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Bioresource Technology 82 (2002) 87±93

E€ect of pH on hydrogen production from glucose
by a mixed culture
Herbert H.P. Fang *, Hong Liu
Centre for Environmental Engineering Research, Department of Civil Engineering, University of Hong Kong, Pokfulam Road, Hong Kong
Received 6 March 2001; received in revised form 15 June 2001; accepted 7 July 2001

The e€ect 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 4.0±7.0, producing biogas and an e‚uent 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 speci®c production rate of 4:6  0:4 l-H2 /(g-VSS day). The e‚uent was composed of acetate (15.3±34.1%) and
butyrate (31.2±45.6%), 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). Ó 2002 Published by Elsevier
Science Ltd.
Keywords: Acidi®cation; 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: acidi®cation and
methane production. Each stage is carried out by a
number of microorganisms through syntrophic interactions. Acidi®cation 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 acidi®cation stage of anaerobic
treatment, leaving the remaining acidi®cation products
for further methanogenic treatment (Mizuno et al.,
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


Corresponding author. Tel.: +852-2859-2660; fax: +852-2559-5337.
E-mail address: (H.H.P. Fang).

Lindblad, 1998; Matsunaga et al., 2000). The former is
generally preferred, because it does not rely on the
availability of light sources and the transparency of the
mixed liquor (Hart, 1997). Production of hydrogen by
fermentation has been studied for a large group of pure
fermentative bacteria, such as Clostridia (Heyndrickx
et al., 1991; Fumiaki et al., 1993) and Enterobacteria
(Rachman et al., 1997, 1998; Kumar and Das, 2000).
However, studies of hydrogen production by mixed
cultures have attracted research attention only recently.
Batch experiments have been conducted to produce
hydrogen from solid wastes (Lay et al., 1999; Mizuno
et al., 2000b) and cellulose wastewater (Ueno et al.,
1995; Lay and Noike, 1999), and continuous experiments from glucose (Nakamura et al., 1993; Majizat
et al., 1997) and sugary wastewater (Ueno et al., 1996).
Results so far indicated that the control of pH is crucial
to the hydrogen production, due to the e€ects of pH on
the hydrogenase activity (Dabrock et al., 1992) and/or
on the metabolism pathways (Lay, 2000). But, the
reported optimal pH value for hydrogen production is
con¯icting, varying from pH 9.0 for batch fermentation
of sucrose (Lee et al., 1999) to pH 4.0±4.5 and pH 4.7±
5.7, respectively, for the continuous fermentation of
sucrose (Ren et al., 1995) and starch (Lay, 2000).
This study was thus conducted to investigate the
e€ects of pH on the continuous production of hydrogen

