Continuous Biohydrogen Production from Starch with Granulated

Energy & Fuels 2008, 22, 93–97

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Continuous Biohydrogen Production from Starch with Granulated Mixed Bacterial Microflora† Ching-Hsiung Wang‡ and Jo-Shu Chang*,§ Department of Biological Engineering, Yung Ta Institute of Technology and Commerce, Pingtung, Taiwan, and Department of Chemical Engineering, National Cheng Kung UniVersity, Tainan, Taiwan ReceiVed May 27, 2007. ReVised Manuscript ReceiVed August 21, 2007

This work demonstrates a continuous-flow biohydrogen producing system able to produce H2 from starch at a high volumetric rate of over 4 L h–1 L–1. Using phosphate-buffered medium containing cassava starch (15 g/L) as the feed, sludge granulation occurred within 15 days after start-up while operating at a hydraulic retention time (HRT) of 2.2 h, enabling efficient biomass retention in the bioreactor. Operation at a progressively decreasing HRT of 5.3–0.5 h gave rise to a H2 content of nearly 50% (i.e., a CO2/H2 ratio of 1.0) and a H2 yield of 0.97–1.43 mol of H2/mol of hexose, which is 26–37% from the theoretical value. Operation at 0.5 h of HRT gave the highest H2 production rate of 4.12 L h–1 L–1, respectively, while further shortening of the HRT resulted in a washout of cells. The H2 production rate obtained from this work appears to be much higher than those indicated in comparable studies using starch to produce H2 via dark fermentation. The soluble metabolites were dominated by butyric acid (54–72% of total soluble products), followed by ethanol and acetic acid. The results indicate the feasibility of using starch as an inexpensive carbon substrate for high-rate and low-cost production of bio-H2 via a granular-sludge-based continuous-flow bioreactor.

1. Introduction Hydrogen gas (H2) is a clean energy carrier that has been recognized as the most promising alternative to fossil fuels.1,2 A cost-effective and sustainable supply of H2 is a crucial component in the infrastructure of H2 energy implementation. Biological H2 production is of particular interest because it is clean, environmentally friendly, and recyclable.1 Fermentative H2 production from converting organic substrates into H2 enables the production of H2 through waste reduction. Dark H2 fermentation could achieve a much higher H2 production rate than the other bio-H2 systems,1 thereby being considered the most economically feasible way of producing bio-H2. One of the key factors determining the commercial viability of bio-H2 is the production cost. It is well-known that sugars, such as glucose and sucrose, are the most efficient substrates for dark fermentative H2 production.1,2 As a result, the majority of H2 fermentation studies concentrated on the conversion of carbohydrates into H2.3–8 However, sugars are apparently not † Presented at the International Conference on Bioenergy Outlook 2007, Singapore, April 26–27, 2007. * To whom correspondence should be addressed. Fax: +886-6-2357146. E-mail: [email protected]. ‡ Yung Ta Institute of Technology and Commerce. § National Cheng Kung University. (1) Das, D.; Verziroglu, T. N. Hydrogen production by biological processes: A survey of literature. Int. J. Hydrogen Energy 2001, 26, 13– 28. (2) Kapdan, I. K.; Kargi, F. Bio-hydrogen production from waste materials. Enzyme Microb. Technol. 2006, 38, 569–582. (3) Lee, K. S.; Wu, J. F.; Lo, Y. S.; Lo, Y. C.; Lin, P. J.; Chang, J.-S. Anaerobic hydrogen production with an efficient carrier-induced granular sludge bed bioreactor. Biotechnol. Bioeng. 2004, 87, 648–657. (4) Chang, F. Y.; Lin, C. Y. Biohydrogen production using up-flow anaerobic sludge blanket reactor. Int. J. Hydrogen Energy 2004, 29, 33– 39. (5) Logan, B. E.; Oh, S. E.; Kim, I. S.; van Ginkel, S. W. Biological hydrogen production measured in batch anaerobic respirometers. EnViron. Sci. Technol. 2002, 36, 2530–2536.

