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Biological hydrogen production by Clostridium acetobutylicum in an unsaturated flow reactor
Husen Zhanga,1, Mary Ann Brunsb, Bruce E. Logana,Ã
a b

Department of Civil and Environmental Engineering, The Pennsylvania State University, 212 Sackett Bldg, University Park, PA 16802, USA Department of Crop and Soil Sciences, The Pennsylvania State University, USA

ar t ic l e i n f o
Article history: Received 29 June 2005 Received in revised form 6 October 2005 Accepted 27 November 2005 Available online 19 January 2006 Keywords: Biohydrogen Trickle bed Trickling filters Industrial wastewater Clostridium


A mesophilic unsaturated flow (trickle bed) reactor was designed and tested for H2 production via fermentation of glucose. The reactor consisted of a column packed with glass beads and inoculated with a pure culture (Clostridium acetobutylicum ATCC 824). A defined medium containing glucose was fed at a flow rate of 1.6 mL/min (0.096 L/h) into the capped reactor, producing a hydraulic retention time of 2.1 min. Gas-phase H2 concentrations were constant, averaging 7473% for all conditions tested. H2 production rates increased from 89 to 220 mL/h L of reactor when influent glucose concentrations were varied from 1.0 to 10.5 g/L. Specific H2 production rate ranged from 680 to 1270 mL/g glucose per liter of reactor (total volume). The H2 yield was 15–27%, based on a theoretical limit by fermentation of 4 moles of H2 from 1 mole of glucose. The major fermentation by-products in the liquid effluent were acetate and butyrate. The reactor rapidly (within 60–72 h) became clogged with biomass, requiring manual cleaning of the system. In order to make long-term operation of the reactor feasible, biofilm accumulation in the reactor will need to be controlled through some process such as backwashing. These tests using an unsaturated flow reactor demonstrate the feasibility of the process to produce high H2 gas concentrations in a trickle-bed type of reactor. A likely application of this reactor technology could be H2 gas recovery from pre-treatment of high carbohydrate-containing wastewaters. & 2005 Elsevier Ltd. All rights reserved.



H2 is being considered for widespread use as a green energy carrier in the future primarily for transportation. The advantage of using H2 is that the only by-product of reacting H2 with oxygen is water, and no carbon dioxide or other green house gases are produced. Most H2 is currently produced from nonrenewable sources such as oil, natural gas, and coal (Benemann, 1996; Van Ooteghem et al., 2002). H2 can also be produced from renewable sources such as biomass, but yields are low. If H2 conversion efficiency could reach 60–80%, based
ÃCorresponding author. Tel.: 814 863 7908; fax: 814 863 7304.

upon a maximum theoretical conversion of 12 mol H2/mol hexose, H2 production from wastewater could have great potential for economical near-term H2 production from renewable resources (Benemann, 1996). There are no known fermentation pathways that can achieve a conversion efficiency of greater than 4 mol H2/mol hexose (Thauer et al., 1977). However, it has recently been discovered that H2 can be produced from a fermentation end product (acetate) by modifying a microbial fuel cell by applying a small potential to that generated by the bacteria (Liu et al., 2005b). High H2 yields in this new process would still require a

E-mail addresses: (H. Zhang), (B.E. Logan). 1 Present address: Division of Geological and Planetary Sciences, California Institute of Technology. 0043-1354/$ - see front matter & 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2005.11.041

