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Enhancement of hydrogen production from glucose by nitrogen gas sparging
Bioresource Technology 73 (2000) 59±65
Enhancement of hydrogen production from glucose by nitrogen gas sparging Osamu Mizunoa, Richard Dinsdaleb, Freda R. Hawkesc, Dennis L. Hawkesb,*, Tatsuya Noikea a b
Department of Civil Engineering, Graduate School of Engineering, Tohoku University, Sendai, Aoba 980-8597, Japan School of Design and Advanced Technology, University of Glamorgan, Pontypridd, Mid-Glamorgan CF37 1DL, UK c School of Applied Sciences, University of Glamorgan, Pontypridd, Mid-Glamorgan CF37 1DL, UK Received 2 July 1999; received in revised form 13 August 1999; accepted 18 August 1999
Abstract The eect on hydrogen yield of N2 sparging was investigated in non-sterile conditions using a hydrogen-producing mixed culture previously enriched from soya bean meal. A continuous stirred-tank reactor (CSTR) at 35°C and pH 6.0 was operated on a mineral salts-glucose (10 g lÿ1 ) medium at a hydraulic retention time (HRT) of 8.5 h, and organic loading rate of 27.02 g glucose litre reactorÿ1 dayÿ1 . Results are reported from an 8 week period of continuous operation, and the enrichment culture gave stable results over an extended period. A hydrogen yield of 0.85 moles H2 /mole glucose consumed was obtained after 5 HRT, the gas produced being 53.4% H2 . With N2 sparging at a ¯ow rate approximately 15 times the hydrogen production rate, the hydrogen yield was 1.43 moles H2 /mole glucose consumed. The speci®c hydrogen production rate increased from 1.446 ml hydrogen minÿ1 gÿ1 biomass to 3.131 ml hydrogen minÿ1 gÿ1 biomass under sparging conditions. It is suggested that hydrogen partial pressure in the liquid phase was an important factor aecting hydrogen yield. Energy could be recovered as hydrogen from processes generating volatile fatty acids for ®ne chemicals and liquid bio-fuels or from acidi®cation reactors preceding normal anaerobic biological treatment of sugary wastewaters. Ó 2000 Elsevier Science Ltd. All rights reserved. Keywords: Hydrogen partial pressure; Biological hydrogen production; Hydrogen yield; N2 sparging
1. Introduction Hydrogen gas is a clean fuel, producing H2 O as its only by-product as it burns, not contributing CO2 , NOx , sulphur or particulates to global atmospheric pollution. It has a high energy content per unit weight (122 kJ gÿ1 ) and thus would have considerable possibilities as a fuel if the cost were low enough. With the development of storage technologies (e.g. as metal hydrides) it can be a multipurpose fuel. For nations such as Japan which import petroleum-based fuels, research on hydrogen production is particularly signi®cant, and has become a focus of governmental support (Benemann, 1996; Ueno et al., 1996; Lay et al., 1999). Hydrogen can be generated in a number of ways, for example through fossil fuel processing, or by electrolysis using solar power. However, these processes are energy intensive and therefore expensive. Biological production
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Corresponding author.
of hydrogen however is potentially more attractive, especially if wastewater or other biomass could be used as the raw material. The two biological routes to hydrogen production, photosynthetic and fermentative, have been extensively reviewed by Nandi and Sengupta (1998) and Benemann (1996). These and other workers (e.g. Yokoi et al., 1995) point to the advantages of the anaerobic fermentative route since H2 can be generated using less complex plant from a large number of sources such as refuse or waste products. Glucose or other carbohydrates are the preferred carbon source for the fermentations, which give rise to acetic and butyric acids together with hydrogen gas: C6 H12 O6 2H2 O ! 2CH3 COOH 2CO2 4H2
1
C6 H12 O6 ! CH3 CH2 CH2 COOH 2CO2 2H2
2
The euent from fermentative hydrogen generation, rich in organic acids, could be further exploited; e.g. by methanogenesis.
0960-8524/00/$ - see front matter Ó 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 9 9 ) 0 0 1 3 0 - 3
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From the ratios of acetic and butyric acids often formed, a hydrogen yield of approximately 2.5 mol H2 / mol hexose degraded can be expected, or approximately 0.3 m3 kgÿ1 carbohydrate utilised, with about 60% H2 in the o-gas. However it is dicult to establish a high hydrogen yield because the amount of fermentation products is signi®cantly in¯uenced by various factors such as nutrient levels (Bahl and Gottschalk, 1984; Dabrock et al., 1992), stirring (Lamed et al., 1988), and levels of carbon dioxide (Tanisho et al., 1998). Hydrogen production by a wide range of bacterial species, both pure and mixed, de®ned, cultures grown on sterile medium, and unde®ned enrichment cultures grown in non-sterile conditions, has been reviewed by Nandi and Sengupta (1998). From an engineering point of view a process using a stable enrichment culture yielding hydrogen from non-sterile organic wastes is required. Roychowdhury et al. (1988) demonstrated hydrogen production by enrichment cultures from cane juice, corn pulp and sacchari®ed cellulose, but not in continuous culture. Mizuno et al. (1997) reported hydrogen production from tofu manufacturing waste in batch culture from the culture used in the experiment reported here. Kalia et al. (1994) studied hydrogen production from damaged wheat grains by Bacillus licheniformis in continuous culture over a 40 day period, though low yields were reported The experiments of Ueno et al. (1996) on hydrogen production from sugar factory wastewater by a mixed micro¯ora in chemostat culture are the most successful known to the authors. Operation at a hydraulic retention time (HRT) of 0.5 days for 20 days is reported, giving a good hydrogen yield. Hydrogen partial pressure in the liquid phase is one of the key factors aecting hydrogen production. Many controversial observations regarding the in¯uence of hydrogen gas on the anaerobic breakdown of saccharides have been reported (Ruzicka, 1996). Tanisho et al. (1998) on sparging with argon obtained an increase in residual NADH, which might be expected to give an increased hydrogen production, although the hydrogen production was not actually measured. These authors found the same eect on NADH when sparging with hydrogen, and attributed this to CO2 removal. In this study we examined the eect of nitrogen sparging on hydrogen yield in a continuous culture of mixed anaerobic micro¯ora operating on a glucosemineral salts non-sterile medium. 2. Methods 2.1. Inoculum The anaerobic micro¯ora (predominantly Clostridium sp.) was obtained from fermented soybean-meal
(ESPRIT, 1989; Lay et al., 1999) and maintained in the laboratories of Tohoku University on a sucrose mineral salts medium in continuous culture at 35°C and a 10 h HRT at an uncontrolled pH of between 4.7 and 5.0, stirred by gas recirculation. After one week in transit to the UK at ambient temperature, the 50 ml culture was used to inoculate 500 ml glucose-mineral salts medium in a stoppered 1 l ¯ask at 35°C without pH control, with a gas outlet under water. An addition of 2 g lÿ1 of Na2 CO3 was made to buer the medium. This culture produced hydrogen after 3 days incubation, and the pH dropped by the fourth day to pH 4.5. The 500 ml culture was used to inoculate 1 l of growth medium and after 1 dayÕs growth this was used to inoculate the continuous stirred tank reactor (CSTR). 2.2. Glucose-mineral salts de®ned medium The medium contained the following ingredients in 1 l of tap water (modi®ed from Mizuno et al., 1998): NH4 Cl, 2600 mg; K2 HPO4 , 250 mg; MgCl2 á 6H2 O, 125 mg; FeSO4 á 7H2 O, 5 mg; CoCl2 á 6H2 O, 2.5 mg; MnCl2 á 4H2 O, 2.5 mg; KI, 2.5 mg; Na2 MoO4 á 2H2 O, 0.5 mg; H3 BO4 , 0.5 mg; NiCl2 á 6H2 O, 0.5 mg; ZnCl2 , 0.5 mg. Glucose was added to a ®nal concentration of 10 g lÿ1 . To prevent excessive foaming, anti-foam agent (Dehysan Z 2111, Henkel KGaA, Dusseldorf, Germany) was added to the nutrient reservoir to a ®nal concentration of 0.3 ml lÿ1 . Medium was made up freshly every day (weekdays) and 2.5 days (weekends) at 13 times the ®nal concentration, and diluted by tap water at the point of delivery to the reactor. The concentrated mineral salts nutrient solution was adjusted to below pH 2 with HCl. The 130 g lÿ1 glucose solution and the mineral salts medium were not sterilised or refrigerated, and microbial degradation did not take place under the conditions described here. 2.3. Operation of CSTR An anaerobic CSTR of 2.5 l capacity with 2.3 l working volume and a 8.5 h retention time, mixed by mechanical stirring at 100 rpm, was used. A schematic of the experimental apparatus is given in Fig. 1. The reactor was ®tted with a diuser for use in sparging experiments. Circulation through the water jacket was by a Grant FH15 ¯ow heater, (Cambridge Instruments, Cambridge, UK) adjusted to maintain the reactor temperature at 35°C 1°C. The pH value was kept constant at 6.0 by automatic titration (Kent EIL 9142 pH controller (ABB Kent-Taylor Ltd., Cambridge, England)) with a Watson-Marlow 101U pump (Falmouth, England) using 6M sodium hydroxide. The culture was maintained in these steady state conditions for 50 days before the experiments reported here. Gas production rate was measured continuously by a low ¯ow gas meter
O. Mizuno et al. / Bioresource Technology 73 (2000) 59±65
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Fig. 1. Anaerobic continuously stirred tank reactor.