economically feasible substrates for bio-H2 commercialization.9 Other inexpensive, abundant, and efficient feedstocks must be explored for fermentative H2 production.2 Starch and cellulosic substances, especially those deriving from agriculture wastes, appeared to be good candidates for energy feedstock.2,10–16 Early investigations on converting those organic wastes to H2 mainly focused on pure cultures aiming at achieving a high yield,12,14,17,18 while in some cases, mixed cultures were also used to produce H2 from organic wastes.8,19,20 (6) Chen, W. M.; Tseng, Z. J.; Lee, K. S.; Chang, J.-S. Fermentative hydrogen production with Clostridium butyricum CGS5 isolated from anaerobic sewage sludge. Int. J. Hydrogen Energy 2005, 30, 1063–1070. (7) Hussy, I.; Hawkes, F. R.; Dinsdale, R.; Hawkes, D. L. Continuous fermentative hydrogen production from a wheat starch co-product by mixed microflora. Biotechnol. Bioeng. 2003, 84, 619–626. (8) Khanal, S. K.; Chen, W. H.; Li, L.; Sung, S. Biological hydrogen production: Effects of pH and intermediate products. Int. J. Hydrogen Energy 2004, 29, 1123–1131. (9) van Ginkel, S. W.; Oh, S. E.; Logan, B. E. Biohydrogen gas production from food procession and domestic wastewaters. Int. J. Hydrogen Energy 2005, 30, 1535–1542. (10) Han, S. K.; Shin, H. S. Biohydrogen production by anaerobic fermentation of food waste. Int. J. Hydrogen Energy 2004, 29, 569–577. (11) Fan, Y. T.; Zhang, Y. H.; Zhang, S. F.; Hou, H. W.; Ren, B. Z. Efficient conversion of wheat straw waste into biohydrogen gas by cow dung compost. Bioresour. Technol. 2006, 97, 500–505. (12) Evvyernie, D.; Morimoto, K.; Karita, S.; Kimura, T.; Sakka, K.; Ohmiya, K. Conversion of chitinous waste to hydrogen gas by Clostridium paraputrificum M-21. J. Biosci. Bioeng. 2001, 91, 339–343. (13) Yu, H.; Zhu, Z.; Hu, W.; Zhang, H. Hydrogen production from rice winery wastewater in an upflow anaerobic reactor by using mixed anaerobic cultures. Int. J. Hydrogen Energy 2002, 27, 1359–1365. (14) Yokoi, H.; Saitsu, A.; Uchida, H.; Hirose, J.; Hayashi, S.; Takasaki, Y. Microbial hydrogen production from sweet potato starch residue. J. Biosci. Bioeng. 2001, 91, 58–63. (15) Yokoi, H.; Maki, R.; Hirose, J.; Hayashi, S. Microbial production of hydrogen from starch-manufacturing wastes. Biomass Bioenergy 2002, 22, 389–395. (16) Valdez-Vazquez, I,; Sparling, R.; Risbey, D.; Rinderkneccht-Seijas, N.; Poggi-Varaldo, H. M. Hydrogen generation via anaerobic fermentation of paper mill wastes. Bioresour. Technol. 2005, 96, 1907–1913.

10.1021/ef700274z CCC: $40.75  2008 American Chemical Society Published on Web 09/28/2007

