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Anaerobic Biohydrogen Production by the Mixed Culture with
Mesoporous Fe3O4 Nanoparticles Activation
Wei Zhao1,a, Jie Zhao1,b, Guodong Chen1,c, Rui Feng1,d, Jian Yang1,e, Yanfang Zhao2,f, Qin Wei2,g, Bin Du*1,2,h and Yongfang Zhang*1,i
1School of Resources and Environment, University of Jinan (UJN), Jinan 250022, P R China
2School of Chemistry and Chemical Engineering, University of Jinan (UJN), Jinan 250022, P R China
[email protected], *correspondence author,
[email protected],*correspondence author
Keywords: anaerobic fermentation, biohydrogen, mesoporous Fe3O4 nanoparticle, alkaline shock.
Abstract. It was the first time to study the catalytic effect of mesoporous magnetic Fe3O4
nanoparticles on the biohydrogen production. The mixed culture used in this study just suffered from
an alkaline shock and lost its bioactivity of hydrogen production. We use mesoporous Fe3O4
nanoparticles and ferrous ions as activators to recover the bioactivity of the mixed culture. The results
indicate that the improvement of biohydrogen yield by mesoporous Fe3O4 was obvious larger than
that by ferrous ions. The maximum yield of cumulative hydrogen production was obtained at the
mesoporous Fe3O4 nanoparticles of 400 mg·L-1
, which is 26% higher than that of the blank. The lag
phases for hydrogen production in the tests added with mesoporous Fe3O4 nanoparticles were
decreased to 12 h, which are 50 h less than those of the corresponding ferrous ions and blank tests.
Introduction
The recent rise in oil and natural gas prices may drive the current economy towards alternative energy
sources. Hydrogen offers tremendous potential as a clean, renewable energy currency for the future
[1]. Biological production of hydrogen, using microorganisms, stands out as an environmentally
harmless process under mild operating conditions because of low-cost, environmentally benign and
resource renewable [2]. In spite of these advantages, satisfactory stability for the hydrogen production
in the practical applicability has not been obtained. Much work should be done on how to enhance the
bioactivity of the microorganisms and to make hydrogen production stable over a long period.
Nanoparticles are of great scientific interest as they are effectively a bridge between bulk materials
and atomic or molecular structures [3]. Magnetic nanoparticles, especially, nanoparticles of iron
oxide such as Fe3O4 and γ -Fe2O3 have been among the most widely used nanoparticle in science. One
highly useful application is to remove many toxic metal ions from wastewater by adsorption and
redox reaction [4,5]. Moreover, it is rather attractive to investigate the effect of Fe3O4 nanoparticle on
the dark fermentation, because it has been reported that the in vivo activity of the hydrogenase
decreases with external iron depletion [6,7]. The mixed anaerobic culture used in this study was
shocked by high strength of alkaline influent (10 g·L-1
sodium carbonate), and lost the bioactivity of
hydrogen production. The purpose of this work was to investigate the recovery effect of mesoporous
Fe3O4 nanoparticles in small batch reactors for hydrogen production.
Experimental Section
Reagents. FeCl3·6H2O, FeSO4·7H2O, ethylenediamine were purchased from Shanghai Chemical
Reagent Co. China. Glucose, ethylene glycol, sodium acetate, Na2CO3, NH4Cl, K2HPO4·3H2O,
CuSO4·5H2O, MgCl2·6H2O, MnSO4·4H2O, FeSO4·7H2O, and CoCl2·6H2O were purchased from
Beijing Yili Fine Chemical Co., Ltd. China. Unless noted otherwise, reagents and solvents were
analytical pure and used as purchased without further purification. Doubly distilled, deionized water
was used for preparation of all aqueous solutions.
Advanced Materials Research Vols. 306-307 (2011) pp 1528-1531Online available since 2011/Aug/16 at www.scientific.net© (2011) Trans Tech Publications, Switzerlanddoi:10.4028/www.scientific.net/AMR.306-307.1528
All rights reserved. No part of contents of this paper may be reproduced or transmitted in any form or by any means without the written permission of TTP,www.ttp.net. (ID: 132.174.255.116, University of Pittsburgh, Pittsburgh, United States of America-10/07/14,14:52:08)
Experimental procedures. In a typical synthesis of monodisperse Fe3O4 nanoparticles with
mesoporous structure [8], FeCl3·6H2O (1 g) was dissolved in ethylene glycol (27.5 mL), followed by
the addition of sodium acetate (3 g) and ethylenediamine (10 mL). The mixture was stirred vigorously
for 30 min and sealed in a teflon lined stainless-steel autoclave, which was heated at 200 °C for 8 h
and then cooled to room temperature. Fig. 1 shows SEM and TEM micrographs of the used
mesoporous Fe3O4 nanoparticles whose average diameters were approximately 40 nm.
