Characteristics of hydrogen production from food waste and waste activated sludge

This study was conducted for microbial hydrogen production from food waste and sewage sludge. Thirty three batch tests with different VS concentration (from 0.5 to 5.0 %, w/v) and mixing ratio of food waste to sewage sludge (from 0:100 to 100:0) were performed at 35°C. Heat-treated anaerobic sludge was used to seed the serum bottles. In all the tests, cumulative hydrogen production reached the maximum values within 2.5 days. n-Butyrate was produced simultaneously with hydrogen production, of which the amount was proportional to that of nbutyrate. Clostridium sp. are, therefore, considered to be the dominant microorganisms in this study because these microorganisms are responsible for n-butyrate fermentation. The hydrogen production potential of food waste was found over 34.0 mL/g VS at all the VS concentration. The maximum potential of 59.2 mL/g VS was found at 3.0 % of VS concentration. The potential decreased as sewage sludge composition increased due to the methanogens contained in sewage sludge and low carbohydrate concentration; however, the addition of sewage sludge to food waste enhanced hydrogen yield because of sufficient protein. The maximum hydrogen yield of 1.01 mole H2/mole hexoseadded was achieved at the food waste to sewage sludge ratio of 80:20 at the VS concentration of 3.0 %. The specific hydrogen production rate increased up to 22.6 mL H2/g VSS/h as both food waste composition and VS concentration increased

Journal of Water and Environment Technology, Vol.1, No.2, 2003 Characteristics of hydrogen production from food waste and waste activated sludge *Hang-Sik Shin1, Sang-Hyoun Kim1 and Byung-Chun Paik2 Department of Civil and Environmental Engineering, Korea Advanced Institute of Science and Technology, 373-1 Guseong-dong, Yuseong-gu, Daejeon, 305-701, Korea Division of Civil and Environmental Engineering, Yosu National University, San 96-1, Dundeok-dong, Yeosu-si, Jeollanam-do, 550-749, Korea ABSTRACT This study was conducted for microbial hydrogen production from food waste and sewage sludge Thirty three batch tests with different VS concentration (from 0.5 to 5.0 %, w/v) and mixing ratio of food waste to sewage sludge (from 0:100 to 100:0) were performed at 35°C Heat-treated anaerobic sludge was used to seed the serum bottles In all the tests, cumulative hydrogen production reached the maximum values within 2.5 days n-Butyrate was produced simultaneously with hydrogen production, of which the amount was proportional to that of nbutyrate Clostridium sp are, therefore, considered to be the dominant microorganisms in this study because these microorganisms are responsible for n-butyrate fermentation The hydrogen production potential of food waste was found over 34.0 mL/g VS at all the VS concentration The maximum potential of 59.2 mL/g VS was found at 3.0 % of VS concentration The potential decreased as sewage sludge composition increased due to the methanogens contained in sewage sludge and low carbohydrate concentration; however, the addition of sewage sludge to food waste enhanced hydrogen yield because of sufficient protein The maximum hydrogen yield of 1.01 mole H2/mole hexoseadded was achieved at the food waste to sewage sludge ratio of 80:20 at the VS concentration of 3.0 % The specific hydrogen production rate increased up to 22.6 mL H2/g VSS/h as both food waste composition and VS concentration increased Key Words: Food waste, hydrogen, mixing ratio, sewage sludge, VS concentration INTRODUCTION Due to the finite quantities and pollutants emission (CO2, CO, CnHm, Sox, NOx, ashes, etc.), fossil fuels should be alternated by renewable and non-polluting energy sources in recent - 177 - Journal of Water and Environment Technology, Vol.1, No.2, 2003 future (Momirlan and Veziroğlu, 1999) As a sustainable energy source with minimal or zero use of hydrocarbons, hydrogen is a promising alternative to fossil fuels With high energy yield (122 kJ/g), hydrogen is clean and renewable In addition, hydrogen can be directly used to produce electricity through fuel cells (Rifkin, 2002) Since conventional physico-chemical production methods (e.g