and upflow granulated reactors (Liu and Fang.. 2002. 0. 2.. 2002). 1999). 1993).8. 2003). 0. Cells were grown anaerobically on glucose (1 g/L) in a defined medium (pH ¼ 6.. In aerobic wastewater treatment systems.. fed-batch (Chin et al. Methods Medium and culture conditions C. the large energy input needed for wastewater aeration can be avoided by using an unsaturated flow. Logan et al. 0.. 2003).2.. 2. 0. MnCl2 Á 4H2O.. Biogas transfer out of the liquid phase is limited in aqueous reactors without intensive stirring (Rachman et al.2. This trickle-bed reactor operation promotes high rates of gas transfer into the biofilm due to the thin fluid film and the high gas diffusivity of oxygen (Logan. KH2PO4. 2004). 2001a. In batch tests. Oh et al. ZnCl2. 2001. CaCl2. 0. . Instead of a configuration where gas was injected into the reactor. 2002).4 mol H2/mol glucose (Lay.7 mol H2/ glucose using a mixed culture of Clostridium butyricum and Enterobacter aerogenes (Yokoi et al.2) containing (g/L): NH4Cl. MgSO4 Á 7H2O. In one study. type of reactor.. 0. 2002. the gas line was modified to allow gas to be continuously released from the reactor. Palazzi et al. 2002. under mesophilic conditions.. In this system. While maintaining a pure culture for a waste stream would not be likely. The main chamber of Medium in Gas out to a bubble meter Syringe sampling port Glass beads for biofilm buildup Stainless steel mesh (A) Zip-loc bag for collecting waste (B) Fig.40. 2.. Na2BO7 Á H2O. Clostridium acetobutylicum ATCC 824. 0. K2HPO4. wastewater is applied over a packing medium at a rate that creates a thin fluid film over the biofilm growing on the medium.01..015.02. 1. 2002.2. 0. Cells used to inoculate the reactor were harvested during late-log growth based on optical density at 600 nm (OD600) of ca. Unsaturated flow reactors have recently been tested for biological H2 production under thermophilic conditions (Oh et al.01.015. 2003). 1 – (A) Photograph and (B) schematic of the unsaturated flow reactor. but so far no reactors have been tested under mesophilic conditions. continuous-flow stirred tank (Fang and Liu.011. 0. Fang and Liu. 2002).ARTICLE IN PRESS WAT E R R E S E A R C H 40 (20 06) 72 8 – 734 729 pre-fermentation stage so that H2 could be recovered in a separate process from the fermentation of the sugars.. Typical conversion efficiencies using continuous-flow reactors are 1.. 1998. it has been found that continuous release of the biogas from the system can increase H2 yields by as much as 40% (Logan et al. Various types of bioreactors have been used for H2 production. or trickling filter.. We reasoned that this unsaturated flow condition could similarly facilitate rapid H2 gas evolution out of the biofilm under anaerobic conditions. Reactor design and operation The unsaturated flow (trickle bed) column reactor (Fig.. CoCl2 Á 6H2O.1.1. Hussy et al. Yokoi et al. 2. it is well known that Clostridia are predominant H2-producing bacteria. continuous H2 production achieved a yield of 2. The energy needed for mixing in anaerobic wastewater treatment reactors ranges from 85 to 105 kW/1000 m3 (Grady et al.9–2. Ueno et al.While stirred reactors facilitate gas release from the liquid phase. acetobutylicum ATCC 824 was stored at À80 1C. 0. CuCl2 Á 2H2O. 1) used for H2 production studies was developed by modifying the gas flow of an anaerobic biofilm reactor developed to transfer H2 from the gas phase into a biofilm (Logan and LaPoint. FeCl3. saturated packed-bed column reactors (Rachman et al. The use of a well-characterized pure culture was therefore helpful for testing the concept of this new type of trickling filter reactor. 2001. 1998). Chang et al.. We therefore tested the feasibility of this concept for H2 production using a laboratory-scale reactor and a pure culture of a H2-producing bacterium. 2002). Lee et al. 1997. Our results with this unsaturated flow reactor are then compared to the performance of stirred tank reactors on the basis of H2 production per unit volume of the reactor.01. 2003). continuous stirring of the reactor consumes considerable electric power. including batch (Van Ginkel et al. 2003).