developed in this laboratory (Guwy et al., 1995) calibrated using a bubble ¯ow meter (Phase Separations, UK). The data were logged to a Viglen 486DX 33 computer ®tted with a RTI data acquisition board down-loading ®les to Excel for data processing. In duplicate experiments, normal conditions were followed by gas sparging. Nitrogen (GC grade, Distillers Company, UK) containing less than 2 ppm O2 was used as sparging gas at a ¯ow rate of 110 2.6 ml minÿ1 measured using a bubble ¯ow meter (Phase Separations, UK). After 50 days of normal operation, nitrogen was sparged for a 49 h period, followed by a return to normal conditions for 5 days. A second period of sparging lasted for 4.5 days. 2.4. Chemical analysis Hydrogen was determined with a gas chromatograph (Varian 6500) equipped with a thermal conductivity detector and a 2.0 m (1/4 in. inside diameter) steel column ®lled with Porapak Q (50/80 mesh). Nitrogen was used as the carrier gas at a ¯ow rate of 40 ml minÿ1 . A 1 ml sample was injected except during calibration. A three-point calibration was performed daily for analysis
of the gas produced in normal operation, when 1.0, 0.5 and 0.1 ml of 100% H2 were used. For sparging conditions, 0.1 and 0.05 ml of 100% hydrogen and 5 ml of 0.2117% hydrogen in nitrogen (BOC Gases, Guildford, UK) were used. Carbon dioxide and methane were determined with a gas chromatograph (Varian 3400cx) equipped with a thermal conductivity detector and a 2 m (1/8 inch inside diameter) steel column ®lled with Poropak T (80±100 mesh). The temperatures at the injection port, column and TCD were 110°C, 60°C and 200°C, respectively. Helium was used as the carrier gas at a ¯ow rate of 30 ml minÿ1 . A single point calibration was performed with 41.52% CO2 in methane, supplied by BOC Gases, Guildford UK. The limit of detection was approximately 1%. Volatile fatty acids (VFA, C2 ±C5 ) were determined by gas chromatography as in Peck et al. (1986). Other fermentation end-products were determined by gas chromatography (Pye Unicam 104, Cambridge) ®tted with FID and a Perkin Elmer LCI-100 Integrator (Perkin Elmer, Beacons®eld). Centrifuged aqueous samples were injected on to a 2 m long (2 mm id) glass column packed with 80/120 Carbopack BAW/6.6% PEG
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O. Mizuno et al. / Bioresource Technology 73 (2000) 59±65
20M (Supelco/Sigma Aldrich UK). Helium was used as the carrier gas at a ¯ow of 30 ml minÿ1 and with an oven temperature ramp of 80°C to 200°C at 4°C minÿ1 . The glucose concentration was measured colourimetrically (Dubois et al., 1956). Biomass concentration was determined according to Standard Methods (APHA, 1992). 3. Results and discussion The culture obtained from Japan was easily grown up to form an inoculum despite 7 days in transit at ambient temperature. It recovered from a period of accidental washout in continuous culture, after which the reactor was re-inoculated from growth deposited in the exit U bend tube, recovering steady state biomass levels within 4 days. The culture operated at an 8 h retention time and was white in colour. Where biomass was stagnant anaerobically, a black colour appeared. While the culture is predominantly Clostridium sp., studies at Tohoku University have shown the presence of both sulphatereducing and methanogenic bacteria in this enrichment culture, and growth at longer HRTs allows these to develop. The biomass concentration in the reactor was for non-sparging conditions on average 1.45 0.25 g lÿ1 , and for sparging conditions after 5 HRTs 1.06 0.08 g lÿ1 . A change in the state of the biomass was visible after several days sparging, from a ¯occulating appearance throughout the reactor to a homogeneous culture. Possibly the increased mixing on sparging disrupted the ¯ocs. The decrease in biomass on sparging might possibly suggest the ¯ocs were incompletely suspended by the stirrer device and some biomass retention was allowed to occur which was not possible in the sparging
situation. Alternatively, physiological changes could lead to a lower growth yield in spaxrging conditions. It should be noted that the nitrogen gas used was 99.998% pure, and that no pretreatment was used to remove the O2 which could have been present. Possibly these traces of O2 may have aected the growth of the anaerobic biomass. Low biomass yields may be advantageous in the production of VFA for chemical re®ning as the feedstock may be directed to volatile products. The organic loading rate to the reactor was 27.02 g glucose lÿ1 dayÿ1 . Euent glucose levels during the whole period of operation reported here were low (Table 1), averaging 76.9 mg lÿ1 in non-sparging and 45.4 mg lÿ1 in sparging conditions. Thus over 99% of the added glucose was converted to fermentation end products. The speci®c organic load to the biomass was 18.63 g glucose gÿ1 biomass dayÿ1 for sparging conditions after 5 HRT, and 25.37 g glucose gÿ1 biomass dayÿ1 in sparging conditions. Average gas production rate from the reactor during non-sparging operation after 5 HRT was 8.9 ml minÿ1 . Average hydrogen measured in the gas was 53.4% during this period of non-sparging operation, and 5.3% measured after 5 HRT during sparging (Table 2). Measurements of the rate of ¯ow of the nitrogen sparging gas showed that this averaged 110.0 2.6 ml minÿ1 . Allowing for dilution by this background level of nitrogen sparging gas, the rate of gas production during sparging by the whole reactor was on average 33.2 ml minÿ1 . From these measurements the hydrogen concentration in the biogas produced during sparging was 22.9%. The hydrogen yield during steady-state operation, based on the average gas ¯ow and average hydrogen concentration, was 0.85 moles hydrogen/mole glucose utilised. Similar calculations for the hydrogen yield
Table 1 Liquid phase parameters ÿ1 a
Residual glucose (mg l ) Glucose removal %a Biomass (g lÿ1 )a Speci®c loading rate (g glucose gÿ1 biomass dayÿ1 ) a
Non-sparging
Sparging
76.9 35.4 99.2 0.3 1.45 0.25 18.38
45.4 13.6 99.5 0.1 1.06 0.08 25.49
Four samples measured.