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Although starch is one of the most abundant organic compounds on earth and is much cheaper than sugars, there is surprisingly little information in the literature regarding the use of starch for bio-H2 production.18–20 Therefore, in the present study, starch was selected as the feedstock for H2 production. To achieve mass production of H2, a continuously feeding process is more feasible from an engineering aspect and was used in this work. In continuous culture, faster substrate feeding usually leads to a higher H2 production rate, but cell washout would occur when the dilution rate is too fast. In particular, starch is a complicated substrate requiring additional hydrolysis steps before converting to a more assimilable reducing sugar, resulting in slow cell growth for H2-producing bacteria. The feature of the low cell growth rate would challenge the operation of continuous H2 fermentation, because a low dilution rate [or a long hydraulic retention time (HRT)] may need to be used to avoid cell washout. The slow substrate feeding rate would thus restrict the H2 production rate. To overcome this limitation, several engineering approaches could be adopted. For instance, better cell retention may be achieved by cell immobilization via matrix entrapment,21–23 carrier attachment,24–26 or membrane restriction.27 Furthermore, the formation of granular sludge was also shown to be an effective way of improving cell retention against the hydraulic dilution arising from medium feeding.3,28–30 In particular, we recently developed an efficient granular sludge bed system able to retain over 50 g of volatile suspended solids (VSS)/L of biomass in the reactor at a short HRT of 0.5 h, leading to a high H2 production rate of 7.3 L h–1 L–1.3 In this work, continuous H2 production was conducted under a progres(17) Nielsen, A. T.; Amandusson, H.; Bjorklund, R.; Dannetun, H.; Ejlertsson, J.; Ekedahl, L. G.; Lundstrom, I.; Svensson, B. H. Hydrogen production from organic waste. Int. J. Hydrogen Energy 2001, 26, 547– 550. (18) Zhu, H.; Ueda, S.; Asada, Y.; Miyake, J. Hydrogen production as a novel process of wastewater treatment—Studies on tofu wastewater with entrapped R. sphaeroides and mutagenesis. Int. J. Hydrogen Energy 2002, 27, 1349–1357. (19) Zhang, T.; Liu, H.; Fang, H. H. P. Biohydrogen production from starch in wastewater under thermophilic condition. J. EnViron. Manage. 2003, 69, 149–156. (20) Liu, G.; Shen, J. Effects of culture and medium condition on hydrogen production from starch using anaerobic bacteria. J. Biosci. Bioeng. 2004, 98, 251–256. (21) Wu, S. Y.; Lin, C. N.; Chang, J. S.; Chang, J.-S. Biohydrogen production with anaerobic sludge immobilized by ethylene–vinyl acetate copolymer. Int. J. Hydrogen Energy 2005, 30, 1375–1381. (22) Wu, S. Y.; Hung, C. H.; Lin, C. N.; Chen, H. W.; Lee, A. S.; Chang, J.-S. Fermentative hydrogen production and bacterial community structure in high-rate anaerobic bioreactors containing silicone-immobilized and selfflocculated sludge. Biotechnol. Bioeng. 2006, 93, 934–946. (23) Zhu, H.; Suzuki, T.; Tsygankov, A. A.; Asada, Y.; Miyake, J. Hydrogen production from tofu wastewater by Rhodobacter sphaeroides immobilized in agar gels. Int. J. Hydrogen Energy 1999, 24, 305–310. (24) Chang, J.-S.; Lee, K. S.; Lin, P. J. Biohydrogen production with fixed-bed bioreactors. Int. J. Hydrogen Energy 2002, 27, 1167–1174. (25) Tanisho, S.; Ishiwata, Y. Continuous hydrogen production from molasses by fermentation using urethane foam as a support of flocks. Int. J. Hydrogen Energy 1995, 20, 541–545. (26) Kumar, N.; Das, D. Continuous hydrogen production by immobilized Enterobacter cloacae T-BT 08 using lignocellulosic materials as solid matrices. Enzyme Microb. Technol. 2001, 29, 280–287. (27) Lee, K. S.; Lin, P. J.; Fangchiang, K.; Chang, J.-S. Continuous hydrogen production by anaerobic mixed microflora using a hollow-fiber microfiltration membrane bioreactor. Int. J. Hydrogen Energy 2007, published online,, 10.1016/j.ijhydene.2006.09.018. (28) Kim, J. O.; Kin, Y. H.; Ryu, J. Y.; Song, B. K.; Kim, I. H.; Yeo, S. H. Immobilization methods for continuous hydrogen gas production biofilm fortion versus granulation. Process Biochem. 2005, 43, 1331–1337. (29) Lee, K. S.; Lo, Y. C.; Lin, P. J.; Chang, J.-S. H2 production with anaerobic sludge using activated-carbon supported packed-bed bioreactors. Biotechnol. Lett. 2003, 25, 113–138. (30) Oh, S. U.; Iyer, P.; Bruns, M. A.; Logan, B. E. Biological hydrogen production using membrane bioreactor. Biotechnol. Bioeng. 2004, 87, 119– 127.