and 10. The specific H2 production rate was calculated as S ¼ 1000RHV/(180RGV). 1200 m2/m3 calculated projected surface area) supported by a stainless-steel mesh.771.771. Cole Palmer Corp.25 mL) were periodically taken using a gas-tight syringe. where V is the reactor volume.5 14. . Conversion efficiency was calculated as (RHM/ 4RG) Â 100%.470. The liquid effluent line was connected directly to a sealed Zip-Loc bag (1.5 cm inside diameter.3 g/L (C).3 (3..9 22078 8.3 1. Effluent samples were taken from the effluent line at a point before the flow entered the collection bag.6 16378 6.5 L) containing sodium chloride (150 g) to halt biological activity in the collection system. The reactor was operated at 30 1C in a constant temperature room. Based on a flow rate of 0. In some tests (as noted).5 and 10 g/L. Water was pumped (Masterflex 7523-30. 2000. The molar H2 production rate.570.0 15 1040 45.270. Oxygen was initially removed from the column by purging with nitrogen gas for 30 s. These influent glucose concentrations were chosen based on sugar concentrations representative of food processing wastewaters (Van Ginkel and Logan.2 15. was calculated from the total gas production rate and the concentration of H2 in the headspace.170.3.2 3.7 12. After collection. OD600 ¼ 0. and verified by measurements demonstrating constant effluent glucose and volatile acids concentrations measured four times (over at least 24 h).271.1 3171 7972 5.570. 2. 2. The fluid was then drained from the reactor.4 (10. 3. Then. aqueous (influent and effluent) samples were immediately centrifuged for 1 min (8000g) and the supernatant was stored at 4 1C for subsequent analysis. Calculations The volumetric H2 production rate.7 0.0.0 5.8 17 760 18. was calculated using the ideal gas law as RHM ¼ RHV =ðRTÞ. B. the beads were gently mixed in the column using an ethanol-wiped spatula to remove excess biofilm.5 2.6 20378 8.0 (4. Otherwise. and the reactor re-started with fresh medium.3. H2 production rates were measured in three trials at an influent glucose concentration of 3. RHV (mL/h L of reactor) Molar H2 production rate. and C). Nitrogen-sparged sterile medium containing glucose was fed at a flow rate of 1. where RG (mmol glucose/h) is the glucose consumption rate (Table 1). Conversion efficiency for 3 g/L A and B.0 15d 680 5.3 g/L (tests A.096 L/h.6 1. 2001) the following stoichiometry: C6 H12 O6 þ 2H2 O ! 2CH3 COOH þ 4H2 þ 2CO2 C6 H12 O6 ! CH3 ðCH2 Þ2 COOH þ 2H2 þ 2CO2 (1) (2) Table 1 – Summary of H2 production rates and conversion efficiencies (based upon a maximum production of 4 moles of H2 from every mole of glucose) System conditions 58. total volume of 0.5) 9.871.123 L) was packed with autoclaved glass beads (3 mm diameter.7 8. The maximum theoretical production of H2 and CO2 were calculated using measured acetic and butyric acid concentrations assuming (Muller.3a) 4. and for an influent glucose concentration of 4. RHM (mmol H2/h).170.570.6 8978 3. the reactor feed was changed to a new glucose concentration after purging the gas phase with nitrogen gas. for 3. where R ¼ 0:0821 (L atm)/(mol K). purged with nitrogen gas.6 (1.5.2 3770 7474 4.3 g/L.5 1. the conversion efficiency was 19% due to the increased gas production rate. First. AR). mM (g/L) 25. Steadystate operation was indicated by constant gas production and a stable headspace H2 concentration. RHM (mmol/h L of reactor) Conversion efficiency (%)c Specific H2 production (mL H2/L g glucose) a b c d Influent glucose concentration. In order to demonstrate reproducibility of the results.1 2771 7472 5. The reactor was then inoculated with cells (70 mL. 4. 2005) and concentrations used in previous H2 production tests (Mizuno et al. Four different glucose concentrations (1. The gas flow through the port on the top of the reactor was monitored using a respirometer system (Challenge Environmental Systems AER-200 respirometer.ARTICLE IN PRESS 730 WA T E R R E S E A R C H 40 (2006) 728– 734 the reactor (25 cm long. Fang and Liu.2) and operated in fedbatch mode (two complete cycles) until gas production was observed. the reactor was switched to a continuous liquid flow mode.170. Headspace gas samples (0. The reactor was cleaned and reinoculated for two of these experiments (3.) into the top of the reactor where it dripped onto the reactor packing.6 mL/ min into the top of the reactor.0) 0.0 0. Fayetteville.270.0 1670 7071 5. 2002.370.570. 2004). RHV (mL H2/h). At this time..5) Effluent glucose concentration (mmol/L) Consumed glucose (mmol/L) Glucose loading rate (g/h L of reactor) Glucose consumption rate (RG)b (mmol/L) Biogas production rate (mL/h) H2 (%) Effluent pH Volumetric H2 production rate. excess biomass was removed from the reactor via a method meant to simulate reactor backwashing. Iyer et al.8 22 1270 From experiment A. Based on 4 mol H2/mol glucose.5 g/L) were used in defined medium for the influent flow.970. tests A and C). and T ¼ 303 K. fresh medium was pumped through the effluent line into the reactor.