Table 2 Gas phase parameters ÿ1
ÿ1
Biogas production rate (ml min l ) Hydrogen %a Hydrogen production rate (ml minÿ1 lÿ1 ) Hydrogen yield (mol molÿ1 glucose) Speci®c hydrogen production rate (ml minÿ1 gÿ1 biomass) a
Six samples measured.
Non-sparging
Sparging
3.895 1.15 53.4 1.7 2.08 0.85 0.32 1.434
14.499 3.035 5.3 0.2 3.31 1.43 0.12 3.126
O. Mizuno et al. / Bioresource Technology 73 (2000) 59±65
under sparging conditions gave 1.43 moles hydrogen/ mole glucose utilised. The hydrogen partial pressure in the reactor liquid during sparging conditions might have been an order of magnitude lower. Traces of methane were found in the biogas, 2% methane with 41% carbon dioxide in non-sparging conditions. As the amount of CO2 in the biogas increased from 46% to 77% during sparging (by dierence, assuming the biogas was chie¯y H2 and CO2 ), changes in fermentation end-products might have been expected. The levels of volatile metabolic products in the reactor euent are shown in Table 3. From the distribution of products in Table 3 it can be seen that the products were principally volatile fatty acids, with virtually no solvent production: i.e. the culture was acidogenic in nature under the conditions described. The principal VFA was n-butyric, as also found by Zoetemeyer et al. (1982) and Cohen et al. (1979) in anaerobic acidogenic reactors. The main dierence between the non-sparging conditions and the sparging conditions was a small increase in n-butyric concentration in the sparging conditions from an average of 1742 mg lÿ1 to 1929 mg lÿ1 . No major shift in metabolic pathways, i.e. from acids to acetone-butanol production, was seen. In hydrogen production, conditions are sought maximising acetic acid production as this gives the maximum hydrogen yield (Eq. (1)). The concept of fermentative hydrogen production is contrary to the more well-studied solvent producing acetone-butanol fermentation in which the production of molecular hydrogen and acetate is unnecessary and decreases solvent recovery. End products such as H2 , CO2 , acetate and butyrate are the result of side reactions in the acetonebutanol fermentation process (Kim et al., 1984). Thus a study of the conditions detrimental to solvent production will give information on those conditions favouring hydrogen and acetate production. Glucose is the fundamental resource for hydrogen production. Glucose is fermented via the EMP pathway to pyruvate. Pyruvate oxidation to acetyl coenzyme A requires ferredoxin (Fd) reduction. Reduced Fd is oxidized by hydrogenase, which generates Fd and releases
electrons as molecular hydrogen. Therefore hydrogen production is the means by which bacteria lose excess electrons. The reaction is reversible and depends on hydrogen partial pressure (pH2 ), suggesting that hydrogen yield is signi®cantly in¯uenced by pH2 . Therefore it is important to control pH2 in the liquid phase for enhancement of hydrogen yield. Gas sparging is a useful method for decreasing pH2 although industrially a method of hydrogen utilisation, e.g. a fuel cell or metal hydrides, would be employed (Levy et al., 1981). The eects of hydrogen on the metabolism and the fermentative pattern of the anaerobic bacteria have been demonstrated in previous studies. Clostridium cellobioparum produces more hydrogen when it is removed by hydrogen-consuming methanogens (Chung, 1976). The quantitative composition of the fermentation products depends on the pH2 . Van Andel et al. (1985) demonstrated that sparging a pure culture of Clostridium butyricum with nitrogen increased the rate of acetate production both absolutely and relative to the rate of butyrate production. The production of acetate and hydrogen by Clostridium thermocellum has been considered an obstacle to the use of this organism in ethanol production (Lamed et al., 1988) and stirring the batch cultures favoured hydrogen and acetate production. This was attributed to accumulation of hydrogen at supersaturated concentrations in unstirred conditions inhibiting acetate production. The CSTR reactor used here was already well stirred. Other workers who have grown enrichment cultures on glucose-mineral salts medium in continuous culture include Nakamura et al. (1993) who studied the eect of HRT from 2±10 h on an enrichment from sewage sludge. These workers do not report the euent glucose concentrations, but assuming these were low, a hydrogen yield of 0.07 moles H2 /mole glucose consumed was achieved at a 2 h HRT, which gave the highest hydrogen % and total gas production. Ueno et al. (1996) also used a sewage sludge enrichment grown in continuous culture on a sugary industrial wastewater. The maximum hydrogen yield reported was 2.52 moles H2 /mole carbohydrate consumed (assuming carbohydrate has the formula C6 H12 O6 ) at a 12 h HRT and pH 6.8. Kumar et
Table 3 Volatile metabolic products in the liquid phasea
a
Product
Non-Sparging (mg lÿ1 )
Sparging (mg lÿ1 )
Acetic Propionic i-butyric n-butyric i-valeric n-valeric Acetone Ethanol Butanol
773 48 (3) 114 6 (3) 29 15 (3) 1742 31 (3) ND (3) 10 9 (3) ND (3) 58 20 (3) 4 1 (3)
785 79 (3) 74 1 (3) 13 2 (3) 1929 170 (3) 2 2 (3) 13 2 (3) 1 1 (3) 30 17 (3) 6 2 (3)
ND Not Detectable; ( ) number of samples.
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Table 4 Hydrogen yields reported in the literature Mixed culture this study Mixed culture this study Enterobacter aerogenes (Tanisho et al., 1998) Enterobacter aerogenes (Tanisho et al., 1998) Enterobacter aerogenes (Yokoi et al., 1995) Clostridium butyricum (Van Andel et al., 1985) Clostridium sp. strain No.2 (Taguchi et al., 1995) Citrobacter intermedius (Brosseau and Zajic, 1982) Clostridium pasteurianum (Brosseau and Zajic, 1982) Clostridium beijerinckii AM21B (Taguchi et al., 1995)
Condition
Substrate
H2 yield (mol/mol-glucose)
N2 sparging, continuous continuous Ar sparging, batch batch continuous continuous continuous batch batch batch
glucose glucose molasses molasses glucose glucose glucose glucose glucose glucose
1.43 0.85 1.58 0.52 1.00 1.60 1.61±2.36 1.00 1.50 1.3±2.0
al. (1995) using a strain of Bacillus licheniformis isolated from cattle dung report hydrogen yields in semi-continuous culture over 60 days of 0.37 moles/mole glucose utilised, though yields of up to 1.1 moles H2 /mole glucose were reported in shorter term batch-type experiments. It should be noted that in each case the gas volume was measured by the water displacement method, which may be less accurate than the gas meter measurements available in the present study. Hydrogen yields from pure culture studies reviewed by Nandi and Sengupta (1998) vary from 0.7 to 2.4 moles H2 /mole glucose, and a summary of yields from pure culture work is given in Table 4. The speci®c hydrogen production rate found here of 3.84 mmol hÿ1 gÿ1 dry weight of biomass (Table 2) compares well with rates for Clostridium pasteurianum of 2.5 mmol hÿ1 gÿ1 biomass in log growth phase in batch culture and 1.2 mmol hÿ1 gÿ1 biomass in stationary phase (Brosseau and Zajic, 1982). However, Taguchi et al. (1995) obtained speci®c hydrogen production rates with Clostridium sp. strain No. 2 grown on glucose in continuous culture of between 7.8 and 32.2 mmol hÿ1 gÿ1 dry weight of biomass depending on the retention time. It appears possible that at an industrial scale, energy could be recovered as hydrogen from an acidogenic reactor as part of a typical biological treatment for carbohydrate-containing wastewaters or from an acidogenic stage used to produce volatile fatty acids for chemical or liquid bio-fuel production (Levy et al., 1981). The euent from the hydrogen producing stage, already at 35°C, could then pass to a methanogenic stage for which the fermentation end products are ideal substrates, or the volatile products could be extracted to supply chemicals or be used as liquid bio-fuels (Levy et al., 1981; Brosseau and Zajic, 1982). The mixed culture used here grows also on sucrose and starch, tofu manufacturing waste, wheat bran and rice bran, in batch culture (Mizuno et al., 1997) so that this process could be of wide application to food industry wastewaters, which would then be seen as a resource for high grade renewable energy production.