0960-8524/02/$ - see front matter Ó 2002 Published by Elsevier Science Ltd.
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5 increments. i-valerate and caproate. Shimazu) was used to measure the organic content in the feed solution and the e‚uent. Liu / Bioresource Technology 82 (2002) 87±93 by mixed culture in a fermentor and on the responsible microbial communities. The morphology of hydrogen-producing bacteria in this study was analyzed using a scanning electron microscope (SEM) (Cambridge Stereoscan 360). plus the following nutrients (in mg/l): NaHCO3 1000. The feed solution was composed of 7000 mg/l of glucose. ZnCl2 23.2 phosphate-bu€er solution. 1992). valerate. Seed sludge The seed sludge of mixed culture was taken from a continuous stirred tank reactor.. propanol and butanol.88 H. The contents of hydrogen. and e‚uent quality. 1994).5% glutaraldehyde for 2 h. NiSO4 32.15 l of hydrogen. Fang. which had been operated for six months. 2. i-butyrate. Braun Biotech). ethanol. A 50 ll sludge sample was diluted to 50 ml with a 0.2. KH2 PO4 250. MgSO4  7H2 O 320. …NH4 †6 Mo7 O24  4H2 O 14. A total organic carbon (TOC) analyzer (TOC-5000A. before increasing the pH to the next level. 1. steady state was reached within 14 days. acidi®ed by formic acid and measured for free acids. The concentration of glucose in both in¯uent and e‚uent was measured using the phenol±sulfuric acid method (Herbert et al.H. CoCl  6H2 O 21. which produced hydrogen by fermentation of sucrose at 36 °C and pH 5:0  0:5.5 min. About 2±5 ml of the diluted sample was ®ltered through a 0. was adjusted stepwise from 4. as illustrated in Fig. 1.. E‚uent samples were ®rst ®ltered through a 0.1. 1971). Methods 2. judging from the constant glucose degradation. The fermentor was stirred at a constant 200 rpm to ensure a thorough mixing and to facilitate rapid di€usion of hydrogen. whereas alcohols included methanol. A level probe was used to control the mixed liquor volume at 1. .P. propionate. 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 (80±100 mesh). The sludge on the membrane was then ®xed in the phosphate-bu€er solution with 2.0 to 7. respectively. Glucose was used as the model substrate for carbohydrates. H. The reactor. Na2 BO7  H2 O 7. MnCl2  4H2 O 30.0 g-VSS/l and produced a biogas comprising 50% hydrogen. K2 HPO4 250.7 l.2 lm nucleopore membrane. The temperatures of the injector and detector were 200 and 250 °C. pH 7. The pH of the mixed liquor was controlled automatically by feeding NaOH (6 M) and HCl (4 M) solutions via respective peristaltic pumps. FeCl3 50. The fermentor was kept in the dark by wrapping with aluminium foil to prevent the growth of photosynthetic bacteria. At each pH level. Five sets of thorough analyses were conducted on the reactor performance during days 14±21.0 with 0. Injector.3. contained 1.2 lm membrane. Experiments of hydrogen production Hydrogen production experiments were conducted in a three liter fermentor (Biostat B. 2. Helium was used as the carrier gas at a ¯ow rate of 25 ml/min. NH4 Cl 500. However. 180 and 50 °C. An additional 50 ml of the original seed sludge was added into the fermentor each time that the pH was adjusted. detector and column temperatures were kept at 57. Experimental setup. The concentrations of volatile fatty acids (VFA) and alcohols in the e‚uent 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 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. butyrate. The pH of the mixed liquor in the fermentor Fig. at 36 °C and 6 h hydraulic retention. 2. The VFA analyzed included acetate. CuCl2  2H2 O 10. The biomass was measured by volatile suspended solid (VSS) according to the Standard Methods (APHA. hydrogen production. CaCl2 50. the e€ect of mixing was not studied. Analyses The amount of biogas produced was recorded daily using the water displacement method. respectively. and criticalpoint dried with carbon dioxide (Fang et al. The ®xed sample was dehydrated stepwise in a graded series of water/ethanol solutions. Degrading each gram of sucrose produced 0.1 M. B.