Glucose was measured by using a phenol-sulfuric method for reducing sugars (Dubois et al. and measuring the conductivity of the reactor effluent (YSI 600XL.3g/L(A) 3. column cleaning is essential for maximizing H2 production. Fig.0g/L 3.3 g/L(C). 2 – Biogas productions over time..0 g/L. 90% 80% 70% 60% H2 50% 40% 30% 20% 10% 0% 0 10 20 30 Time (h) 40 50 60 10g/L 4.1 min based on a salt tracer test in the presence of the biofilm (Fig. . 2004).5.3 g/L glucose concentration) increased the conversion efficiency from 15% (A) to 19% (C). Acetate. and 1.5. the effluent pH was 4.0g/L The reactor’s hydraulic retention time (HRT) was determined by spiking the feed line with a concentrated KCl solution (16 g/ L). 1999).. CA) equipped with a thermal conductivity detector and a molecular sieve column (Alltech 5A 80/100) with nitrogen gas as the carrier gas. Torrence. Carbon dioxide and methane were similarly analyzed except that a different column was used (Alltech Porapak Q 80/100) with helium as the carrier gas. are used for a linear regression. and then steady at 240 1C for 3 min. 2003. Chin et al. The reactor’s retention time was determined to be 2. and 7071% for glucose concentrations of 10. The rate of gas production increased from 1670 to 3771 mL/h when the glucose loading rate was increased over the range of 0.3 mL/h for 3. YSI Incorporated. 3. as previously described (Min et al. see also Miller and Logan.e. 2000). propionate. The oven temperature was programmed as follows: 60–120 1C increased at 20 1C/min.. In a full-scale system.5g/L 3. When only the feed bottle was changed to a new glucose concentration.32 mm  0. At all glucose loading rates. and ethanol were determined by gas chromatography (Agilent 6890N) and a fused-silica capillary column (DB-FFAP 30 m  0. Fang and Liu.3g/L(B) 3. 7S. The conversion efficiencies for producing H2 from glucose ranged from 15% to 22%. 2001b.6 at the other glucose concentrations. Oh et al.6 for 3. Solid symbols. respectively (Fig. 2. 1956). The average H2 headspace concentra- tions were 7473%. then 240 1C at 30 1C/min. These conversion efficiencies are slightly less than those of 24% and 23% reported in batch studies (Logan et al. based on a theoretical stoichiometry of 4 moles H2 from 1 mole of glucose (Table 1).3g/L(A) 3. 2002) and 48–55% (Ueno et al. 28.3 g/h L of reactor (Table 1). At 10 g/L.5g/L 3.E. 2003) achieved in stirred reactors (fed-batch or continuous flow). biomass can be cleaned by backwashing the medium (Min et al. Thus.9 but it was 5. there was a high rate of biogas production (Fig. 2). Gas production rates were reproducible when the reactor inoculation conditions were the same.5 mL/min (240 1C).970.3 g/L (B).3 g/L (A) vs. 2). Hussy et al..25 mL injection volume) and a gas chromatograph (SRI instruments. The effluent pH varied as a function of glucose feed concentration.4. 2004..371. Lag times for H2 production varied primarily as a result of procedures used for setting a new glucose concentration in the influent. The retention time was determined by using the front half of the tracer curve with the mean of the distribution defined as the HRT.. 7972%. with helium as a carrier gas at flow rates of 3. lag times were only 6–12 h.7–8.5 mm) with injector and flame ionization detector temperatures of 250 1C. which represent a region of constant biogas production. OH. butyrate. which is not obtained in a biofilm system due to slow diffusion of the tracer out of the biofilm (Logan. Determination of hydraulic retention time 900 800 700 Biogas (mL) 600 500 400 300 200 100 0 0 10 20 30 Time (h) 40 50 60 10g/L 4.5. 2003). Cleaning the column (i. The slopes of the regression lines represent gas production rates and are summarized in Table 1.3g/L(C) 1.ARTICLE IN PRESS WAT E R R E S E A R C H 40 (20 06) 72 8 – 734 731 2. or overall averaged 7473%. 4). 3). as shown by the similar gas production rates (28. of the slope shown in Fig. removing excess biomass) increased the H2 production rate from 2071 (A) to 2671 mL/ h (C). 7472%. Results and discussion Fig.3g/L(B) 3. 4. or the reactor was cleaned to remove excess biomass. The operation with column cleaning is indicated as 3. USA). 2002. Cleaning the column (3. 3 – Headspace H2 concentrations over time. 2002. and much less than 68% (Yokoi et al.3... Analytical procedures H2 was measured using a gas-tight syringe (0.. The trailing edge of the tracer curve is not used because the method requires an assumption of a normal distribution of data.3g/L(C) 1. Steady-state headspace H2 concentrations in the gas phase were all in the range of 70–79% and did not show a trend with glucose concentration.5 mL/min (60 1C) to 1. Yellow Springs. The lag time for a sterile reactor that was inoculated with fresh cells was 32–40 h.

