Beruflich Dokumente
Kultur Dokumente
Abstract
The eect of pH on the conversion of glucose to hydrogen by a mixed culture of fermentative bacteria was evaluated. At 36 C,
six hours hydraulic retention, over 90% of glucose was degraded at pH ranging 4.07.0, producing biogas and an euent comprising
mostly fatty acids. At the optimal pH of 5.5, the biogas comprised 64 2% of hydrogen with a yield of 2:1 0:1 mol-H2 /molglucose and a specic production rate of 4:6 0:4 l-H2 /(g-VSS day). The euent was composed of acetate (15.334.1%) and
butyrate (31.245.6%), plus smaller quantities of other volatile fatty acids and alcohols. The diversity of microbial communities
increased with pH, based on 16S rDNA analysis by denaturing gradient gel electrophoresis (DGGE). 2002 Published by Elsevier
Science Ltd.
Keywords: Acidication; Anaerobic; Fermentation; Glucose; Hydrogen; pH
1. Introduction
Organic pollutants are converted into methane in the
conventional anaerobic treatment of wastewater (Hulsho Pol and Lettinga, 1986; Fang and Liu, 2000) and
solid wastes (Iglesias et al., 1998, 2000). The process can
be divided into two distinct stages: acidication and
methane production. Each stage is carried out by a
number of microorganisms through syntrophic interactions. Acidication produces hydrogen as a by-product,
which in turn is used as an electron donor by many
methanogens at the second stage of the process. However, hydrogen itself is of high commercial value. It can
be used as a raw material in a variety of industrial applications (Kirk et al., 1985), as well as a clean energy
source for fuel cells (Hart, 1997). It might be feasible to
harvest hydrogen at the acidication stage of anaerobic
treatment, leaving the remaining acidication products
for further methanogenic treatment (Mizuno et al.,
2000a).
Microorganisms are capable of producing hydrogen
via either fermentation (Fumiaki et al., 1996; Yokoi et al.,
1997) or photosynthesis (Lichtl et al., 1997; Hansel and
88
2. Methods
2.1. Seed sludge
The seed sludge of mixed culture was taken from a
continuous stirred tank reactor, which produced hydrogen by fermentation of sucrose at 36 C and pH
5:0 0:5. The reactor, which had been operated for six
months, contained 1.0 g-VSS/l and produced a biogas
comprising 50% hydrogen. Degrading each gram of
sucrose produced 0.15 l of hydrogen.
2.2. Experiments of hydrogen production
Hydrogen production experiments were conducted in
a three liter fermentor (Biostat B, B. Braun Biotech), as
illustrated in Fig. 1, at 36 C and 6 h hydraulic retention.
The feed solution was composed of 7000 mg/l of glucose,
plus the following nutrients (in mg/l): NaHCO3 1000;
NH4 Cl 500; KH2 PO4 250; K2 HPO4 250; MgSO4 7H2 O
320; FeCl3 50; NiSO4 32; CaCl2 50; Na2 BO7 H2 O 7;
NH4 6 Mo7 O24 4H2 O 14; ZnCl2 23; CoCl 6H2 O 21;
CuCl2 2H2 O 10; MnCl2 4H2 O 30.
The fermentor was stirred at a constant 200 rpm to
ensure a thorough mixing and to facilitate rapid diusion of hydrogen. However, the eect of mixing was not
studied. The pH of the mixed liquor was controlled
automatically by feeding NaOH (6 M) and HCl (4 M)
solutions via respective peristaltic pumps. A level probe
was used to control the mixed liquor volume at 1.7 l.
The fermentor was kept in the dark by wrapping with
aluminium foil to prevent the growth of photosynthetic
bacteria. The pH of the mixed liquor in the fermentor
was adjusted stepwise from 4.0 to 7.0 with 0.5 increments. At each pH level, steady state was reached within
14 days, judging from the constant glucose degradation,
hydrogen production, and euent quality. Five sets of
thorough analyses were conducted on the reactor performance during days 1421, before increasing the pH
to the next level.
An additional 50 ml of the original seed sludge was
added into the fermentor each time that the pH was
adjusted.
2.3. Analyses
The amount of biogas produced was recorded daily
using the water displacement method. The contents of
hydrogen, carbon dioxide and methane in the biogas
were analyzed by a gas chromatograph (GC) (Hewlett
Packard 5890 II) equipped with a thermal conductivity
detector and a 2 m 2 mm (inside diameter) stainlesssteel column packed with Porapak N (80100 mesh).
