of the electrons and plant in Paju, Korea) was added under anaerobic conditions by
made the overall process for reducing Au3þ to Au0 and later sparging N2 gas for 5 min. All media, including the bacterial
for forming hydrogen gas very fast. The as-synthesized (þ) inoculum and substrate, were changed every two days under
AuNPs catalyzed and facilitated hydrogen production in an anaerobic conditions. This was repeated for two weeks. The
Electrochemically Active Biofilm Reactor (EABR) under living EAB with a mixed culture formed on the stainless steel
anaerobic and inert atmosphere without any external energy mesh was used to synthesize the (þ)AuNPs, whereas the EAB
input. To the best of the authors’ knowledge, this is the first formed on a stainless steel mesh and carbon paper was tested
report of the use of an EAB made on a stainless steel mesh for hydrogen production.
for the extracellular synthesis of (þ)AuNPs as well as for in-
situ hydrogen production. In this report, a hybrid system 2.4. Synthesis of (þ)AuNPs
was made by integrating EAB, nanoparticles and catalysis,
which appears to be successful for catalytic molecular Briefly, 0.5 mM, 1 mM and 2.0 mM HAuCl4 aqueous solutions
hydrogen production at the initial stage. (200 mL) were prepared. In each solution, 0.2 g of sodium
 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 3 8 ( 2 0 1 3 ) 5 2 4 3 e5 2 5 0 5245

acetate was added as an electron donor and the pH was by GC. The results were reconfirmed using a fuel cell attached
maintained at pH 4 using 0.1 N NaOH. The initial color of the to a multimeter to record the voltage generated by produced
reaction mixtures was golden yellow. The anaerobic condi- hydrogen.
tions were maintained by sparging N2 gas for 5 min. The EAB Setup B: 1 mM (þ)AuNPs were synthesized as mentioned above
on the stainless steel mesh was hung by plastic wire and the in the EABR. The remaining set-up was the same as setup A.
system was sealed. The reaction mixture was stirred with Setup C: 2 mM (þ)AuNPs were synthesized as mentioned above
magnetic stirring at 30  C. Within 15 min, the initial golden in the EABR. The remaining set-up was the same as setup A.
yellow color changed to a ruby red color, which indicated the Setup D: 1 mM (þ)AuNPs were synthesized as mentioned
formation of gold nanoparticles. The reaction mixture was above. Instead of an EAB on stainless steel mesh, an EAB on
stirred for a further 15 min to complete and stabilize the re- carbon paper was challenged in the EABR. The remaining set-
action. Therefore, (þ)AuNPs were synthesized within 30 min. up was the same as in setup A.
Finally, the UVevisible spectrum was measured and an Setup E: In this case, the AuNPs were synthesized using the
absorbance maximum was observed at 535 nm (Fig. 1S), which well-known sodium citrate reduction method (Turkevich
is characteristic of (þ)AuNPs. Finally, the reaction mixture was method) and used in the EABR. The remaining set-up was the
centrifuged, and powdered (þ)AuNPs were isolated for further same as in setup A.
characterization. The synthesis of (þ)AuNPs at pH 4 was Setup F: 1 mM (þ)AuNPs were synthesized as setup A. Instead
chosen because it was the optimum condition. An attempt of an EAB on stainless steel mesh, only the stainless steel
was made to synthesize (þ)AuNPs at pH 4, 7 and 9 but pH 4 was mesh was challenged in the EABR. The remaining set-up was
found to provide better results than at pH 7 and 9, as indicated the same as in setup A.
by the progress of the reaction i.e. color of the reaction mixture Setup G: (þ)AuNPs were not used in this setup. The remaining
and UVevisible spectra of (þ)AuNPs. set-up was the same as in setup A.