water electrolysis or chemical cracking of hydrocarbons) require electricity derived from fossil fuel combustion, interest in biohydrogen production has increased significantly (BenneHawkes et al., 2002) Between two biological processes, fermentative process that uses refuse or organic wastes seems technically simpler than photosynthetic process Clostridium species (sp.) are the representative anaerobic fermentative hydrogen producing bacteria (Hawkes et al., 2002) Due to the ability to produce endospore, they can be easily selected from natural environments such as anaerobic sludge, compost and soil by inhibiting other bacteria using heat, acid/base, ultrasound, chemicals, freezing/thawing, etc (Sparling et al., 1997; Van Ginkel et al., 2001; Chen et al., 2002; Wang et al., 2003) Clostridium sp are also able to use wide range of biopolymers with various extracellular enzymes or enzyme complexes (Mitchell, 2001) Carbohydrates are the preferred organic carbon source for hydrogen producing fermentation Stoichiometrically, Clostridium sp can produce moles of hydrogen with mole of n-butyrate or moles of hydrogen with mole of acetate from mole of hexose In most cases using soluble defined substrates, hydrogen production yield and major byproduct were 0.7-2.1 mole/hexoseconsumed and n-butyrate, respectively (Mizuno et al., 2000; Fang and Liu, 2002) However, hydrogen was hardly produced from protein and lipids (Okamoto et al., 2000; Noike and Mizuno, 2000) Up to now, fermentative hydrogen production was studied using organic wastes such as high-strength wastewater (Ueno et al., 1996), lignocellulosic waste (Sparling et al., 1997), municipal solid waste (Lay et al., 1999; Okamoto et al., 2000; Lay et al., 2003), food manufacturing waste (Noike et al., 2000; Noike et al., 2002) and waste acitivated sludge (Wang et al., 2003a; Wang et al., 2003b) The maximum hydrogen production potentials were in the range of 10-70 mL H2/g VS However, systematic studies of the anaerobic fermentation of solid wastes are still lacking, although hydrogen yield and hydrogen production rate may significantly depend on the characteristics of organic wastes, such as water content, carbohydrate composition, carbohydrate/nutrient balance, etc Food waste and sewage sludge are the most abundant and problematic organic solid wastes in Korea The generation of food waste reaches 11,237 tons per day in Korea, accounting for 23.2 % of municipal solid wastes (Ministry of environment, 2002) Food waste is the major source of decay, odor, and leachate in collection and transportation due to the high volatile solids (VS; 80~90%) and moisture content (75~85%) Food waste, consolidated in landfills with other wastes, has resulted in serious environmental problems such as odor emanation, vermin attraction, toxic gas emission and groundwater contamination However, food waste - 178 - Journal of Water and Environment Technology, Vol.1, No.2, 2003 might be suitable for anaerobic hydrogen production, because it is the carbohydrate-rich, and easily hydrolysable waste (Han and Shin, 2002) 5,689 tons of digested and dewatered sewage sludge cakes are generated per day (Ministry of environment, 2002) Now, 72 % of them are disposed by ocean dumping, however it will be prohibited according to London Convention in recent future The enhancement of anaerobic digester is, therefore, urgent to reduce the amounts of the sludge cakes and improve the quality for reuse Phase separation and/or codigestion with carbon-rich waste is known as an economic and feasible approach to retrofit the conventional digester (Lafitte-Trouquē and Forster, 2000; Schafer and Farrell, 2000) If hydrogen can be produced in acidogenesis of sewage sludge or sewage sludge/food waste codigestion, sewage sludge will be the important source for hydrogen production due to its amounts Thus, in this work, food waste and sewage sludge were used for fermentative production of hydrogen Effects of VS concentrations and mixing ratio of two substrates were investigated by serum bottle tests MATERIALS AND METHODS Seed The seed sludge was taken from an anaerobic digester in a local wastewater treatment plant and heat-shocked at 90°C for 10 to inhibit