Injector, detector and column temperatures were kept at
57, 180 and 50 C, respectively.
The concentrations of volatile fatty acids (VFA) and
alcohols in the euent were analyzed by a second GC of
the same model equipped with a ame ionization detector and a 10 m 0:53 mm HP-FFAP fused-silica
capillary column. The VFA analyzed included acetate,
propionate, butyrate, i-butyrate, valerate, i-valerate and
caproate, whereas alcohols included methanol, ethanol,
propanol and butanol. Euent samples were rst ltered through a 0.2 lm membrane, acidied by formic
acid and measured for free acids. The initial temperature
of the column was 70 C for 3 min followed with a ramp
of 10 C/min and a nal temperature of 180 C for 4.5
min. The temperatures of the injector and detector were
200 and 250 C, respectively. Helium was used as the
carrier gas at a ow rate of 25 ml/min.
A total organic carbon (TOC) analyzer (TOC-5000A,
Shimazu) was used to measure the organic content in the
feed solution and the euent. The concentration of
glucose in both inuent and euent was measured using
the phenolsulfuric acid method (Herbert et al., 1971).
The biomass was measured by volatile suspended solid
(VSS) according to the Standard Methods (APHA,
1992).
The morphology of hydrogen-producing bacteria in
this study was analyzed using a scanning electron microscope (SEM) (Cambridge Stereoscan 360). A 50 ll
sludge sample was diluted to 50 ml with a 0.1 M, pH 7.2
phosphate-buer solution. About 25 ml of the diluted
sample was ltered through a 0.2 lm nucleopore membrane. The sludge on the membrane was then xed in
the phosphate-buer solution with 2.5% glutaraldehyde
for 2 h. The xed sample was dehydrated stepwise in a
graded series of water/ethanol solutions, and criticalpoint dried with carbon dioxide (Fang et al., 1994).
89
(a)
(b)
(c)
(d)
90
Table 1
Comparison of hydrogen yield using glucose as substrate
Microorganism
pH
Mixed culture
Mixed culture
Mixed culture
E. aerogenes
E. cloacae
C. butyricum
a
5.5
5.7
Unspecied
5.56.0
5.06.0
6.7
Reference
This study
Lin and Chang (1999)
Roychowdhury et al. (1988)
Tanisho et al. (1989)
Kumar and Das (2000)
Kataoka et al. (1997)
2:1 0:1
1.7
0.7
1.0
2.2
1.42.3
Mean S.D. n 5.
1
2
Table 2
Product distribution (on carbon basis) in euent
pH
TOC (C-mg/l)
Glucose (%)
Butyrate (%)
Acetate (%)
Ethanol (%)
Lactate (%)
Caproate (%)
Propionate (%)
4.0
4.5
5.0
5.5
6.0
6.5
7.0
1312
1452
1356
1337
1317
1487
1281
20.7
7.2
6.8
1.5
1.1
1.4
2.7
41.4
45.6
38.3
35.1
32.4
31.5
31.2
15.3
23.1
23.4
29.2
29.5
33.1
34.1
6.5
6.9
9.0
9.8
10.1
5.9
4.6
2.4
3.1
3.8
4.1
4.6
2.8
2.0
1.4
4.0
5.8
2.4
0.5
0.8
2.5
1.2
1.0
0.9
1.2
2.9
4.9
15.9
91
Table 3
Carbon balance for each liter of inuent
pH
Inuent
Euent
Biogas
TOC (mg)
IC (mg)
TOC (mg)
IC (mg)
CO2 (mg)
CH4 (mg)
4.0
4.5
5.0
5.5
6.0
6.5
7.0
2816
2834
2803
2846
2867
2859
2866
143
152
150
170
151
150
154
1312
1451
1355
1336
1317
1486
1281
170
136
137
113
181
330
827
753
687
632
479
516
384
257
0
0
0
0
37
39
38
Biomass (mg)
Recovery (%)
522
615
705
785
795
815
835
93.1
96.8
95.8
90.0
94.3
101.5
107.2
of H2 CO3 and HCO3 . The carbon content in the biomass was calculated assuming the composition of
C5 H7 O2 N. Table 3 shows that the overall carbon balance was 90.0107%.
Table 3 also shows that the sludge yield increased
with pH. Degrading one gram of glucose produced 0.15
g of VSS at pH 4.0, 0.21 g VSS at the optimal pH of 5.5
and 0.22 g VSS at pH 7.0.