The methane content increased from 3  1% at pH 6. The hydrogen content increased from 40  2% at pH 4..P. in correspondence to the glucose degradation. (b) biogas content. Treating a wastewater of concentrated glucose (18720 mg/l) at 35 °C. E. In order to analyze the complexity of the hydrogenproducing microbial communities.7 mol-H2 /mol-glucose.. (1993).0 to 7. 2000). 1995) with an annealing temperature of 54 °C (Zhang and Fang. the 16S rDNA fragments were ampli®ed by polymerase chain reaction (PCR). A 6% (w/v) acrylamide solution was used to cast a gel with denaturant gradients ranging 40±60%. The biogas was free of methane at pH 5. pH 5.0. and remained nearly constant (98.0 to 99:3  0:9% at pH 5. The extracted DNA was used as the template in PCR ampli®cation (Muyzer et al.0. Electrophoresis was conducted in a 1xTAE bu€er solution at 200 V and 60 °C for 5 h. 1996).0. pH e€ects on (a) glucose degradation. For DNA extraction.H. coli position 968-984) with GC-clamp (Muyzer et al.H. (c) hydrogen yield. At pH 7. Liu / Bioresource Technology 82 (2002) 87±93 Lastly. 2(b) illustrates that the biogas comprised mostly hydrogen and carbon dioxide. coli position 1392-1406) (Ferris et al.5. It shows that the maximum yield of 2:1  0:1 mol-H2 /mol-glucose observed in this study was substantially higher than the yields reported for other mixed cultures. E.5%) for pH ranging 5. each sludge sample was washed with a pH 7. Fig. (c) hydrogen yield.0. Lin and Chang (1999) reported a yield of 1. of hydrogenotrophic methanogens.5. the DNA in the sludge sampled at each pH level was extracted. The total community DNA from the sludge was extracted in three steps: cell lysis. followed by centrifugation at 4000 rpm for 10 min.8±99. Production of hydrogen Wastewater containing 7000 mg/l of glucose was treated in all experiments at 36 °C. Fig.. Five sets of extensive analysis were conducted at each pH level under steady-state condition.5 or lower.5. 6 h hydraulic retention and pH varying from 4. Further increase of pH drastically lowered the hydrogen content. Fang. accompanied by the decrease of hydrogen content. Fig. H. Fig.0 to 64  2% at pH 5. The carbon dioxide content in biogas followed an opposite trend of hydrogen... 89 (a) (b) (c) (d) 3. 1997). and (d) speci®c hydrogen production rate. the dried sample was sputter coated with gold prior to SEM observation. 1989). 2(c) illustrates that the hydrogen yield reached the optimum at pH 5. due to the bioactivity Fig.7 and 6 h hydraulic retention using a mixed culture. the biogas comprised only 35  1% of hydrogen.7%) .5±7. The DGGE was performed following the method of Muyzer et al.0 to 9  1% at pH 7. and (d) speci®c hydrogen production rate. The low yield is likely due to the poor degradation of glucose (81. (b) biogas composition. due to the suppression of methanogenic activity under acidic condition. 2 illustrates the pH effects on: (a) glucose conversion. phenol/chloroform/ isoamyl-alcohol extraction. 2. and precipitation by isopropanol and ethanol (Liu et al. One set of primers was used for PCR ampli®cation: 968F (bacteria domain speci®c forward primer.1. Table 1 lists the hydrogen yields in the literature from glucose at similar pH for comparison. But considerable quantities of methane were produced as pH further increased. Results and discussion 3.4 phosphatebu€er. The 16S rDNA bands on the gel were then stained with silver nitrate (Riesner et al. 2(a) illustrates that glucose degradation increased from 90:3  1:0% at pH 4. 1995) and 1392R (universal reverse primer. and separated by denaturing gradient gel electrophoresis (DGGE).