the bioactivity of hydrogen consumers and to harvest spore-forming anaerobic bacteria (Hawkes et al., 2002) The pH value, alkalinity, and volatile suspended solids (VSS) concentration of the sludge were 7.6, 2.83 g CaCO3/L, and 5.5 g/L, respectively Substrate The feed was a mixture of food waste and sewage sludge, representing typical Korean food waste and sewage sludge Food waste, sampled from a dining hall, was crushed by an electrical blender under anaerobic condition Sewage sludge was sampled from a local wastewater treatment plant All the substrates were filtered through a stainless steel sieve (U.S Mesh No 10 with corresponding sieve opening of 2.00 mm) The characteristics of the substrate were summarized in Table Operating procedure The experiments were conducted using 415 mL Wheaton media lab bottles A total of 33 bottles with different volatile solids (VS) concentrations and mixing ratios of food waste and sewage sludge were simultaneously operated Total VS concentrations were controlled to 0.5, 1.0, 1.5, 2.0, 3.0, and 5.0 % The mixing ratios of food waste to sewage sludge were designed 100:0, 80:20, 60:40, 40:60, 20:80, and 0:100 on VS basis; however, the experiments at 20:80 - 179 - Journal of Water and Environment Technology, Vol.1, No.2, 2003 and 0:100 for 3.0 and 5.0 % of total VS concentrations could not be conducted because of low VS concentration of sewage sludge 20 mL of seed sludge and appropriate amounts of food waste and sewage sludge were added in individual bottles Each bottle was supplemented with 200 mg of KH2PO4, 14 mg of MgCl2•4H2O, mg of Na2MoO4•4H2O, mg of CaCl2•2H2O, 2.5 mg of MnCl2•6H2O, and 10 mg of FeCl2•4H2O, which was modified from Lay et al (1999) NaHCO3 was also added to adjust total carbohydrate : alkalinity ratio to 1.0±0.1 Each bottle was then filled to 200 mL with distilled water and pH value was adjusted to 6.0 using either M HCl or M KOH Subsequently, the headspaces of the bottles were flushed with N2 gas for and the bottles were tightly sealed using open-top screw caps with rubber septum The bottles were then placed in a reciprocating shaker at 35oC and 100 rpm The biogas production was determined using a glass syringe of 20-200 mL (Owen et al., 1979) At the same time, gas composition was measured and the sample from the supernatant was taken to analyze pH and organic concentrations If the pH value was out of the range from 5.0 to 6.0, it was re-adjusted using injection of either M HCl or M KOH by syringes Table Characteristics of substrate Parameter Total solids Volatile solids Total COD Soluble COD Total carbohydrate Soluble carbohydrate Total protein Soluble protein Total Kjeldahl nitrogen pH Alkalinity Unit Food waste Sewage sludge %, w/v %, w/v g/L mg/L g COD/L mg COD/L g COD/L mg COD/L g N/L 15.92 15.17 158.4 50,300 84.9 33,100 37.7 5,793 4.4 4.6 0.4 5.01 2.53 31.9 143.1 5.0 71.4 18.4 68.2 2.3 7.5 4.7 g CaCO3/L Analytical methods Hydrogen content in biogas was measured by a gas chromatography (GC, Gow Mac series 580) using a thermal conductivity detector and a 1.8 m × 3.2 mm stainless-steel column packed with molecular sieve 5A with N2 as carrier gas The contents of CH4, N2, and CO2 were measured using a GC of the same model noted previously with a 1.8 m × 3.2 mm stainless-steel column packed with porapak Q (80/100 mesh) using helium as a carrier gas The temperatures of injector, detector, and column were kept at 80, 90, and 50°C, respectively, in both GCs VFA (C2-C6), and lactate were analyzed by a high performance liquid chromatograph (Spectrasystem P2000) with an ultraviolet (210 nm) detector and an 300 mm × - 180 - Journal of Water and Environment Technology, Vol.1, No.2, 2003 7.8 mm Aminex HPX-97H column using H2SO4 of 0.005 M as mobile phase Aliphatic alcohol was determined using another high performance liquid chromatograph (DX-600, Dionex) with an electrochemical detector (ED50A) and an 250 mm × mm Dionex CarboPac PA10 column using NaOH of 0.01 M as mobile phase The liquid samples were pretreated with 0.45 µm membrane filter before injection to both HPLCs Chemical oxygen demands (COD), Suspended solids (SS), VSS, TKN, ammonia, and pH were determined according to Standard Methods (APHA, 1998) Carbohydrate was determined by the colorimetric method of Dubois et al (1956) with UV wavelength at 480, 484 and 490 nm using glucose as standard Soluble protein was also measured by the colorimetric method at a wavelength of 562 nm with bovine serum albumin as standard (Smith et al., 1985) Total protein was calculated from organic nitrogen (9.375 g COD/g organic nitrogen) (Miron et al., 2000) RESULTS AND DISCUSSION Fermentation characteristics In all the tests, cumulative hydrogen production reached the maximum values within 2.5 days as shown in Fig The hydrogen production curve was fitted to a modified Gompertz equation (1), which has been used as a suitable model for describing the hydrogen production in batch tests (Lay, 2001; Lee et al., 2001; Chen et al., 2002) H = P ∗ exp[− exp{ Rm (λ − t )e + 1}] P (1) where H was cumulative hydrogen production (mL), P was hydrogen production potential (mL), Rm was hydrogen production rate (mL/day), λ was lag-phase time (days), and e was exponential All the correlation coefficients, R2, were larger than 0.98 Additionally, all the t-values for parameters were larger than t0.975, = 2.571 (table value) The specific hydrogen production potential (mL/g VS) was obtained by dividing P by the substrate weight (g VS), while the specific hydrogen production rate was calculated by dividing Rm by the inoculum weight (g VSS) Hydrogen production yield (mole/mole hexose) was determined by dividing P by 22,400 (mL/mole) and by either carbohydrate added (mole hexose) or carbohydrate consumed (mole hexose) Lag-phase times (λ) for hydrogen production were not longer than 0.8 day, which was shorter than reported values (2-4 days) in batch tests with heat-treated inocula (Lay et al., 1999; Lay 2001) It was seemed that environmental conditions such as substrate, amino acids, inorganic nutrients, and pH were sufficient for spore germination in this study (Hawkes et al., 2002) - 181 - Journal of Water and Environment Technology, Vol.1, No.2, 2003 Carbohydrate (mg COD/L) Cumulative CH4 production (mL, STP) Cumulative H2 production (mL, STP) 250 200 150 100 50 30 25 20 15 10 12000 Food waste composition 1.00 Food waste composition 0.80 Food waste composition 0.60 Food waste composition 0.40 Food waste composition 0.20 Food waste composition 0.00 Organic acids (mg COD/L) 10000 8000 6000 4000 2000 12000 10000 8000 6000 4000 2000 n-Butyrate (mg COD/L) 5000 4000 3000 2000 1000 Alcohols (mg COD/L) 5000 4000 3000 2000 1000 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Time (days) Fig Development of biogas (H2 and CH4), carbohydrate and soluble products with time at 2.0 % of VS concentration Methane was observed in all the bottles where sewage sludge was added, due to methanogenic bacteria in sewage sludge (Wang et al., 2003) However, the amount of methane was less than 8.1 mL/g VS, which was much lower than reported values (17.5 L/g VS) in which the methanogenic bacteria was externally dosed (Chu et al., 2002) Carbohydrate degradation and organic acids production almost ceased as hydrogen production ended up In most cases, nbutyrate was produced simultaneously with hydrogen production Simultaneous nbutyrate production with hydrogen was also reported in anaerobic hydrogen fermentation (Lay et al., 1999; Noike et al., 2000), meaning that Clostridium sp were related with hydrogen production in this study (Payot et al., 1998; Yokoi et al., 1998) H2/VFA production was followed by alcohols production In normal batch culture, Clostridium sp form H2/VFAs during the exponential growth phase, and alcohols in the late growth phase (Lay et al., 1999; Ueno et al., 2001) Alcohols represent hydrogen that has not been liberated as a gas (Hawkes et al., 2002) Ethanol was the most abundant alcohol, and small amounts of 2propanol, butanol and 2-pentanol were also detected Effects of VS concentration and mixing ratios on hydrogen fermentation The hydrogen production potential of food waste was found over 34.0 mL/g VS at all the VS concentrations as shown in Fig It is known that hydrogen production using concentrated substrates higher than 1% TS is needed for suitable energy production system - 182 - Journal of Water and Environment