4. Conclusions The hydrogen-producing enrichment culture was extremely stable when grown at pH 6.0 and a HRT of 8.5 h, re-growing well after dormancy and accidental washout. A ¯occulant biomass of concentration 1.5 g lÿ1 dry weight was obtained in the CSTR without sparging, a non-¯occulant biomass of a lower concentration resulting during sparging. Hydrogen yields of 0.85 and 1.43 mol/mol glucose were obtained under non-sparging and sparging conditions, respectively; sparging with nitrogen resulted in a 68% increase in hydrogen yield. No signi®cant change in fermentation end products was observed. Acknowledgements The authors wish to thank Dr. A.J. Guwy and Miss H. Forsey for their expert technical assistance, and the UK EPSRC for an equipment grant to DLH and FRH (GR/M38346). References Andel, J.G., Zoutberg, G.R., Crabbendam, P.M., Breure, A.M., 1985. Glucose fermentation by Clostridium butyricum grown under a self generated gas atmosphere in chemostat culture. Applied Microbiology and Biotechnology 23, 21±26. APHA, 1992. Standard Methods for the Examination of Waste and Wastewater, 18th ed. American Public Health Association, Washington, USA. Bahl, H., Gottschalk, G., 1984. Parameters aecting solvent production by Clostridium acetobutylicum in continuous culture. Biotechnology and Bioengineering Symposium 14, 215±223. Benemann, J., 1996. Hydrogen biotechnology: progress and prospects. Nature Biotechnology 14, 1101±1103. Brosseau, J.D., Zajic, J.E., 1982. Hydrogen-gas production with Citrobacter intermedius and Clostridium pasteurianum. Journal Chemical Technology and Biotechnology 32, 496±502. Chung, K.T., 1976. Inhibitory eects of H2 on growth of Clostridium cellobioparum. Applied and Environmental Microbiology 31, 342±348. Cohen, A., Zoetemeyer, R.J., Van Deursen, A., Van Andel, J.G., 1979. Anaerobic digestion of glucose with separated acid production and methane formation. Water Research 13, 571±580.
O. Mizuno et al. / Bioresource Technology 73 (2000) 59±65 Dabrock, B., Bahl, H., Gottschalk, G., 1992. Parameters aecting solvent production by Clostridium pasteurianum. Applied and Environmental Microbiology 58, 1233±1239. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Analytical Chemistry 28, 350±356. ESPRIT, 1989. Research Report of the explosion of soybean-meal in a silo. ESPRIT Co. Ltd. (In Japanese). Guwy, A.J., Hawkes, D.L., Hawkes, F.R., 1995. On-line low ¯ow high-precision gas metering systems. Water Research 29, 977±979. Kalia, V.C., Jain, S.R., Kumar, A., Joshi, A.P., 1994. Fermentation of bio-waste to H2 by Bacillus licheniformis. World Journal of Microbiology and Biotechnology 10, 224±227. Kim, B.H., Bellows, P., Datta, R., Zeikus, J.G., 1984. Control of carbon and electron ¯ow in Clostridium acetobutylicum fermentations: utilization of carbon monoxide to inhibit hydrogen production and to enhance butanol yields. Applied and Environmental Microbiology 48, 764±770. Kumar, A., Jain, S.R., Sharma, C.B., Joshi, A.P., Kalia, V.C., 1995. Increased H2 production by immobilized microorganisms. World Journal of Microbiology and Biotechnology 11, 156±159. Lamed, R.J., Lobos, J.H., Su, T.M., 1988. Eect of stirring and hydrogen on fermentation products of Clostridium thermocellum. Applied and Environmental Microbiology 54, 1216±1221. Lay, J.J., Lee, Y.J., Noike, T., 1999. Feasibility of biological hydrogen production from organic fraction of municipal solid waste. Water Research 33 (11), 2579±2586. Levy, P.F., Sanderson, J.E., Kispert, R.G., Wise, D.L., 1981. Biore®ning of biomass to liquid fuels and organic chemicals. Enzyme Microbiol Technology 3, 207±215. Mizuno, O., Li, Y.Y., Noike, T., 1998. The behavior of sulfatereducing bacteria in acidogenic phase of anaerobic digestion. Water Research 32, 1626±1634. Mizuno, O., Ohara, T., Noike, T., 1997. Hydrogen Production from Food Processing Waste by Anaerobic Bacteria. Journal of Envi-
65
ronmental Systems and Engineering, JSCE (573/VII-5), 111±118 (in Japanese). Nakamura, M., Kanabe, H., Matsumoto, J., 1993. Fundamental studies on hydrogen production in the acid-forming phase and its bacteria in anaerobic treatment processes-the eects of solids retention time. Water Science Technology 28 (7), 81±88. Nandi, R., Sengupta, S., 1998. Microbial production of hydrogen: an overview. Critical Reviews in Microbiology. 24 (1), 61±84. Peck, M.W., Skilton, J.M., Hawkes, F.R., Hawkes, D.L., 1986. Eects of temperature shock treatments on the stability of anaerobic digesters operated on separated cattle slurry. Water Research 20 (4), 453±462. Roychowdhury, S., Cox, D., Levandowsky, M., 1988. Production of hydrogen by microbial fermentation. International Journal of Hydrogen Energy 13, 407±410. Ruzicka, M., 1996. The eect of hydrogen on acidogenic glucose cleavage. Water Research 30, 2447±2451. Taguchi, F., Mizukami, N., Saito-Taki, T., Hasegawa, K., 1995. Hydrogen production from continuous fermentation of xylose during growth of Clostridium sp. strain No.2.. Canadian Journal of Microbiology 41, 536±540. Tanisho, S., Kuromoto, M., Kadokura, N., 1998. Eect of CO2 removal on hydrogen production by fermentation. International Journal of Hydrogen Energy 23, 559±563. Ueno, Y., Otauka, S., Morimoto, M., 1996. Hydrogen production from industrial wastewater by anaerobic micro¯ora in chemostat culture. Journal of Fermentation and Bioengineering 82, 194±197. Yokoi, H., Ohkawa, T., Hirosse, J., Hayashi, S., Takasaki, Y., 1995. Characteristics of hydrogen production by aciduric Enterobacter aerogen strain HO-39. Journal of Fermentation and Bioengineering 80, 571±574. Zoetemeyer, R.J., Arnoldy, P., Cohen, A., Boelhouwer, C., 1982. In¯uence of temperature on the anaerobic acidi®cation of glucose in a mixed culture forming part of a two stage digestion process. Water Research 16, 313±321.