The H2 yield for an Influent glucose concentration.1 0.4 mM.470..470.2 60 15 Consumed glucose. Acetate or Butyrate (mM) Comparison of H2 production based on reactor volume 15 Glucose (mM) 15 Glucose.2 2.570.3 1.0) 0.570. 2003).1 8. 2000) (Table 3).070.4 (10. 20 20 3.870. and lower than that reported for a saturated reactor of 13..6 (1.370. open circles are symmetrical with the front half of the actual curve.1 2. Acetate and butyrate were the main soluble products of H2 production (Fig. Theoretical CO2 and H2 yields were calculated from volatile acid concentrations measured in the reactor effluent and from Eqs. indicating that there was no loss of H2 via methanogenesis. (1) and (2).170. assuming the production of 2 moles of H2 with the production of 1 mole of acetate or butyrate.0 2.270.1 0. 5 – Glucose.9 mmol H2/(L h).52 mmol H2/(L h) (Fang and Liu.270.570.370.670.570.ARTICLE IN PRESS 732 WA T E R R E S E A R C H 40 (2006) 728– 734 4500 4000 Conductivity (uS/cm) 3500 3000 2500 2000 1500 1000 0 60 120 180 Time (sec) 240 300 Fig.170. Table 2 – Summary of volatile fatty acids production System conditions Reactor efficiency is often examined on the basis of the production of the product (H2) normalized to the total reactor volume.270.5) 1. and CO2 (mmol/h) Carbon recovery (%) Predicted H2 (mmol/h)c Predicted conversion efficiency (%)d Observed conversion efficiency (%) a 1. 4 – Determination of reactor retention time by tracer study..470.5 72 22 From experiment A. 1997). However.8 mL H2/(L h) (Iyer et al. the trickle-bed reactor was as effective as a CSTR in terms of overall H2 production normalized to the reactor volume.3a) 1.1 0. with the balance assumed to have been converted to biomass.1 8. Based on Eqs. Total propionic acid and ethanol concentrations were less than 0. assuming the production of 1 mole of CO2 with the production of 1 mole of acetate. 2004) and 7. Based on the H2 production rate of 27.570. with acetate reaching higher concentrations than butyrate (Table 2).4 92 3. d Predicted conversion efficiency calculated as (predicted H2)/(4RG) Â 100%. however. 5). including biological factors such as the loss of H2 via homoacetogenesis (Oh et al. was detected in the gas throughout the tests.570.870. (1) and (2).1 2.1 94 3.2 0. (1) and (2). were much higher than the actual measured H2 conversion efficiencies (15–27%) based on H2s production. acetate. the H2 production rate achieved here was 8.570.9 mL H2/(L h) (Mizuno et al. The predicted conversion efficiencies of H2 production from glucose (60–72%) on the basis of these measured volatile acid concentrations.4 6. No methane.870. 2002). and butyrate concentration profiles during the column operation (experiment B at 3.2 70 15 5.3 80 1.In 10 Glucose.2 7..4 69 17 18. This difference could be due to several factors.6 mmol H2/(L h) (Yokoi et al. and physical factors such as the loss of biogas in the collection bag. This is similar to values obtained in other studies using CSTRs of 8. mM (g/L) 58. RG (mmol/h) Consumed carbon (mmol/h) Acetate (mmol/h) Butyrate (mmol/h) Predicted CO2 (mmol/h)b Carbon recovered in acetate.Out Acetate Butyrate 10 5 5 0 0 10 20 Time (h) 30 0 40 Fig. butyrate.0 1. The carbon recovery ranged from 80% to 94% based on volatile acid production.2 mL/h for an influent glucose concentration of 10 g/L.570.5) 25.671.0 (4.1.371. however.3 7.0 3.1 0.570.0 87 4. Solid symbols are measured conductivity.1 0. c Based on Eqs. the H2 production rate in the unsaturated flow reactor is higher than that reported in another study of 2.1 7.7 1. This suggests that on the basis of reactor volume.3 (3.3 g/L influent glucose concentration).971. b . and 2 moles of CO2 with the production of 1 mole of butyrate.