Technology, Vol.1, No.2, 2003 (Hawkes et al., 2002) In this study, hydrogen production potential increased as VS concentration increased up to 3.0 % (3.15 % as TS) The maximum potential was 59.2 mL/g VS, which was in the range of reported maximum potential of carbohydrate-rich biomass such as rice bran, carrot, cabbage (Okamoto et al., 2000; Noike et al., 2000) The hydrogen production potential decreased as VS concentration increased further Product inhibition by H2 and VFAs might cause the decrease in the hydrogen production potential at 5.0 % of VS concentration (Van Ginkel et al., 2001; Lay, 2001) The hydrogen production potential decreased as sewage sludge composition increased Hydrogen production over 2.2 mL/g VS could be achieved only when food waste composition was higher than 20% The reasons of insignificant hydrogen production from sewage sludge might be the methanogens contained in sludge and low carbohydrate concentration (Wang et al., 2003a) 5 15 25 30 35 However, the maximum hydrogen yield of 1.01 mole H2/mole hexoseadded (1.12 mole 45 35 50 15 H2/mole hexoseconsumed) was found at the 25 30 40 20 55 mixing ratio of 80:20 (food waste:sewage 10 45 50 35 sludge) and at the VS concentration of 3.0 % 55 30 40 15 25 as shown in Fig Addition of sewage 50 20 45 sludge to 20 % of total VS enhanced 10 35 40 20 30 25 hydrogen yield (based on carbohydrate) at 15 20 35 VS concentrations ranging 1.0 to 5.0 % It 10 30 25 15 20 was reported that adequate control of 20 40 60 80 100 Food waste composition (%, VS basis) inorganic nutrient can enhance the hydrogen Fig Constant hydrogen production production (Hawkes et al., 2002) In this potential (mL H2/g VS) contour lines study, however, the concentrations of against food waste composition and nutrients such as phosphorus and iron were VS concentration sufficiently supplemented (Lee et al., 2001; Fang et al., 2002) Nitrogen was not externally dosed, but carbohydrate to nitrogen ratio was less than 19.3 g carbohydrate-COD/g TKN-N, which meant nitrogen was also sufficient (Mizuno et al., 2000; Van Ginkel et al., 2001; Lin and Chang, 2003) Protein would be a better explanation for the synergic effect It was well known that protein such as peptone or yeast extract was better a nitrogen source than ammonium salts or urea for activation and growth of Clostridium sp (Mitchell, 2001) Addition of protein was helpful or even indispensable, sometimes, for the hydrogen production in both pure and mixed culture (Taguchi et al., 1996; Ueno et al., 2001; Yokoi et al., 2001) Food waste is a carbohydrate-rich waste (0.56 g carbohydrate-COD/g VS and 0.25 g protein-COD/g VS), while sewage sludge is a protein-rich waste (0.20 g carbohydrate-COD/g VS and 0.73 g protein-COD/g VS) Addition of sewage sludge from to 20 % of total VS decreased carbohydrate to protein ratio 20 40 VS concentration (%) 10 - 183 - Journal of Water and Environment Technology, Vol.1, No.2, 2003 from 2.24 to 1.85 g carbohydrate-COD/g protein-COD The specific hydrogen production rate increased as both food waste composition and VS concentration increased as shown in Fig It was reported that the production rate was less inhibited than the production yield (Lay, 2001; Van Ginkel et al., 2001) The maximum hydrogen production rate was 22.6 mL H2/g VSS/h, which was in the range of reported values in serum bottle tests using organic wastes (Lay et al., 1999; Lay, 2001) 5 0.10 0.20 0.60 0.30 10 0.40 0.50 14 20 4 0.60 0.10 0.20 0.30 0.80 0.70 0.80 0.50 0.40 0.90 0.90 0.90 0.60 0.10 0.20 0.70 0.30 0.80 0.50 0.40 0.80 0.60 VS concentration (%) VS concentration (%) 18 0.70 22 16 12 16 12 10 18 14 16 12 10 0.70 14 0.30 0.10 0.20 20 40 0.50 0.40 0.60 0.30 0.20 0.10 12 4 10 60 2 0.50 0.40 80 100 Food waste composition (%, VS basis) Fig Constant hydrogen yield (mole H2/mole hexoseadded) contour lines against food waste composition and VS concentration 20 40 60 80 100 Food waste composition (%, VS basis) Fig Constant specific hydrogen production rate (mL H2/g VSS./h) contour lines against food waste composition and VS concentration CONCLUSIONS Food waste and sewage sludge at various VS concentration (from 0.5 to 5.0 %, w/v) and mixing ratio of food waste to