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sively decreasing HRT, using mixed microflora originating from municipal sewage sludge as the inoculum. The behavior of granular sludge formation was monitored during the course of fermentation. The effect of HRT on H2 production and byproduct formation was also investigated. This study aims to identify feasible operation strategies for continuous H2 production from starch. 2. Experimental Section 2.1. H2-Producing Sludge and Culture Medium. The seed sludge used in this work was obtained from the final sedimentation tank of a municipal wastewater treatment plant located in central Taiwan.3,29 The sludge was thermally treated (70 °C for 1 h) to eliminate the methanogenic activity and was then acclimated in a continuous culture operated at 35 °C and a hydraulic retention time of 6–12 h using a phosphate-buffered medium, containing 20 g of chemical oxygen demand (COD)/L of starch as the sole carbon source.3 The acclimated sludge was inoculated into a fermentor with a 5% inoculum. The fermentation medium was a phosphate-buffered medium31 containing cassava starch (15 g/L) and the following nutrients: 11.867 g/L Na2HPO4, 4.5 g/L NH4H2PO4, 0.125 g/L K2HPO4, 0.100 g/L MgCl2 · 6H2O, 0.015 g/L MnSO4 · 6H2O, 0.025 g/L FeSO4 · 7H2O, 0.005 g/L CuSO4 · 5H2O, 1.25 × 10-4 g/L CoCl2 · 5H2O, and 0.100 g/L CaCl2. 2.2. Bioreactor Setup, Start-up, and Operation. The main body of the bioreactor was a cylindrical glass vessel with a diameter of 8 cm, a height of 40 cm, and a working volume of 0.8 L. The influent medium was fed upward from the bottom of the reactor. The effluent of the bioreactor was introduced to a gas/liquid separator, where gas and liquid was collected separately. After inoculation with the seed sludge, the bioreactor was operated in batch mode for 16 h, followed by a 48 h pulse feeding (30 min feeding at 150 mL/h every 2 h), and then switched to a continuous mode. The continuous culture was started-up with a HRT of 5.3 h. When steady-state operation (on the basis of the H2 production rate with less than 5% variation with time) was reached, the operating HRT was shortened progressively from 5.3 to 0.5 h. The operating temperature was 37 °C, and the culture pH was controlled within the range of 5.0–5.5. The amount and composition of biogas produced were monitored with time. Liquid samples were also taken from the culture at designated time intervals to analyze the composition of soluble metabolites and the residual carbon substrate concentration. 2.3. Analytical Methods. The amount of biogas produced was measured by a gas-meter (type TG1; Ritter, Inc., Germany). The gas products (H2 and CO2) was analyzed by gas chromatography (GC-14B, Shimazu, Tokyo, Japan) using a thermal conductivity detector. The volatile fatty acids and ethanol were also detected by GC using a flame ionization detector. The procedures, conditions, and columns used for GC analysis were identical to those reported in our recent work.3,29 The residual starch concentration was measured by the KI–I2 colorimetric method,32 in which starch content in the sample was quantified by the absorbance at 680 nm. The residual sugar concentration of the culture was measured by the phenol–sulfuric acid method.33

3. Results and Discussion 3.1. HRT-Dependent Bio-H2 Production under a Progressive HRT Decrease. The continuous dark H2 fermentation from a starch-based influent was started-up at a HRT of 5.3 h, which is much shorter than comparable studies using a start-up (31) Wang, C. H.; Lin, P. J.; Chang, J.-S. Fermentative conversion of sucrose and pineapple waste into hydrogen gas in phosphate-buffered culture seeded with a municipal sewage sludge. Proc. Biochem. 2006, 41, 1353– 1358. (32) Yoo, Y. J.; Hong, J.; Hatch, R. T. Comparison of R-amylase activities from different assay methods. Biotechnol. Bioeng. 1987, 30, 147– 151. (33) Rao, P.; Pattabiraman, T. N. Reevaluation of the phenol–sulfuric acid reaction for the estimation of hexoses and pentoses. Anal. Biochem. 1989, 181, 18–22.

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Figure 2. Time course profiles of the (a) H2 content in biogas and (b) CO2/H2 ratio during continuous H2 fermentation from starch.

Figure 1. Effect of HRT on the (a) H2 production rate and (b) H2 yield during continuous H2 fermentation from starch.