.3 mol-H2 / mol-glucose. The inorganic carbon (IC) in the in¯uent was attributed to the added NaHCO3 . instead of acetate.0 1.1 l-H2 /(g-VSS day) at pH 4.90 H.0 5.9 9.8 2.0 9. The hydrogen yield found in this study is also higher than the 1.4% on carbon basis).4 0.2 15.0 to 7. followed by acetate (15.0 mol-H2 /mol-glucose reported for the pure culture of Enterobacter aerogenes (Tanisho et al.8 2. but comparable to the yields of two other pure cultures. Other VFA and alcohols in the e‚uent included caproate (0.5± 5.1 mol-H2 /mol-glucose observed in this study re¯ects the observation that most of the glucose was converted to butyrate.5 6. At pH 5.4±32.0.5 31.1 1. and the dissociation constants C6 H12 O6 ‡ 2H2 O ! 2CH3 COOH ‡ 4H2 ‡ 2CO2 C6 H12 O6 ! CH3 …CH2 †2 COOH ‡ 2H2 ‡ 2CO2 …1† …2† converting 1 mole of glucose into acetate would produce 4 moles of hydrogen.0. 1999).5 5.9 1.1%) and butyrate (31.8 4. the reason is unknown because little information regarding the experimental condition was provided.5 33.2. 1997). Ethanol (4.D.0±6...7 mol-H2 /mol-glucose (Roychowdhury et al.9 4.5±6. plus minute but detectable amounts of methanol.8 2.9 15.7 Reference This study Lin and Chang (1999) Roychowdhury et al.P.0 4.1 34.0 5. Carbon balance and sludge yield Table 2 lists the distribution of the key VFA and alcohols in the e‚uents at various pH.7 0.5±31.5 0. butyricum a Hydrogen yield (mol-H2 /mol-glucose) a 5.6 2.1 6.1 5. (1989) Kumar and Das (2000) Kataoka et al.2 1.e. plus those in the e‚uent. Carbon in the in¯uent was converted into biomass.0 6. Increase of pH from 4. The TOCs of in¯uent were calculated from the glucose concentration.6±10.3 Mean  S.8 1. i-butyrate.3 35. and the IC content in the e‚uent was the sum of CO2 (aq). acetate (33. H.4 2.0 2. …n ˆ 5†.7 7. the maximum production of ethanol occurred at pH 5.4±2. calculated from the partial pressures of carbon dioxide.3 23. butanol..3. 1988). valerate and i-caproate. Another study using a mixed culture reported a low yield of 0. resulting from the high concentration of glucose in the wastewater.5%).4 4. i. the e‚uent contained mostly butyrate (41. according to Henry's law.4 45. 1989).9±15. According to the stoichiometry of the two following reactions: 3.5.6 2.7 1.0.4±2.1± 34. whereas those of the e‚uent were measured.1%) was the third most abundant of all in the e‚uent. Fig. Production of VFA and alcohols 3.4 3. At pH 6.0 6. Liu / Bioresource Technology 82 (2002) 87±93 Table 1 Comparison of hydrogen yield using glucose as substrate Microorganism pH Mixed culture Mixed culture Mixed culture E.7 41.9 .4 29.5 6.H.1 3.5 and 7. 2000) and 1.5 5. At pH 4. accompanied by the increased production of methane. propanol.5±5. 2. Table 3 summarizes the overall carbon mass balance.0 1312 1452 1356 1337 1317 1487 1281 20. cloacae (Kumar and Das. The yield of 2.8%) and propionate (0.0 resulted in the decrease of butyrate but in the increase of acetate. HCO3 and CO23 .3 mol-H2 /mol-glucose for Clostridium butyricum (Kataoka et al. aerogenes E. i-valerate.1±2. Production of propionate was suppressed at low pH as observed by others (Inanc et al. Results of this study and those reported for the pure cultures seem to suggest that the maximum hydrogen yield by glucose fermentation is about 2.5 1.0±6. (1988) Tanisho et al. 2(d) illustrates that the speci®c hydrogen production rate ranged 4. It shows that butyrate and acetate were the two most abundant species in the e‚uent.1 4.2 1.7 Unspeci®ed 5.5 7.. Whereas converting one mole of glucose into butyrate would produce only 2 moles of hydrogen.5 and 7.2 2.8 10.0 0.5±5. Table 2 Product distribution (on carbon basis) in e‚uent pH TOC (C-mg/l) Glucose (%) Butyrate (%) Acetate (%) Ethanol (%) Lactate (%) Caproate (%) Propionate (%) 4. cloacae C.0±6. carbon dioxide in the biogas.30±29.5. but it increased drastically at pH 6. Fang. the speci®c hydrogen production rate was 4:6  0:4 l-H2 /(g-VSS day).2 mol-H2 /mol-glucose for E.4 31. (1997) 2:1  0:1 1.2 29.0 5.1 32.9 4.2 6.9%).2%) became about equally abundant.5 1.6 38.1 23.0.

0. 4 illustrates the DGGE pro®les of the 16S rDNA gene fragment ampli®ed from the sludges sampled from pH 4.4.5 and 0.H. and (b) pH 5.0 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. 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 90.0.3 101.5 5.0 to 7.5 and (b) pH 5.5 were creamy white.8 95.P. 0. of H2 CO3 and HCO3 .8 90.2 Fig.0 4.0. Microbiology Sludges sampled at pH 4. Fig.0±5.H. Each band on the DGGE pro®le corre- Fig. . due to the increased sul®dogenic activity. Degrading one gram of glucose produced 0.0 94.5 7.5 6.5 were mostly composed of bacilli of various lengths.15 g of VSS at pH 4.5. Liu / Bioresource Technology 82 (2002) 87±93 91 Table 3 Carbon balance for each liter of in¯uent pH In¯uent E‚uent Biogas TOC (mg) IC (mg) TOC (mg) IC (mg) CO2 (mg) CH4 (mg) 4. Morphologies of hydrogen-producing bacteria at (a) pH 4. 3 illustrates that hydrogen-producing bacteria at (a) pH 4.5.1 96.0±107%.22 g VSS at pH 7. Fig. 4. 3.21 g VSS at the optimal pH of 5.0 5. plus some diplobacilli and streptobacilli. At pH > 6:0.0 6. and the color was darkened with the increase of pH. Fang. H. 3. sulfate-reducing bacteria converted the sulfate into sul®de which reacted with metals forming dark precipitates. DGGE pro®les for hydrogen-producing communities. Table 3 also shows that the sludge yield increased with pH.5 107.

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