2.5. Hydrogen production in Electrochemically Active In setups AeC only the concentration of (þ)AuNPs were
Biofilm Reactor (EABR) changed with the other conditions kept constant. Setups DeG
were controlled setups, where one or more parameters were
Batch experiments were conducted in a 250 mL EABR with a checked/altered in comparison to setups AeC. Table 1 shows
working volume of 200 mL (Fig. 3S). Seven different setups were that out of seven setups, only four setups (A, B, C and D)
arranged in the EABR. In each setup, the (þ)AuNPs were syn- showed hydrogen production. Of these, setup B showed the
thesized, as mentioned above using an EAB on a stainless steel maximum level of hydrogen production, which was
mesh under anaerobic conditions except for setup E, where the confirmed by GC and reconfirmed by the voltage obtained
AuNPs were synthesized using the well-known sodium citrate from the fuel cell. Fig. 2 shows the cyclic hydrogen production
reduction method [26]. In-situ hydrogen production was by setups A, B, C and D, whereas Fig. 5 shows the relative
observed using an EAB on a stainless steel mesh or carbon voltage obtained by the fuel cell after utilizing the hydrogen
paper, sodium acetate (a carbon source for bacteria) and (þ) produced by setups A, B, C and D, which was measured using a
AuNPs. Sodium acetate acts as the electron and proton donor multimeter. The hydrogen produced by different setups were
[25,27]. Table 1 shows representative results of independent first analyzed by GC and reconfirmed by the voltage obtained
experiments performed in triplicate for each setup as follows: by the fuel cell. In each case, the hydrogen produced and
voltage obtained were in good agreement with each other,
Setup A: 0.5 mM (þ)AuNPs were synthesized, as mentioned which further confirms the hydrogen production by the EAB in
above in the EABR. In-situ fresh EAB on a stainless steel mesh presence of the (þ)AuNPs and sodium acetate.
was challenged with 0.5 mM (þ)AuNPs and 0.2 g sodium ace-
tate under anaerobic conditions by sparging with nitrogen gas.
The EABR was sealed and left for magnetic stirring at 30  C. 3. Results and discussions
Initially, all the present gases (air) were removed from the
head-space using a syringe to ensure there was no oxygen or An EAB is a well-known biogenic system that provides elec-
other gases. The hydrogen gas produced was collected using a trons and protons by decomposing sodium acetate as a car-
glass syringe [20,28] (100 mL and 25 mL) every 24 h for analysis bon source [14,18,25,27]. Stainless steel is also well known for

Table 1 e Arrangements made in the EABR by varying the different parameters.

Parameters setup (þ)AuNPs EAB on stainless steel EAB on carbon paper No EAB only S. steel H2 produced

Setup A O0.5 mM O e e O
Setup B O1.0 mM O e e O
Setup C O2.0 mM O e e O
Setup D O1.0 mM e O e O
Setup E O1.0 mMa O e e e
Setup F O1.0 mM e e O e
Setup G e O e e e

a AuNPs were synthesized by well-known sodium citrate reduction method [26].

5246 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 3 8 ( 2 0 1 3 ) 5 2 4 3 e5 2 5 0