Enhancement of hydrogen production from glucose by nitrogen gas sparging Osamu Mizunoa, Richard Dinsdaleb, Freda R. Hawkesc, Dennis L. Hawkesb,*, Tatsuya Noikea a b
Department of Civil Engineering, Graduate School of Engineering, Tohoku University, Sendai, Aoba 980-8597, Japan School of Design and Advanced Technology, University of Glamorgan, Pontypridd, Mid-Glamorgan CF37 1DL, UK c School of Applied Sciences, University of Glamorgan, Pontypridd, Mid-Glamorgan CF37 1DL, UK Received 2 July 1999; received in revised form 13 August 1999; accepted 18 August 1999
Abstract The eect on hydrogen yield of N2 sparging was investigated in non-sterile conditions using a hydrogen-producing mixed culture previously enriched from soya bean meal. A continuous stirred-tank reactor (CSTR) at 35°C and pH 6.0 was operated on a mineral salts-glucose (10 g lÿ1 ) medium at a hydraulic retention time (HRT) of 8.5 h, and organic loading rate of 27.02 g glucose litre reactorÿ1 dayÿ1 . Results are reported from an 8 week period of continuous operation, and the enrichment culture gave stable results over an extended period. A hydrogen yield of 0.85 moles H2 /mole glucose consumed was obtained after 5 HRT, the gas produced being 53.4% H2 . With N2 sparging at a ¯ow rate approximately 15 times the hydrogen production rate, the hydrogen yield was 1.43 moles H2 /mole glucose consumed. The speci®c hydrogen production rate increased from 1.446 ml hydrogen minÿ1 gÿ1 biomass to 3.131 ml hydrogen minÿ1 gÿ1 biomass under sparging conditions. It is suggested that hydrogen partial pressure in the liquid phase was an important factor aecting hydrogen yield. Energy could be recovered as hydrogen from processes generating volatile fatty acids for ®ne chemicals and liquid bio-fuels or from acidi®cation reactors preceding normal anaerobic biological treatment of sugary wastewaters. Ó 2000 Elsevier Science Ltd. All rights reserved. Keywords: Hydrogen partial pressure; Biological hydrogen production; Hydrogen yield; N2 sparging
1. Introduction Hydrogen gas is a clean fuel, producing H2 O as its only by-product as it burns, not contributing CO2 , NOx , sulphur or particulates to global atmospheric pollution. It has a high energy content per unit weight (122 kJ gÿ1 ) and thus would have considerable possibilities as a fuel if the cost were low enough. With the development of storage technologies (e.g. as metal hydrides) it can be a multipurpose fuel. For nations such as Japan which import petroleum-based fuels, research on hydrogen production is particularly signi®cant, and has become a focus of governmental support (Benemann, 1996; Ueno et al., 1996; Lay et al., 1999). Hydrogen can be generated in a number of ways, for example through fossil fuel processing, or by electrolysis using solar power. However, these processes are energy intensive and therefore expensive. Biological production
*
Corresponding author.
of hydrogen however is potentially more attractive, especially if wastewater or other biomass could be used as the raw material. The two biological routes to hydrogen production, photosynthetic and fermentative, have been extensively reviewed by Nandi and Sengupta (1998) and Benemann (1996). These and other workers (e.g. Yokoi et al., 1995) point to the advantages of the anaerobic fermentative route since H2 can be generated using less complex plant from a large number of sources such as refuse or waste products. Glucose or other carbohydrates are the preferred carbon source for the fermentations, which give rise to acetic and butyric acids together with hydrogen gas: C6 H12 O6 2H2 O ! 2CH3 COOH 2CO2 4H2
1
C6 H12 O6 ! CH3 CH2 CH2 COOH 2CO2 2H2
2
The euent from fermentative hydrogen generation, rich in organic acids, could be further exploited; e.g. by methanogenesis.
0960-8524/00/$ - see front matter Ó 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 9 9 ) 0 0 1 3 0 - 3
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From the ratios of acetic and butyric acids often formed, a hydrogen yield of approximately 2.5 mol H2 / mol hexose degraded can be expected, or approximately 0.3 m3 kgÿ1 carbohydrate utilised, with about 60% H2 in the o-gas. However it is dicult to establish a high hydrogen yield because the amount of fermentation products is signi®cantly in¯uenced by various factors such as nutrient levels (Bahl and Gottschalk, 1984; Dabrock et al., 1992), stirring (Lamed et al., 1988), and levels of carbon dioxide (Tanisho et al., 1998). Hydrogen production by a wide range of bacterial species, both pure and mixed, de®ned, cultures grown on sterile medium, and unde®ned enrichment cultures grown in non-sterile conditions, has been reviewed by Nandi and Sengupta (1998). From an engineering point of view a process using a stable enrichment culture yielding hydrogen from non-sterile organic wastes is required. Roychowdhury et al. (1988) demonstrated hydrogen production by enrichment cultures from cane juice, corn pulp and sacchari®ed cellulose, but not in continuous culture. Mizuno et al. (1997) reported hydrogen production from tofu manufacturing waste in batch culture from the culture used in the experiment reported here. Kalia et al. (1994) studied hydrogen production from damaged wheat grains by Bacillus licheniformis in continuous culture over a 40 day period, though low yields were reported The experiments of Ueno et al. (1996) on hydrogen production from sugar factory wastewater by a mixed micro¯ora in chemostat culture are the most successful known to the authors. Operation at a hydraulic retention time (HRT) of 0.5 days for 20 days is reported, giving a good hydrogen yield. Hydrogen partial pressure in the liquid phase is one of the key factors aecting hydrogen production. Many controversial observations regarding the in¯uence of hydrogen gas on the anaerobic breakdown of saccharides have been reported (Ruzicka, 1996). Tanisho et al. (1998) on sparging with argon obtained an increase in residual NADH, which might be expected to give an increased hydrogen production, although the hydrogen production was not actually measured. These authors found the same eect on NADH when sparging with hydrogen, and attributed this to CO2 removal. In this study we examined the eect of nitrogen sparging on hydrogen yield in a continuous culture of mixed anaerobic micro¯ora operating on a glucosemineral salts non-sterile medium. 2. Methods 2.1. Inoculum The anaerobic micro¯ora (predominantly Clostridium sp.) was obtained from fermented soybean-meal
(ESPRIT, 1989; Lay et al., 1999) and maintained in the laboratories of Tohoku University on a sucrose mineral salts medium in continuous culture at 35°C and a 10 h HRT at an uncontrolled pH of between 4.7 and 5.0, stirred by gas recirculation. After one week in transit to the UK at ambient temperature, the 50 ml culture was used to inoculate 500 ml glucose-mineral salts medium in a stoppered 1 l ¯ask at 35°C without pH control, with a gas outlet under water. An addition of 2 g lÿ1 of Na2 CO3 was made to buer the medium. This culture produced hydrogen after 3 days incubation, and the pH dropped by the fourth day to pH 4.5. The 500 ml culture was used to inoculate 1 l of growth medium and after 1 dayÕs growth this was used to inoculate the continuous stirred tank reactor (CSTR). 2.2. Glucose-mineral salts de®ned medium The medium contained the following ingredients in 1 l of tap water (modi®ed from Mizuno et al., 1998): NH4 Cl, 2600 mg; K2 HPO4 , 250 mg; MgCl2 á 6H2 O, 125 mg; FeSO4 á 7H2 O, 5 mg; CoCl2 á 6H2 O, 2.5 mg; MnCl2 á 4H2 O, 2.5 mg; KI, 2.5 mg; Na2 MoO4 á 2H2 O, 0.5 mg; H3 BO4 , 0.5 mg; NiCl2 á 6H2 O, 0.5 mg; ZnCl2 , 0.5 mg. Glucose was added to a ®nal concentration of 10 g lÿ1 . To prevent excessive foaming, anti-foam agent (Dehysan Z 2111, Henkel KGaA, Dusseldorf, Germany) was added to the nutrient reservoir to a ®nal concentration of 0.3 ml lÿ1 . Medium was made up freshly every day (weekdays) and 2.5 days (weekends) at 13 times the ®nal concentration, and diluted by tap water at the point of delivery to the reactor. The concentrated mineral salts nutrient solution was adjusted to below pH 2 with HCl. The 130 g lÿ1 glucose solution and the mineral salts medium were not sterilised or refrigerated, and microbial degradation did not take place under the conditions described here. 2.3. Operation of CSTR An anaerobic CSTR of 2.5 l capacity with 2.3 l working volume and a 8.5 h retention time, mixed by mechanical stirring at 100 rpm, was used. A schematic of the experimental apparatus is given in Fig. 1. The reactor was ®tted with a diuser for use in sparging experiments. Circulation through the water jacket was by a Grant FH15 ¯ow heater, (Cambridge Instruments, Cambridge, UK) adjusted to maintain the reactor temperature at 35°C 1°C. The pH value was kept constant at 6.0 by automatic titration (Kent EIL 9142 pH controller (ABB Kent-Taylor Ltd., Cambridge, England)) with a Watson-Marlow 101U pump (Falmouth, England) using 6M sodium hydroxide. The culture was maintained in these steady state conditions for 50 days before the experiments reported here. Gas production rate was measured continuously by a low ¯ow gas meter
O. Mizuno et al. / Bioresource Technology 73 (2000) 59±65
61
Fig. 1. Anaerobic continuously stirred tank reactor.