sewage sludge (from 0:100 to 100:0) were used for fermentative production of hydrogen After lag-phase shorter than 0.8 day, hydrogen was produced rapidly The metabolic results indicated that the characteristics of the heat-shocked digester sludge converting the organic wastes were similar to those of anaerobic spore-forming bacteria, Clostridium sp The hydrogen production potential of food waste was found over 34.0 mL/g VS at all the VS concentrations The maximum potential of 59.2 mL/g VS was found at 3.0 % of VS concentration The potential decreased as sewage sludge composition increased The maximum hydrogen yield of 1.01 mole H2/mole hexoseadded was, however, achieved at the sewage sludge composition of 20 % and at the VS concentration of 3.0 % Increase of protein concentration by adding sewage sludge might cause the synergic effect The specific - 184 - Journal of Water and Environment Technology, Vol.1, No.2, 2003 hydrogen production rate increased up to 22.6 mL H2/g VSS/h as both food waste composition and VS concentration increased Use of food waste and sewage sludge as the main and the auxiliary substrates seems feasible way to produce hydrogen ACKNOWEDGEMENT This work was supported by grant No M1-0203-00-0063 from the National Research Laboratory Program of the Korean Ministry of Science and Technology REFERENCES APHA, AWWA, WEF, 1998 Standard Methods for the examination of Water and Wastewater, 20 th ed., APHA, Washington, D.C Chen, C.-C., Lin, C.-Y and Lin, M.-C., 2002 Acid-base enrichment enhances anaerobic hydrogen production process, Appl Microbiol Biotechnol., 58, 224-228 Chu, C P., Lee, D J., Chang, B.-V and You, C S., 2002 Weak ultrasonic pretreatment on anaerobic digestion of polyelectrolyte flocculated activated biosolids Wat Res 36, 26812688 Dubois, M., Gilles, K A., Hamilton, J K., Rebers, P A and Smith, F., 1956 Colorimetric method for determination of sugars and related substances Anal Chem., 28, 350-356 Fang, H H P and Liu, H., 2002 Effect of pH on hydrogen production from glucose by a mixed culture, Bioresource Technol., 82, 87-93 Hallenbeck, P C and Benemann, J R., 2002 Biological hydrogen production; fundamentals and limiting processes, Int J Hydrogen Energy, 27, 1185-1193 Han, S.-K and Shin, H.-S., 2002 Enhanced acidogenic fermentation of food waste in a continuous-flow reactor, Waste Manage Res., 20, 110-118 Hanaki, K., Matsuo, T and Nagase, M, 1981 Mechanism of inhibition caused by long-chain fatty acids in anaerobic digestion process, Biotechnol Bioeng., 23, 1591-1610 Hawkes, F R., Dinsdale, R., Hawkes, D L., Hussy, I., 2002 Sustainable fermentative hydrogen production: challenges for process optimization Int J hydrogen energy, 27, 1339-1347 Lafitte-Trouquē, S and Forster, C F., 2000 Dual anaerobic co-digestion of sewage sludge and confectionery waste, Bioresource Technol., 71, 77-82 Lay, J.-J., 2000 Modeling and optimization of anaerobic digested sludge converting starch to hydrogen, Biotech Bioeng., 68(3), 269-278 Lay, J.-J., 2001 Biohydrogen generation by mesophilic anaerobic fermentation of microcrystalline cellulose, Biotechnol Bioeng., 74(4), 280-287 Lay J.-J., Lee, Y J and Noike, T., 1999 Feasibility of biological hydrogen production from - 185 - Journal of Water and Environment Technology, Vol.1, No.2, 2003 organic fraction of municipal solid waste Wat Res 33(11), 2579-2586 Lay, J.-J., Fan, K.-S., Chang, J.-1, Ku and C.-H., 2003 Influence of chemical nature of organic wastes on their conversion to hydrogen by heat-shock digested sludge Int J Hydrogen Energy, 28, 1361-1367 Lee, Y J., Miyahara, T and Noike, T., 2001 Effect of iron concentration on hydrogen fermentation Bioresource Technol., 80, 227-231 Lin, C Y and Lay, C H., 2003 Carbon/nitrogen-ratio effect on fermentative hydrogen production by mixed microflora, Int J Hydrogen Energy (accepted) Ministry of Environment, 2002 The state of solid waste generation and treatment in 2001, Seoul, Korea: Ministry of Environment, Korea Miron, Y., Zeeman, G, Van Lier, J B and Lettinga, G., 2000 The role of sludge retention time in the hydrolysis and acidification of lipids, carbohydrates and proteins during digestion of primary sludge in CSTR systems, Wat Res., 