HRT of 2734 and 18 h,7 respectively. The short start-up HRT could eliminate a prolonged adaptation period commonly involved in most starch-feeding bio-H2-producing systems and also enhance the H2 production rate by increasing the substrateloading rate. Even with the high substrate feeding rate, the starch conversion was able to maintain at 80–93%, indicating an efficient starch hydrolytic activity of the bacterial culture. As indicated in Figure 1a, the H2 production rate slightly increased with a progressive decrease of HRT from 5.3 to 2.2 h. While operating at a HRT shift-down from 2.2 to 0.5 h, the H2 production rate increased rapidly from 1.04 to 4.12 L h–1 L–1. The drastic improvement on the H2 production rate is considered to have close connection with the formation of granular sludge commencing at a culture time of ca. 360 h, when the system was operated at a HRT of 2.2 h (representing a starch loading rate of 5.0 g L–1 h–1) (Figures 1 and 2). The sludge granulation led to efficient biomass retention in the bioreactor against an increasing substrate-loading rate (i.e., a decrease of HRT from 2.2 to 0.5 h), resulting in a higher H2 production rate. Our recent work using sucrose as a carbon source and a carrier-induced granular sludge bed bioreactor3 showed that sludge granulation occurred within 80 h (at an organic loading rate of 10 g of COD h–1 L–1), boosting the H2 production rate to as high as 7.3 L h–1 L–1 at 0.5 h of HRT.3,29 However, there is little information regarding the use of granular sludge to produce H2 from starch. In fact, to our knowledge, this work seems to be the first report describing sludge granulation during fermentation of starch for H2 production. The quick formation of granular sludge (in 360 h (34) Lay, J. J. Modeling and optimization of anaerobic digested sludge converting starch to hydrogen. Biotechnol. Bioeng. 2000, 68, 269–278.

after start-up) also gains benefits to the bioreactor operation for high-rate bio-H2 production. When HRT was further shortened to 0.3 h, severe cell washout occurred, leading to a poor H2 production performance and eventually a failure of the bioreactor operation. Thus, the fastest medium feeding rate for stable operation was approximately at a HRT of 0.5 h. Unlike a marked dependence of the H2 production rate on HRT, the H2 yield did not vary significantly with HRT. The highest yield (1.43 mol of H2/mol of hexose) occurred at the longest HRT (5.3 h) used but decreased slightly to 0.97 mol of H2/mol of hexose at HRT ) 2.6 h and was maintained essentially constant at 1.06–1.22 mol of H2/mol of hexose after further HRT decreases from 2.2 to 0.5 h (Figure 1b). It is known that the H2 yield usually decreases when HRT is shortened.3,4,24,35 However, the bioreactor in the present work appeared to give a similar H2 yield regardless of the operation at a decreasing HRT from 5.3 to 0.5 h. Because the theoretical yield of H2 production from starch is 4 mol of H2/mol of hexose [assuming the complete hydrolysis of starch into hexose (glucose)],1 the H2 yield obtained from this work is in the range of 26–37% from the theoretically maximum yield. 3.2. Composition of Gaseous and Soluble Metabolites. The time course profile of the H2 content in the biogas is depicted in Figure 2a. Except for a high H2 content of around 62% in the early stage of operation (HRT ) 5.3 h), the H2 content was maintained fairly stable in the range of 48–51% during the continuous operation at a HRT of 3.6-0.5 h. Assuming that all starch completely converts to glucose, the highest H2 content would be 66.7% based on the following stoichiometry equation: C6H12O6 f 2CH3COOH + 4H2 + 2CO2

(1)

(35) van Ginkel, S. W.; Logan, B. Increased biological hydrogen production with reduced organic loading. Water Res. 2005, 39, 3819–3826.

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Table 1. Effect of the HRT on the Soluble Metabolite Production during Continuous Dark H2 Fermentation from Starcha HRT (h)

HBu/SMP (%)

HAc/SMP (%)

EtOH/SMP (%)

butandiol/SMP (%)

TVFAs (mg of COD/L)

SMP (mg of COD/L)

TVFAs/SMP (%)