its reduction behavior via the corrosion of its surface by the 3.2. Hydrogen production in an Electrochemically Active
penetration of Cl ions, which provide electrons that can be Biofilm Reactor (EABR)
used for reduction purposes [23,24]. These two phenomena
were combined as an EAB on a stainless steel mesh to take A novel and simple approach was developed for hydrogen
advantage of both properties. After the successful formation production in an EABR using the EAB as an electron and pro-
of an EAB on the stainless steel mesh surface, it was ton donor by oxidizing acetate and (þ)AuNPs as a catalyst. The
exploited for the synthesis of gold nanoparticles and in-situ catalytic activities of the gold nanoparticles are well known
hydrogen production. The (þ)AuNPs were synthesized and [6,7,16,29]. In the present system, the EAB on a stainless steel
characterized, as reported earlier [22]. The magnitude of the mesh produces electrons and protons by decomposing so-
positive charge found on the (þ)AuNPs was þ32.72 mV. High dium acetate under anaerobic conditions in the EABR. The (þ)
resolution transmission electron microscopy (HRTEM) AuNPs catalyzed and combined these protons and electrons at
revealed the morphology of the as-synthesized (þ)AuNPs the charged surface to produce hydrogen gas [16]. The
(Fig. 1), which were 15e21 nm in size and mostly spherical. hydrogen gas produced was collected in the head-space of the
The electronic supplementary information (Figs. 1S and 2S) EABR, and sampled every 24 h using glass syringes with
shows the results of the remaining characterization tech- different capacities for detection and confirmation by GC and
nique’s results (UVevisible and X-ray diffraction). the fuel cell, respectively. After adding sodium acetate,
initially, an induction period of w24 h before hydrogen pro-
duction started was observed. 0.2 g of sodium acetate per
3.1. Measurement of hydrogen production and its 200 mL of reaction volume was consumed within five days, as
confirmation indicated by the decrease in hydrogen production [20,27].
From setups AeD, after adding sodium acetate again (second
Initially, the head-space of the EABR was made free of oxygen time), hydrogen production accelerated within 24 h, reached
and other gases by removing all the gases by a syringe fol- the maximum rate within 5e6 days, and then began
lowed by flushing with nitrogen. The hydrogen produced was decreasing over a 4e5 day period. This shows that acetate is
removed by glass syringes (100 mL and 25 mL) and measured by needed to produce electrons and protons, which are used for
releasing the gas pressure until the pressure in the syringe the formation of hydrogen gas.
had equilibrated with atmospheric pressure [20,28]. Initially, In the case of setups A, C and D, hydrogen production was
the gas produced was identified and confirmed by a very small observed but the amount was less than that of setup B (Figs. 2
amount of gas sample removed from the headspace using a and 3). The maximum rate of hydrogen production for setups
gastight micro syringe (100 mL), and analyzed by GC using ni- A, B, C and D were 55, 105, 60 and 45  2 mL/L day, respectively,
trogen as the carrier gas. Finally, the remaining gas was within 5e6 days, which again began decreasing over a 4e5 day
collected using another syringe (25 mL) until the back pressure period. For setups A, B and C, all experimental conditions were
was equilibrated with atmospheric pressure [20,28]. The gases the same except for the concentration of (þ)AuNPs. Setup B
collected were tested using a fuel cell, which was attached to a with 1 mM (þ)AuNPs showed the maximum hydrogen pro-
digital multimeter to record the voltage, to reconfirm duction compared to setups A and C with a 0.5 mM and 2 mM
hydrogen production. (þ)AuNPs concentration, respectively. This shows that 1 mM
(þ)AuNPs is the optimum concentration for maximum
hydrogen production. For setup D, the probable reason for the

Fig. 1 e HRTEM image of the as-synthesized 1 mM (D) Fig. 2 e Cyclic measurements of hydrogen production per
AuNPs by the EAB. The inset shows the histogram of the day, which depend on the (D)AuNPs concentration and
particle size distribution of the (D)AuNPs. sodium acetate consumption by the EAB in an EABR.
 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 3 8 ( 2 0 1 3 ) 5 2 4 3 e5 2 5 0 5247