3.4. Microbiology
Sludges sampled at pH 4.05.5 were creamy white,
and the color was darkened with the increase of pH, due
to the increased suldogenic activity. At pH > 6:0, sulfate-reducing bacteria converted the sulfate into sulde
which reacted with metals forming dark precipitates.
Fig. 3 illustrates that hydrogen-producing bacteria at (a)
pH 4.5 and (b) pH 5.5 were mostly composed of bacilli
of various lengths, plus some diplobacilli and streptobacilli.
Fig. 4 illustrates the DGGE proles of the 16S rDNA
gene fragment amplied from the sludges sampled from
pH 4.0 to 7.0. Each band on the DGGE prole corre-
92
Acknowledgements
The authors wish to thank the Hong Kong Research
Grant Council for the nancial support of this project
(HKU7004/98E), and Mr. Tong Zhang for his technical
assistance.
References
APHA, AWWA, WEF, 1992. Standard Methods for the Examination
of Water and Wastewater. 18th ed. American Public Health
Association, Washington, DC (Chapter 2).
Dabrock, B., Bahl, H., Gottschalk, G., 1992. Parameters aecting
solvent production by Clostridium pasteurianum. Appl. Environ.
Microbiol. 58 (4), 12331239.
Fang, H.H.P., Chui, H.K., Li, Y.Y., 1994. Microbial structure and
activity of UASB granules treating dierent wastewaters. Water
Sci. Technol. 30, 8796.
Fang, H.H.P., Liu, Y., 2000. Anaerobic wastewater treatment in (sub-)
tropical regions. In: Proceedings of the Symposium on Establishment and Evaluation of Advanced Water Treatment Technology
Systems Using Functions of Complex Microbial Community,
Tokyo, March 68, Japan, pp. 109118.
Ferris, M.J., Muyzer, G., Ward, D.M., 1996. Denaturing gradient gel
electrophoresis proles of 16S rRNA-dened populations inhabiting a hot spring microbial mat community. Appl. Environ.
Microbiol. 62, 340346.
Fumiaki, T., Chang, J.D., Mizukami, N., Tatsuo, S.T., Katsushige, H.,
1993. Isolation of a hydrogen-producing bacterium Clostridium
beijerinckii strain AM21B, from termites. Can. J. Microbiol. 39,
726730.
Fumiaki, T., Chang, J.D., Mizukami, N., Tatsuo, S.T., Katsushige, H.,
1996. Continuous hydrogen production by Clostridium sp. Strain
No. 2 from cellulose hydrolysate in an aqueous two-phase system.
J. Ferment. Bioeng. 82 (1), 8083.
Hansel, A., Lindblad, P., 1998. Towards optimization of Cyanobacteria as biotechnological relevant producers of molecular hydrogen
a clean and renewable energy source. Appl. Microbiol. Biotechnol.
50, 153160.
Hart, D., 1997. Hydrogen Power: The Commercial Future of the
Ultimate Fuel. Financial Times Energy Publishing, London.
Herbert, D., Philipps, P.J., Strange, R.E., 1971. Carbohydrate analysis. Methods Enzymol. 5B, 265277.
93
transitions, sequence variations, and protein-nucleic acid interactions. Electrophoresis 10, 377389.
Roychowdhury, S., Cox, D., Levandowsky, M., 1988. Production of
hydrogen by microbial fermentation. Int. J. Hydrogen Energy 13,
407410.
Tanisho, S., Kamiya, N., Wakao, N., 1989. Hydrogen evolution of
Enterobacter aerogenes depending on culture pH: mechanism of
hydrogen evolution from NADH by means of membrane-bound
hydrogenase. Biochim. Biophys. Acta 973, 16.
Ueno, Y., Kawai, T., Sato, S., Otsuka, S., Morimoto, M., 1995.
Biological production of hydrogen from cellulose by mixed
anaerobic microora. J. Ferment Bioeng. 79 (4), 395397.
Ueno, Y., Sato, S., Morimoto, M., 1996. Hydrogen production from
industrial wastewater by anaerobic microora in chemostate
culture. J. Ferment. Bioeng. 82 (2), 194197.
Yokoi, H., Tokushige, T., Hirose, J., Hayashi, S., Takasaki, Y., 1997.
Hydrogen production by immobilized cells of aciduric Enterobacter
aerogenes strain HO-39. J. Ferment. Bioeng. 83, 484491.
Zhang, T., Fang, H.H.P., 2000. Digitization of DGGE (denaturing
gradient gel electrophoresis) prole and cluster analysis of microbial communities. Biotechnol. Lett. 22, 399405.