developed in this laboratory (Guwy et al., 1995) calibrated using a bubble ¯ow meter (Phase Separations, UK). The data were logged to a Viglen 486DX 33 computer ®tted with a RTI data acquisition board down-loading ®les to Excel for data processing. In duplicate experiments, normal conditions were followed by gas sparging. Nitrogen (GC grade, Distillers Company, UK) containing less than 2 ppm O2 was used as sparging gas at a ¯ow rate of 110 2.6 ml minÿ1 measured using a bubble ¯ow meter (Phase Separations, UK). After 50 days of normal operation, nitrogen was sparged for a 49 h period, followed by a return to normal conditions for 5 days. A second period of sparging lasted for 4.5 days. 2.4. Chemical analysis Hydrogen was determined with a gas chromatograph (Varian 6500) equipped with a thermal conductivity detector and a 2.0 m (1/4 in. inside diameter) steel column ®lled with Porapak Q (50/80 mesh). Nitrogen was used as the carrier gas at a ¯ow rate of 40 ml minÿ1 . A 1 ml sample was injected except during calibration. A three-point calibration was performed daily for analysis
of the gas produced in normal operation, when 1.0, 0.5 and 0.1 ml of 100% H2 were used. For sparging conditions, 0.1 and 0.05 ml of 100% hydrogen and 5 ml of 0.2117% hydrogen in nitrogen (BOC Gases, Guildford, UK) were used. Carbon dioxide and methane were determined with a gas chromatograph (Varian 3400cx) equipped with a thermal conductivity detector and a 2 m (1/8 inch inside diameter) steel column ®lled with Poropak T (80±100 mesh). The temperatures at the injection port, column and TCD were 110°C, 60°C and 200°C, respectively. Helium was used as the carrier gas at a ¯ow rate of 30 ml minÿ1 . A single point calibration was performed with 41.52% CO2 in methane, supplied by BOC Gases, Guildford UK. The limit of detection was approximately 1%. Volatile fatty acids (VFA, C2 ±C5 ) were determined by gas chromatography as in Peck et al. (1986). Other fermentation end-products were determined by gas chromatography (Pye Unicam 104, Cambridge) ®tted with FID and a Perkin Elmer LCI-100 Integrator (Perkin Elmer, Beacons®eld). Centrifuged aqueous samples were injected on to a 2 m long (2 mm id) glass column packed with 80/120 Carbopack BAW/6.6% PEG
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20M (Supelco/Sigma Aldrich UK). Helium was used as the carrier gas at a ¯ow of 30 ml minÿ1 and with an oven temperature ramp of 80°C to 200°C at 4°C minÿ1 . The glucose concentration was measured colourimetrically (Dubois et al., 1956). Biomass concentration was determined according to Standard Methods (APHA, 1992). 3. Results and discussion The culture obtained from Japan was easily grown up to form an inoculum despite 7 days in transit at ambient temperature. It recovered from a period of accidental washout in continuous culture, after which the reactor was re-inoculated from growth deposited in the exit U bend tube, recovering steady state biomass levels within 4 days. The culture operated at an 8 h retention time and was white in colour. Where biomass was stagnant anaerobically, a black colour appeared. While the culture is predominantly Clostridium sp., studies at Tohoku University have shown the presence of both sulphatereducing and methanogenic bacteria in this enrichment culture, and growth at longer HRTs allows these to develop. The biomass concentration in the reactor was for non-sparging conditions on average 1.45 0.25 g lÿ1 , and for sparging conditions after 5 HRTs 1.06 0.08 g lÿ1 . A change in the state of the biomass was visible after several days sparging, from a ¯occulating appearance throughout the reactor to a homogeneous culture. Possibly the increased mixing on sparging disrupted the ¯ocs. The decrease in biomass on sparging might possibly suggest the ¯ocs were incompletely suspended by the stirrer device and some biomass retention was allowed to occur which was not possible in the sparging
situation. Alternatively, physiological changes could lead to a lower growth yield in spaxrging conditions. It should be noted that the nitrogen gas used was 99.998% pure, and that no pretreatment was used to remove the O2 which could have been present. Possibly these traces of O2 may have aected the growth of the anaerobic biomass. Low biomass yields may be advantageous in the production of VFA for chemical re®ning as the feedstock may be directed to volatile products. The organic loading rate to the reactor was 27.02 g glucose lÿ1 dayÿ1 . Euent glucose levels during the whole period of operation reported here were low (Table 1), averaging 76.9 mg lÿ1 in non-sparging and 45.4 mg lÿ1 in sparging conditions. Thus over 99% of the added glucose was converted to fermentation end products. The speci®c organic load to the biomass was 18.63 g glucose gÿ1 biomass dayÿ1 for sparging conditions after 5 HRT, and 25.37 g glucose gÿ1 biomass dayÿ1 in sparging conditions. Average gas production rate from the reactor during non-sparging operation after 5 HRT was 8.9 ml minÿ1 . Average hydrogen measured in the gas was 53.4% during this period of non-sparging operation, and 5.3% measured after 5 HRT during sparging (Table 2). Measurements of the rate of ¯ow of the nitrogen sparging gas showed that this averaged 110.0 2.6 ml minÿ1 . Allowing for dilution by this background level of nitrogen sparging gas, the rate of gas production during sparging by the whole reactor was on average 33.2 ml minÿ1 . From these measurements the hydrogen concentration in the biogas produced during sparging was 22.9%. The hydrogen yield during steady-state operation, based on the average gas ¯ow and average hydrogen concentration, was 0.85 moles hydrogen/mole glucose utilised. Similar calculations for the hydrogen yield
Table 1 Liquid phase parameters ÿ1 a
Residual glucose (mg l ) Glucose removal %a Biomass (g lÿ1 )a Speci®c loading rate (g glucose gÿ1 biomass dayÿ1 ) a
Non-sparging
Sparging
76.9 35.4 99.2 0.3 1.45 0.25 18.38
45.4 13.6 99.5 0.1 1.06 0.08 25.49
Four samples measured.