Fig. 2 shows the cyclic hydrogen production, which de-

pends on the consumption of sodium acetate. Initially, when
sodium acetate was added, the rate of hydrogen production
accelerated reaching a maximum value of 11, 21, 12 and
9  2 mL/day for setups A, B, C and D, respectively, at 5e6 days.
These results show that the hydrogen production in setup B
was highest compared to setups A, C and D. As the substrate
was consumed, the level of hydrogen production began to
decrease, reaching its minimum value, as shown in Fig. 2.
After adding the substrate again, it re-gained its hydrogen
production behavior in a cyclic manner for setups A, B C and
D. Feeding of the EABR with sodium acetate was kept constant
i.e. 0.2 g/200 mL (1 g/L) [20,27]. Instead, the concentration of (þ)
AuNPs was varied to observe its catalytic effect.
Fig. 3 shows the level of hydrogen productivity measured in
mL of H2 per liter volume of the (þ)AuNPs solution in the
reactor per day (mL/L day) [21]. The maximum hydrogen
Fig. 3 e Hydrogen productivity per day (for one cycle) by the production was w105  2 mL/L day (0.105 m3/m3 day) for setup
EAB in an EABR. B on the fifth day and w45  2 mL/L day (0.045 m3/m3 day) for
setup D on the sixth day. This shows that setup B is more
efficient than setups A, C and D. For setup B, which showed
the maximum hydrogen production, the average H2 produc-
lower amount of hydrogen production might be the EAB, which tion rate (Q) was w0.06 m3/m3 day with an overall H2 yield
was made on carbon paper. The EAB on carbon paper probably ðYH2 ;th ð%ÞÞ of w44%, which is shown in Table 2 and compared
provides fewer electrons compared to the EAB on stainless with other reports [20].
steel mesh. This is because in the case of EAB on carbon paper, Fig. 4 shows the cumulative hydrogen production for
there was no Cl ion penetration, which results fewer elec- setups A, B, C and D. The cumulative H2 production for setups
trons. This could be a limiting factor and is probably the reason A, B, C and D was 53, 102, 61, and 44  2 mL for 10 days (1 cycle)
for lower amount of hydrogen production in setup D. for a 200 mL reactor volume of (þ)AuNPs. This suggests that
Another important and interesting observation was that the overall performance of setup B is much better than that of
in case of setup E, the AuNPs were synthesized using the the setups A, C and D.
citrate reduction method (Turkevich method), resulting in Fig. 5 shows the maximum voltage obtained from the fuel
negatively charged AuNPs [26] and no hydrogen production. cell, which was connected to a multimeter, for the hydrogen
This shows that negatively charged AuNPs cannot catalyze produced each day. The maximum voltage observed for
the production of hydrogen. This suggests that positively setups A, B, C and D were 8.5, 16.5, 9.13 and 6.8 mV, respec-
charged AuNPs facilitate hydrogen production by providing a tively, on the fifthesixth days. In the initial 5e6 days, the
charged surface. voltage increased and then began decreasing as the substrate
In setup G, in absences of (þ)AuNPs hydrogen production was consumed. The voltage obtained by the fuel cell corre-
was not observed though EAB on stainless steel mesh and sponded to the hydrogen produced on each day in the EABR.
acetate were present. This shows that EAB alone cannot Each setup showed similar behavior but the magnitude of the
produce hydrogen and suggests that (þ)AuNPs are required voltage produced in setup B was approximately 2, 1.8 and 2.5
for the hydrogen production and confirms catalytic role of times that of setups A, C and D, respectively. This confirms
(þ)AuNPs in hydrogen production. that setup B is better than setups A, C and D. This further

Table 2 e Comparative performance of these results and MECs reported in other studies.
Study Total reactor Substrate Applied H2 production rate Overall H2 Energy input
liquid volume (L) voltage (V) (Q)(m3/m3 day) yield, YH2 ;th ð%Þ (kW h/m3)

Our resultsa 0.2 Acetate 0.0b 0.06 44 0

Liu et al. 0.03 Acetate 0.45 0.37 61 1.0
Rozendall et al. 6.6 Acetate 0.5 0.02 53 1.9
Cheng & Logan 0.04 Acetate 0.6 1.1 88 1.3
Hu et al. 0.3 Acetate 0.6 0.69 64 1.4
Call and Logan 0.03 Acetate 0.8 3.12 93 1.7
Rozendal et al. 3.3 Acetate 1.0 0.3 23 2.2
Ditzig et al. 0.58 Wastewater 0.58 0.01 9.8 2.5

a Results of this experiment (for setup B).

b The external applied voltage was zero except for the energy required for stirring and temperature control.
5248 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 3 8 ( 2 0 1 3 ) 5 2 4 3 e5 2 5 0

Fig. 6 e Proposed mechanism for the simultaneous

synthesis of (D)AuNPs and hydrogen production by the
EAB, which is catalyzed by the as-synthesized (D)AuNPs.