Fig. 1 SEM (A) and TEM (B) micrographs of mesoporous Fe3O4 nanoparticles.
Batch experiments were conducted in 120-mL vials with a working volume of 80 mL, which
included 30 mL inoculum, 40 mL nutrient solution [9] and 10 mL activator solution ( FeSO4 solution
ranging from 0 to 400 mg·L−1
and mesoporous Fe3O4 nanoparticles in the same moles of iron element
with the the contrasted FeSO4 test). The initial anaerobic condition was established with nitrogen gas
sparging for 5 minutes. The initial pH of the medium was adjusted to 7.0 and operation temperature of
the reactor was controlled at 35 °C. The bottles were placed in a reciprocal shaker (Reciprocation: 5
cm×120 strokes per min). The biogas production was measured by glass syringes, arranging from
10-100 mL [10]. Data shown are average results of independent experiments that were duplicated.
Chemical analysis. The microstructure of Fe3O4 nanoparticles was determined with TEM (Hitachi
H-800 microscope, Japan) and SEM (JEOL JSM-6700F microscope, Japan). The proportion of
hydrogen was determined with a gas chromatograph (GC9790, FuLi Analytical Instrument, China)
with a thermal conductivity detector (TCD) and a 2-m stainless iron column packed with GDX-102
(60/80 mesh). The operating temperatures of the injection port, the oven, and the detector were set at
80, 50 and 100 °C, respectively. Nitrogen gas was used as the carrier gas at a flow rate of 30 mL·min-1
.
The concentrations of volatile suspended solids (VSS) were determined according to the procedures
described in the standard methods [11]. The pH was measured using a pH meter (PHS-3B Shanghai,
China).
Results and Discussion
The mixed cultures used in this study were enriched from the aeration basin of local municipal
wastewater treatment plant, dominated by Clostridium butyricum. Product analysis shows that the
anaerobic fermentation produced a biogas only containing hydrogen and carbon dioxide. Fig. 2
illustrates the influences of different concentration of two activators on hydrogen production at
different fermentation period. For the blank system, the evolution of hydrogen was not observed until
32 h after the fermentation began, which indicated that the bioactivity of anaerobic bacteria after
alkaline-shock could not be recovered without the assistance of activator. As shown in Fig. 2A, for all
the tests the mixed culture started hydrogen production almost at the beginning of the fermentation,
which indicated that Fe3O4 nanoparticles played an important role in the bioactivity recovery of
hydrogen-producing bacteria. With the activator concentration increased, the total yields of hydrogen
were obviously increased, and the maximum yield of hydrogen production reached 83.6 mL (System
A) at the concentration of 400 mg·L-1
.
It is well-known that iron is an indispensable element for bacteria multiplication. Undoubtedly, an
appropriate amount of iron is beneficial to increasing the activity of bacteria. However, as shown in
Fig. 2B, the mixed culture started hydrogen production at 32 h after the incubation and the lag phases
Advanced Materials Research Vols. 306-307 1529
were as long as that of the blank system for all the tests. With the concentration of this activator
increased, the total yields of hydrogen were also increased gradually, but lower than the yield of the
blank system. That is, in the concentration range in this work, ferrous ions could not promote the
bioactivity recovery, unlikely the data we obtained before [12].
0 20 40 60 80 100 120
0
20
40
60
80A: Mesoporous Fe3O4
Cumulative H
ydrogen Yield (mL)
Fermentation Time (h)
Blank
50 mgL-1
100mgL-1
200mgL-1
400mgL-1
0 20 40 60 80 100 120
0
20
40
60
80
Blank
50 mgL-1
100mgL-1
200mgL-1
400mgL-1
B: FeSO4
Cumulative H
ydrogen Yoeld (mL)
Fermentation Time (h)
Fig.2 Cumulative hydrogen yield produced in batch tests with different activator concentrations.
Table 1 shows the comparison of gas products evolution of the two systems. The percentage of
hydrogen and carbon dioxide in the biogas produced was slightly affected by the activator
concentration. The percent of hydrogen ranged from 32.8% to 43.8% and that of carbon dioxide
ranged from 56.2% to 67.2%. The hydrogen percent reached the maximum (43.8%) at the Fe3O4
nanoparticles concentration of 400 mg·L-1
.