Table 2 Gas phase parameters ÿ1
ÿ1
Biogas production rate (ml min l ) Hydrogen %a Hydrogen production rate (ml minÿ1 lÿ1 ) Hydrogen yield (mol molÿ1 glucose) Speci®c hydrogen production rate (ml minÿ1 gÿ1 biomass) a
Six samples measured.
Non-sparging
Sparging
3.895 1.15 53.4 1.7 2.08 0.85 0.32 1.434
14.499 3.035 5.3 0.2 3.31 1.43 0.12 3.126
O. Mizuno et al. / Bioresource Technology 73 (2000) 59±65
under sparging conditions gave 1.43 moles hydrogen/ mole glucose utilised. The hydrogen partial pressure in the reactor liquid during sparging conditions might have been an order of magnitude lower. Traces of methane were found in the biogas, 2% methane with 41% carbon dioxide in non-sparging conditions. As the amount of CO2 in the biogas increased from 46% to 77% during sparging (by dierence, assuming the biogas was chie¯y H2 and CO2 ), changes in fermentation end-products might have been expected. The levels of volatile metabolic products in the reactor euent are shown in Table 3. From the distribution of products in Table 3 it can be seen that the products were principally volatile fatty acids, with virtually no solvent production: i.e. the culture was acidogenic in nature under the conditions described. The principal VFA was n-butyric, as also found by Zoetemeyer et al. (1982) and Cohen et al. (1979) in anaerobic acidogenic reactors. The main dierence between the non-sparging conditions and the sparging conditions was a small increase in n-butyric concentration in the sparging conditions from an average of 1742 mg lÿ1 to 1929 mg lÿ1 . No major shift in metabolic pathways, i.e. from acids to acetone-butanol production, was seen. In hydrogen production, conditions are sought maximising acetic acid production as this gives the maximum hydrogen yield (Eq. (1)). The concept of fermentative hydrogen production is contrary to the more well-studied solvent producing acetone-butanol fermentation in which the production of molecular hydrogen and acetate is unnecessary and decreases solvent recovery. End products such as H2 , CO2 , acetate and butyrate are the result of side reactions in the acetonebutanol fermentation process (Kim et al., 1984). Thus a study of the conditions detrimental to solvent production will give information on those conditions favouring hydrogen and acetate production. Glucose is the fundamental resource for hydrogen production. Glucose is fermented via the EMP pathway to pyruvate. Pyruvate oxidation to acetyl coenzyme A requires ferredoxin (Fd) reduction. Reduced Fd is oxidized by hydrogenase, which generates Fd and releases
electrons as molecular hydrogen. Therefore hydrogen production is the means by which bacteria lose excess electrons. The reaction is reversible and depends on hydrogen partial pressure (pH2 ), suggesting that hydrogen yield is signi®cantly in¯uenced by pH2 . Therefore it is important to control pH2 in the liquid phase for enhancement of hydrogen yield. Gas sparging is a useful method for decreasing pH2 although industrially a method of hydrogen utilisation, e.g. a fuel cell or metal hydrides, would be employed (Levy et al., 1981). The eects of hydrogen on the metabolism and the fermentative pattern of the anaerobic bacteria have been demonstrated in previous studies. Clostridium cellobioparum produces more hydrogen when it is removed by hydrogen-consuming methanogens (Chung, 1976). The quantitative composition of the fermentation products depends on the pH2 . Van Andel et al. (1985) demonstrated that sparging a pure culture of Clostridium butyricum with nitrogen increased the rate of acetate production both absolutely and relative to the rate of butyrate production. The production of acetate and hydrogen by Clostridium thermocellum has been considered an obstacle to the use of this organism in ethanol production (Lamed et al., 1988) and stirring the batch cultures favoured hydrogen and acetate production. This was attributed to accumulation of hydrogen at supersaturated concentrations in unstirred conditions inhibiting acetate production. The CSTR reactor used here was already well stirred. Other workers who have grown enrichment cultures on glucose-mineral salts medium in continuous culture include Nakamura et al. (1993) who studied the eect of HRT from 2±10 h on an enrichment from sewage sludge. These workers do not report the euent glucose concentrations, but assuming these were low, a hydrogen yield of 0.07 moles H2 /mole glucose consumed was achieved at a 2 h HRT, which gave the highest hydrogen % and total gas production. Ueno et al. (1996) also used a sewage sludge enrichment grown in continuous culture on a sugary industrial wastewater. The maximum hydrogen yield reported was 2.52 moles H2 /mole carbohydrate consumed (assuming carbohydrate has the formula C6 H12 O6 ) at a 12 h HRT and pH 6.8. Kumar et
Table 3 Volatile metabolic products in the liquid phasea
a
Product
Non-Sparging (mg lÿ1 )
Sparging (mg lÿ1 )
Acetic Propionic i-butyric n-butyric i-valeric n-valeric Acetone Ethanol Butanol
773 48 (3) 114 6 (3) 29 15 (3) 1742 31 (3) ND (3) 10 9 (3) ND (3) 58 20 (3) 4 1 (3)
785 79 (3) 74 1 (3) 13 2 (3) 1929 170 (3) 2 2 (3) 13 2 (3) 1 1 (3) 30 17 (3) 6 2 (3)
ND Not Detectable; ( ) number of samples.
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Table 4 Hydrogen yields reported in the literature Mixed culture this study Mixed culture this study Enterobacter aerogenes (Tanisho et al., 1998) Enterobacter aerogenes (Tanisho et al., 1998) Enterobacter aerogenes (Yokoi et al., 1995) Clostridium butyricum (Van Andel et al., 1985) Clostridium sp. strain No.2 (Taguchi et al., 1995) Citrobacter intermedius (Brosseau and Zajic, 1982) Clostridium pasteurianum (Brosseau and Zajic, 1982) Clostridium beijerinckii AM21B (Taguchi et al., 1995)
Condition
Substrate
H2 yield (mol/mol-glucose)
N2 sparging, continuous continuous Ar sparging, batch batch continuous continuous continuous batch batch batch
glucose glucose molasses molasses glucose glucose glucose glucose glucose glucose
1.43 0.85 1.58 0.52 1.00 1.60 1.61±2.36 1.00 1.50 1.3±2.0
al. (1995) using a strain of Bacillus licheniformis isolated from cattle dung report hydrogen yields in semi-continuous culture over 60 days of 0.37 moles/mole glucose utilised, though yields of up to 1.1 moles H2 /mole glucose were reported in shorter term batch-type experiments. It should be noted that in each case the gas volume was measured by the water displacement method, which may be less accurate than the gas meter measurements available in the present study. Hydrogen yields from pure culture studies reviewed by Nandi and Sengupta (1998) vary from 0.7 to 2.4 moles H2 /mole glucose, and a summary of yields from pure culture work is given in Table 4. The speci®c hydrogen production rate found here of 3.84 mmol hÿ1 gÿ1 dry weight of biomass (Table 2) compares well with rates for Clostridium pasteurianum of 2.5 mmol hÿ1 gÿ1 biomass in log growth phase in batch culture and 1.2 mmol hÿ1 gÿ1 biomass in stationary phase (Brosseau and Zajic, 1982). However, Taguchi et al. (1995) obtained speci®c hydrogen production rates with Clostridium sp. strain No. 2 grown on glucose in continuous culture of between 7.8 and 32.2 mmol hÿ1 gÿ1 dry weight of biomass depending on the retention time. It appears possible that at an industrial scale, energy could be recovered as hydrogen from an acidogenic reactor as part of a typical biological treatment for carbohydrate-containing wastewaters or from an acidogenic stage used to produce volatile fatty acids for chemical or liquid bio-fuel production (Levy et al., 1981). The euent from the hydrogen producing stage, already at 35°C, could then pass to a methanogenic stage for which the fermentation end products are ideal substrates, or the volatile products could be extracted to supply chemicals or be used as liquid bio-fuels (Levy et al., 1981; Brosseau and Zajic, 1982). The mixed culture used here grows also on sucrose and starch, tofu manufacturing waste, wheat bran and rice bran, in batch culture (Mizuno et al., 1997) so that this process could be of wide application to food industry wastewaters, which would then be seen as a resource for high grade renewable energy production.