5.3 3.6 2.6 2.2 1.6 1.4 1.1 0.9 0.9 0.7 0.6 0.5

69.4 69.2 67.9 68.6 72.0 70.2 67.2 65.4 64.5 60.8 54.1 61.3

7.8 8.3 8.9 7.7 9.2 8.5 11.1 9.4 10.1 11.0 12.3 13.3

14.1 15.9 16.6 18.4 18.8 18.3 18.2 22.0 21.4 22.2 24.0 25.5

8.7 6.7 6.6 5.3 0.0 3.0 3.6 3.3 4.0 6.0 9.6 0.0

7482 ( 12 6836 ( 90 6253 ( 135 6816 ( 66 5654 ( 43 6277 ( 32 6340 ( 110 6375 ( 127 5692 ( 56 5743 ( 66 5879 ( 72 6259 ( 108

9696 ( 232 8825 ( 84 8141 ( 52 8933 ( 92 6960 ( 38 7975 ( 51 8106 ( 102 8531 ( 133 7631 ( 68 7996 ( 62 8857 ( 104 8396 ( 255

77.2 77.5 76.8 76.3 81.2 78.7 78.2 74.7 74.6 71.8 66.4 74.5

a HBu, butyric acid; HAc, acetic acid; EtOH, ethanol; butandiol, 2,3-butandiol; TVFAs (total volatile fatty acids) ) HBu + HAc; SMP (soluble microbial products) ) TVFA + EtOH + 2,3-butandiol.

Thus, our system could produce H2 from starch at 72–93% of the theoretically possible H2 content. Furthermore, because CO2 is an undesired byproduct from the prospective of global warming, a low CO2/H2 ratio is preferable. In the present work, the phosphate-based buffer, instead of the bicarbonate buffer, was used to prevent abiotic production of additional CO2 from the reaction of bicarbonate ion with the acidic soluble metabolites (i.e., acetic acid or butyric acid).31 When the system was started-up at a HRT of 5.3 h, the CO2/H2 was ca. 0.6 (Figure 2b), which is very close to the theoretically lowest CO2/H2 ratio of 0.5.31 After HRT was gradually shortened from 3.6 to 0.5 h, the CO2/H2 ratio slightly increased but was maintained within the range of 0.9–1.1 (Figure 2b). The CO2/H2 ratio obtained from this work is much lower than that of our recent study using bicarbonate-buffered sucrose medium as the feed. Those previous systems gave a high CO2/H2 ratio of 1.6–3.0, despite achieving a high production rate of up to 7.3 L h–1 L–1.3,29 The low CO2/H2 ratio attained from this work not only lowers the production of the major greenhouse gas (i.e., CO2) but could also make the downstream H2 purification practice easier. During the course of dark H2 fermentation, the most abundant soluble metabolite was butyric acid (HBu), followed by ethanol (EtOH) and acetic acid (HAc) (Table 1). In addition, a small amount of 2,3-butandiol was also formed. The production of HBu was predominant, because it accounted for 54–72% of the total soluble microbial products (SMP). Meanwhile, the EtOH and HAc production contributed to 14–26 and 8–14% of SMP, respectively. As the HRT decreased, the production of HBu decreased slightly, while the EtOH and HAc formation increased. The production of 2,3-butandiol only accounted for 0–9.6% of SMP. Table 1 shows that the ratio of total volatile fatty acids (TVFAs) to SMP was very high (72–81%), suggesting that the mixed culture system was metabolically favorable for H2 production, because the alcohol production (the unfavorable production for H2 evolution) was limiting. The composition of soluble metabolites also suggests a butyrate-type fermentation. The major bacterial population is most likely Clostridium species, such as Clostridium butyricum and C. pasteurianum that have been identified in the same seed culture source in our recent work.6,22 Moreover, the formation of 2,3-butandiol also suggests the presence of Enterobacter or Klebsiella species that are also found from the mixed culture in our recent work.22 Previous studies revealed that the soluble metabolite composition resulting from bio-H2 production with starch is highly dependent upon the nitrogen source, pH, starch concentration, and HRT,