Table 1 Comparison of hydrogen production data using different activators. System Concentration
(mg·L-1)
Biogas
(mL)
H2 aConversion
efficiency(%)
bDBW
(gVSS· g-1glucose) (mL) (%) mol· mol-1glucose
Blank 0 168 66.4 39.5 1.21 30.3 0.219
A 50 186 61.8 33.2 1.13 28.2 0.247
100 185 66.6 36.0 1.22 30.4 0.244
200 190 71.2 37.5 1.30 32.5 0.225
400 191 83.6 43.8 1.53 38.2 0.292
B 50 186 61.1 32.8 1.12 27.9 0.309
100 175 59.1 33.8 1.08 27.0 0.293
200 173 58.1 33.6 1.06 26.5 0.289
400 189 63.8 33.8 1.17 29.1 0.315 aSubstrate conversion efficiencies: we assume production of acetate as the organic carbon end product in order to calculate
the indicated maximum production of hydrogen for glucose. bDBW: The dry biomass weight (DBW) is the biomass production yield of 80 mL liquid reagent in each bottle of the tests.
Hydrogen conversion efficiencies for carbohydrates were usually calculated based on the
assumption of a maximum of 4 mole of hydrogen per mole of glucose and 8 mole of hydrogen per
mole of sucrose, assuming a maximum stoichiometric conversion of the substrate to hydrogen and
acetate. As shown in Table 1, the maximum conversion efficiencies of glucose to hydrogen for the
two systems (System A and B) were 38.2 % and 29.1 %, respectively, both in the tests of 400 mg·L-1
.
These results proved again appropriate addition of Fe3O4 nanoparticles would activate the shocked
culture efficiently. It is well-known that iron is an indispensable element for the bacterial growth and
multiplication. The maximum dry biomass weight (DBW) was 0.315 gVSS·g−1
glucose at the ferrous
ion concentration of 400 mg·L-1
(shown in Table 1). From the distributions of the dry biomass weight
and the hydrogen production yield, we concluded that the quantities of the bacteria were not
consistent with the hydrogen production yield. Namely, the addition of ferrous ions in this study only
affected the cultivation of the mixed culture and has little effect on the hydrogen-producing activity of
1530 Emerging Focus on Advanced Materials
the bacteria. Although the ultimate hydrogen yield was relatively lower than those of previous studies
we did [9,12], the alkline-shocked culture in System A made a quick response to the addition of Fe3O4
nanoparticles that the biogas production increased obviously compared with the blank. The net
increase of the yields compared with the blank test were 25.9 % corresponding to 400 mg·L-1
Fe3O4
nanoparticles.
Conclusion
In conclusion, the tests using the Fe3O4 nanoparticles as activators behaved better than the blank
systems and the yield and percentage of hydrogen increased sharply. The maximum yield (1.53
mol/mol glucose) and percent (43.8 %) of hydrogen were obtained at System A with 400 mg·L-1
Fe3O4 nanoparticles. These results indicated that Fe3O4 nanoparticles could be used as activators
when the bioactivity of hydrogen-producing bacteria was inhibited. A better understanding of the
catalytic mechanism does require more experimental and theoretical work, such as genetic
engineering, spectroscopic studies of the hydrogen-producing enzymes frozen in various states. Some
work in these directions is already in progress and will be communicated in due course.
Acknowledgements
We sincerely express our thanks to the Doctor Foundation of Shandong Province (BS2010NJ002), the
Natural Science Foundation of China (No. 21075052), the Natural Science Foundation of Shandong
Province (No. ZR2010BM030, ZR2010ZR063), the Science and Technology Key Plan Project of
Shandong Province (No. 2010GSF10628), Special Research and Development Environmental
Protection Industry of Shandong Province, National Major Projects on Water Pollution Control and
Management Technology (No. 2008ZX07422) and the Science and Technology Development Plan
Project of Jinan City (No. 201004015) for the financial supports.
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Advanced Materials Research Vols. 306-307 1531
Emerging Focus on Advanced Materials 10.4028/www.scientific.net/AMR.306-307 Anaerobic Biohydrogen Production by the Mixed Culture with Mesoporous Fe3O4 Nanoparticles
Activation 10.4028/www.scientific.net/AMR.306-307.1528
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