4. Conclusions The hydrogen-producing enrichment culture was extremely stable when grown at pH 6.0 and a HRT of 8.5 h, re-growing well after dormancy and accidental washout. A ¯occulant biomass of concentration 1.5 g lÿ1 dry weight was obtained in the CSTR without sparging, a non-¯occulant biomass of a lower concentration resulting during sparging. Hydrogen yields of 0.85 and 1.43 mol/mol glucose were obtained under non-sparging and sparging conditions, respectively; sparging with nitrogen resulted in a 68% increase in hydrogen yield. No signi®cant change in fermentation end products was observed. Acknowledgements The authors wish to thank Dr. A.J. Guwy and Miss H. Forsey for their expert technical assistance, and the UK EPSRC for an equipment grant to DLH and FRH (GR/M38346). References Andel, J.G., Zoutberg, G.R., Crabbendam, P.M., Breure, A.M., 1985. Glucose fermentation by Clostridium butyricum grown under a self generated gas atmosphere in chemostat culture. Applied Microbiology and Biotechnology 23, 21±26. APHA, 1992. Standard Methods for the Examination of Waste and Wastewater, 18th ed. American Public Health Association, Washington, USA. Bahl, H., Gottschalk, G., 1984. Parameters aecting solvent production by Clostridium acetobutylicum in continuous culture. Biotechnology and Bioengineering Symposium 14, 215±223. Benemann, J., 1996. Hydrogen biotechnology: progress and prospects. Nature Biotechnology 14, 1101±1103. Brosseau, J.D., Zajic, J.E., 1982. Hydrogen-gas production with Citrobacter intermedius and Clostridium pasteurianum. Journal Chemical Technology and Biotechnology 32, 496±502. Chung, K.T., 1976. Inhibitory eects of H2 on growth of Clostridium cellobioparum. Applied and Environmental Microbiology 31, 342±348. Cohen, A., Zoetemeyer, R.J., Van Deursen, A., Van Andel, J.G., 1979. Anaerobic digestion of glucose with separated acid production and methane formation. Water Research 13, 571±580.
O. Mizuno et al. / Bioresource Technology 73 (2000) 59±65 Dabrock, B., Bahl, H., Gottschalk, G., 1992. Parameters aecting solvent production by Clostridium pasteurianum. Applied and Environmental Microbiology 58, 1233±1239. Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., Smith, F., 1956. Colorimetric method for determination of sugars and related substances. Analytical Chemistry 28, 350±356. ESPRIT, 1989. Research Report of the explosion of soybean-meal in a silo. ESPRIT Co. Ltd. (In Japanese). Guwy, A.J., Hawkes, D.L., Hawkes, F.R., 1995. On-line low ¯ow high-precision gas metering systems. Water Research 29, 977±979. Kalia, V.C., Jain, S.R., Kumar, A., Joshi, A.P., 1994. Fermentation of bio-waste to H2 by Bacillus licheniformis. World Journal of Microbiology and Biotechnology 10, 224±227. Kim, B.H., Bellows, P., Datta, R., Zeikus, J.G., 1984. Control of carbon and electron ¯ow in Clostridium acetobutylicum fermentations: utilization of carbon monoxide to inhibit hydrogen production and to enhance butanol yields. Applied and Environmental Microbiology 48, 764±770. Kumar, A., Jain, S.R., Sharma, C.B., Joshi, A.P., Kalia, V.C., 1995. Increased H2 production by immobilized microorganisms. World Journal of Microbiology and Biotechnology 11, 156±159. Lamed, R.J., Lobos, J.H., Su, T.M., 1988. Eect of stirring and hydrogen on fermentation products of Clostridium thermocellum. Applied and Environmental Microbiology 54, 1216±1221. Lay, J.J., Lee, Y.J., Noike, T., 1999. Feasibility of biological hydrogen production from organic fraction of municipal solid waste. Water Research 33 (11), 2579±2586. Levy, P.F., Sanderson, J.E., Kispert, R.G., Wise, D.L., 1981. Biore®ning of biomass to liquid fuels and organic chemicals. Enzyme Microbiol Technology 3, 207±215. Mizuno, O., Li, Y.Y., Noike, T., 1998. The behavior of sulfatereducing bacteria in acidogenic phase of anaerobic digestion. Water Research 32, 1626±1634. Mizuno, O., Ohara, T., Noike, T., 1997. Hydrogen Production from Food Processing Waste by Anaerobic Bacteria. Journal of Envi-
65
ronmental Systems and Engineering, JSCE (573/VII-5), 111±118 (in Japanese). Nakamura, M., Kanabe, H., Matsumoto, J., 1993. Fundamental studies on hydrogen production in the acid-forming phase and its bacteria in anaerobic treatment processes-the eects of solids retention time. Water Science Technology 28 (7), 81±88. Nandi, R., Sengupta, S., 1998. Microbial production of hydrogen: an overview. Critical Reviews in Microbiology. 24 (1), 61±84. Peck, M.W., Skilton, J.M., Hawkes, F.R., Hawkes, D.L., 1986. Eects of temperature shock treatments on the stability of anaerobic digesters operated on separated cattle slurry. Water Research 20 (4), 453±462. Roychowdhury, S., Cox, D., Levandowsky, M., 1988. Production of hydrogen by microbial fermentation. International Journal of Hydrogen Energy 13, 407±410. Ruzicka, M., 1996. The eect of hydrogen on acidogenic glucose cleavage. Water Research 30, 2447±2451. Taguchi, F., Mizukami, N., Saito-Taki, T., Hasegawa, K., 1995. Hydrogen production from continuous fermentation of xylose during growth of Clostridium sp. strain No.2.. Canadian Journal of Microbiology 41, 536±540. Tanisho, S., Kuromoto, M., Kadokura, N., 1998. Eect of CO2 removal on hydrogen production by fermentation. International Journal of Hydrogen Energy 23, 559±563. Ueno, Y., Otauka, S., Morimoto, M., 1996. Hydrogen production from industrial wastewater by anaerobic micro¯ora in chemostat culture. Journal of Fermentation and Bioengineering 82, 194±197. Yokoi, H., Ohkawa, T., Hirosse, J., Hayashi, S., Takasaki, Y., 1995. Characteristics of hydrogen production by aciduric Enterobacter aerogen strain HO-39. Journal of Fermentation and Bioengineering 80, 571±574. Zoetemeyer, R.J., Arnoldy, P., Cohen, A., Boelhouwer, C., 1982. In¯uence of temperature on the anaerobic acidi®cation of glucose in a mixed culture forming part of a two stage digestion process. Water Research 16, 313±321.