as well as the source and pretreatment of the seed culture.8,13,36 For instance, propionic acid was the major soluble product in the study by Khanal et al.,8 while in the other case, butyric acid was the predominant product while converting starch to H2.13 3.3. Assessment of the Operation Strategy and Comparison to the Literature. The outcome of this work suggest that a HRT shift-down operation of the mixed culture could successfully enable sludge granulation, which appeared to be the key to achieving a high-rate production of H2 from starch. The formation of granular sludge was a common behavior in our recently developed carrier-induced granular sludge bed (CIGSB) system while using sucrose as the carbon substrate,3,29,37 but this has not yet been observed on starch-feeding cultures. The timing of the forming granules seemed to be closely related to the hydraulic feeding rate and organic loading rate, similar to our previous studies when granular sludge started to form when the HRT was shorter than 4 h and the organic loading rate was higher than 10 g of COD h–1 L–1.3,22 In the present work, adjusting to a HRT of 2.2 h (a loading rate of 5 g of starch L–1 h–1) allowed for the granulation of sludge and marked enhancement on the H2 production. The maximum H2 production rate of over 4 L h–1 L–1 obtained from this work is over an order of magnitude higher than the reported values (typically 0.1–0.3 L h–1 L–1) in the literature (Table 2). In addition to obtaining a much higher H2 production rate, our continuous culture also gave a good H2 yield, which is comparable to that of relevant studies (Table 2). Maintaining a satisfactorily high yield during high-rate H2 production is quite important to ensure a good H2-producing performance. In fact, it is hard to achieve a high rate and high yield simultaneously, as many studies reported a decline in the H2 yield in the attempt to increase the H2 production rate by shortening the HRT.3,4,24,35 The features of a significantly higher H2 production rate and a competitive H2 yield of our starch–H2 system warrant a much better H2 productivity over the comparable work (Table 2). This also suggests the potential of applying our dark fermentation process (36) Kawagoshi, Y.; Hino, N.; Nakao, M.; Fujimoto, A.; Nakao, M.; Fujita, Y.; Sugimura, S.; Furukawa, K. Effect of inoculumn conditioning on hydrogen fermentation and pH effect on bactrial community relevant to hydrogen production. J. Biosci. Bioeng. 2005, 5, 524–530. (37) Lee, K. S.; Lo, Y. C.; Lin, P. J.; Chang, J.-S. Improving biohydrogen production in a carrier-induced granular sludge bed by altering physical configuration and agitation pattern of the bioreactor. Int. J. Hydrogen Energy 2006, 31, 1648–1657. (38) Plazzi, E.; Fabino, B.; Perego, P. Process development of continuous hydrogen production by Enterobacter aerogenes in a packed column reactor. Bioprocess Eng. 2000, 22, 205–213. (39) Kanai, T.; Imanaka, H.; Nakajima, A.; Uwamori, K.; Omori, Y.; Fukui, T.; Atomi, H.; Imanaka, T. Continuous hydrogen production by the hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1. J. Biotechnol. 2005, 116, 271–282.

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Table 2. Comparison of the Bio-H2 Production Performance with Relevant Studies Reported in the Literature maximal H2 production rate (L h–1 L–1)

H2 yield (mol of H2/ mol of hexose)

H2 content (%)

reference

microorganisms

substrate

Eneterobacter aerogenes anaerobic-digested sludge mixed microflora

corn starch hydrolysatea starch

packed bed

10

0.25b

1.54

nac

38

CSTR

17

0.07b

na

64

34

rice winery (wastewater) wheat starch starch (5 g/L)

UASB

2

0.16b

1.37–2.14

53–61

13

CFB CSTR

18–12 5

na 0.16

1.3–1.9 na

50 64

7 39

cassava starch (15 g/L)

continuous flow with granular sludge

0.5

4.12

0.97–1.43

48–62

this study

mixed microflora Thermococcus kodakaraensis mixed microflora a

bioreactor

optimal HRT (h)

A total of 85% glucose dry extract. b Calculated from data presented in the paper. c na ) not available.

to a large-scale bio-H2 production from starch. Using starch as the carbon source usually requires a longer retention time, because starch needs to be hydrolyzed to reduce sugars before entering the glycolysis pathway for further degradation. It is believed that the key to an excellent H2 production performance in this system is being able to operate at a much shorter HRT (as low as 0.5 h), gaining kinetic benefits from a higher organic loading rate for elevation of the H2 production rate. The ability to stably operate against a low HRT (i.e., high dilution rate) with effective biomass retention appeared to play a major role in achieving a much higher H2 production rate as well as a comparable H2 yield over the relevant systems. However, the operation HRT of the present system still has a limit, because operation at low HRT (e.g.,