100% [20]. On a molar basis, the value of nth equals the moles
of substrate converted ns multiplied by the stoichiometric
production of hydrogen from 1 mol of substrate. Although the
Fig. 4 e Cumulative hydrogen production (for one cycle) by
H2 production rate was comparatively lower, the fact that
an EAB in an EABR.
there was no energy input (except for magnetic stirring and
temperature control) makes the present system more efficient
compared to other reported systems [20,21].
confirms the optimum conditions for hydrogen production in
EABR. The probable reason for the better performance of setup 3.3. Mechanism of hydrogen production
B could be the optimum concentration of (þ)AuNPs (1 mM)
and EAB, which was formed on the stainless steel mesh that The proposed mechanism (Fig. 6) showed the overall process
provides an excess of electrons [23,24]. These excess of elec- for the successful synthesis of (þ)AuNPs followed by in-situ
trons assists in charging the (þ)AuNPs and reducing the pro- hydrogen production by EAB and (þ)AuNPs. The (þ)AuNPs
tons at the charged surface of the (þ)AuNPs [16]. catalyze this reaction by providing its charged surface where
As per Logan et al.’s report [20], Table 2 shows the the protons and electrons can combine and form hydrogen
comparative performance of hydrogen production for these gas [16,29,30]. The positively charged gold nanoparticles act as
results and the MECs reported in other studies. electropositive electron acceptors and are charged by the
Table 2 shows the comparative data for hydrogen pro- electrons produced by the EAB. The catalytic activity of the (þ)
duction in this system and other studies reported in different AuNPs could be increased by increasing the electron density
systems. These results show that even without energy input, on the gold nanoparticles [31].
the overall H2 yield ðYH2 ;th Þ reached 44% with the reactor liquid The electrons produced by the EAB were adsorbed at the
volume (0.2 L). The hydrogen yield for a specific substrate can surface of the as-synthesized (þ)AuNPs, which makes it
be compared with the theoretical maximum production (nth), charged [16,30e32]. At the charged surface of the (þ)AuNPs,
which is normally reported as a percentage, YH2 ;th ¼ ðnH2 =nth Þ electrons and protons were adsorbed, which assists in the
formation of molecular hydrogen according to the Volmer-
Heyrovsky mechanism, as shown by Reactions (1) and (2)
[29,30]. According to Volmer-Heyrovsky mechanism, protons
are adsorbed on the charged surface of the gold nanoparticles
followed by the instant reduction of a second proton at the
same place and the release of molecular hydrogen [29]. Each
electron captured by the gold nanoparticles should attract a
proton to its surface. This highlights the unique proton cata-
lytic redox chemistry of gold nanoparticles [29,30,33].

Au þ Hþ þ e
 / AueH (1)

AueH þ Hþ þ e
 / AueH2 (2)

Compared to other reports for hydrogen production by the

fermentation of organic matter or acetate, such reactions
generally takes place at approximately 25  C, pressure 1 bar
Fig. 5 e Maximum voltage obtained (for one cycle) after and pH 7 [20,34]. Zhang et al. [19] reported that hydrogen
passing the hydrogen produced into the fuel cell, as production, which was improved and enhanced by the AuNPs,
measured using a digital multimeter. was fermentative hydrogen production that took place at
 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 3 8 ( 2 0 1 3 ) 5 2 4 3 e5 2 5 0 5249

pH 7.2 and 35  C. On the other hand, in the present system, the references
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