34(5), 1705-1713 Mitchell, W J., 2001 Biology and physiology In: Bahl, H and Pűrre, P., Clostrida: Biotechnology and medical applications Weinheim, Germany: Wiley-VCH, pp 53-68 Mizuno, O., Dinsdale, R., Hawkes, F R., Hawkes, D L and Noike, T., 2000 Enhancement of hydrogen production from glucose by nitrogen gas sparging, Bioresource Technol., 73, 59-65 Momirlan, M and Veziroğlu, T., 1999 Recent directions of world hydrogen production, Renew Sust Energ Rev., 3, 219-231 Noike, T and Mizuno, O., 2000., Hydrogen fermentation of organic municipal wastes, Wat Sci Tech., 42(12), 155-162 Noike, T., Takabatake, H., Mizuno, O and Ohba, M., 2002 Inhibition of hydrogen fermentation of organic wastes by lactic acid bacteria, Int J Hydrogen Energy, 27, 13671371 Okamoto, M., Miyahara, T., Minuno, O and Noike, T., 2000 Biological hydrogen potential of materials characteristic of the organic fraction of municipal solids wastes, Wat Sci Tech., 41(3), 25-32 Owen, W F and Stuckey D C., Healy, J B., Jr., Young, L Y and McCarty, P L., 1979 Bioassay for monitoring biochemical methane potential and anaerobic toxicity Wat Res., 13, 485-493 Payot, R., Guedon, E., Cailliez, C., Gelhage, E and Petitdemange, H., 1998 Metabolism of cellobiose by Clostridium celluolyticum growing in continuous culture: evidence for decreased NADH reoxidation as a factor limiting growth, Microbiology, 144, 375-384 Rifkin, J., 2002 The hydrogen economy: the creation of the worldwide energy web and the redistribution of the power on earth New Work, NY, US: Penguin Putnam, pp 15-17 Schafer, P L and Farrell, J B., 2000 Advanced anaerobic digestion systems, Wat Environ Tech., 12(11), 26-32 - 186 - Journal of Water and Environment Technology, Vol.1, No.2, 2003 Smith, P K., Krohn, R I., Hermanson, G T., Mallia, A K., Gartner, F H., Provenzano, M D., Fujimoto, E K., Goeke N M., Olson, B J and Klenk D C., 1985 Measurement of protein using bicinchoninic acid, Anal Biochem., 150, 76-85 Sparling, R., Risbey, D and Poggi-Varaldo, H M., 1997 Hydrogen production from inhibited anaerobic composters, Int J Hydrogen Energy, 22(6), 563-566 Taguchi, F., Yamada, K., Hasegawa, K., Taki-Saito, T and Hara, K., 1996 Continuous hydrogen production by Clostridium sp Strain No from cellulose hydrolysate in an aqueous two-phase system, J Ferment Bioeng., 82(1), 80-83 Ueno, Y., Otauka, S and Morimoto, M., 1996 Hydrogen production from industrial wastewater by anaerobic microflora in chemostat culture, J Ferment Bioeng., 82, 94-207 Ueno, Y., Haruta, S., Ishii, M and Igarashi, Y., 2001 Microbial community in anaerobic hydrogen-producing microflora enriched from sludge compost, Appl Microbiol Biotechnol., 57, 555-562 Van Ginkel, S., Sung, S and Lay, J.-J., 2001 Biohydrogen production as a function of pH and substrate concentration, Environ Sci Technol., 35, 4726-4730 Wang, C C., Chang, C.W., Chu, C P., Lee, D J., Chang, B.-V., Liao, C S and Tay, J H., 2003a Using filtrate of waste biosolids to effectively produce bio-hydrogen by anaerobic fermentation, Wat Res., 37, 2789-2793 Wang, C C., Chang, C W., Chu C P., Lee D J., Chang, B.-V and Liao, C S., 2003b Producing hydrogen from wastewater sludge by Clostridium bifermentans, J Biotechnol., 102, 83-92 Yokoi, H., Tokushige, T., Hirose, J., Hayashi, S and Takasaki, Y., 1998 H2 production from starch by a mixed culture of Clostridium butyricum and Enterobacter aerogenes, Biotechnology Letters, 20(2), 143-147 Yokoi, H., Saitsu, A., Uchida, H., Hirose, J., Hayashi, S and Takasaki, Y., 2001 Microbial hydrogen production from sweet potato starch residue, J Biosci Bioeng., 91(1), 58-63 - 187 - ... for 3.0 and 5.0 % of total VS concentrations could not be conducted because of low VS concentration of sewage sludge 20 mL of seed sludge and appropriate amounts of food waste and sewage sludge. .. produce moles of hydrogen with mole of n-butyrate or moles of hydrogen with mole of acetate from mole of hexose In most cases using soluble defined substrates, hydrogen production yield and major... typical Korean food waste and sewage sludge Food waste, sampled from a dining hall, was crushed by an electrical blender under anaerobic condition Sewage sludge was sampled from a local wastewater