2005a..2.. 2003.L.J.0 10 h 436 17. K. Hawkes. Logan. I. which supports the Penn State Biogeochemical Research Initiative for Education (BRIE). there will be soluble organic matter remaining in solution that will require further treatment.. Chin. 66. H. will likely be needed for achieving higher H2 yields with an unsaturated flow reactor. Marcel Dekker.. (2004) 1.3 mol H2/mol glucose reported in the other studies using CSTR or saturated flow reactors (Yokoi et al. Prog.8 1. C. Gilles. D. Longer HRT. Mizuno et al.9 0. Dinsdale. Reactor clogging due to the biomass build-up was not specifically examined here.. 2000)..3 7. 2004. 87–93. Bruns.S. F. Fedbatch operation using Clostridium acetobutylicum suspension culture as biocatalyst for enhancing H2 production. J.3 2.53 2 8. P. b). 280–287. P.C.. Bioeng.5 h 457 18. 2nd ed. H2-producing bacterial communities from a heat-treated soil inoculum. Biotechnol. C. 3. 2000. Hydrogen biotechnology: progress and prospects. 2001. Grady.. the H2 concentrations achieved here appear to be similar or slightly larger than values achieved in stirred reactors (Iyer et al. Bioresource Technol.. 1999. Hamilton. 82. Biological Wastewater Treatment.9 mol H2/mol glucose reported in a CSTR (Mizuno et al. The energy needed for this step would need to be considered in the overall energy balance. Even then. Lim. Van Ginkel.09 0. Bioeng. anaerobic digestion.5 2. Fang and Liu..123 8. Iyer. 19... and.P.. 1101–1103. Biotechnol.. J. Hawkes.. Hussy.. Lay.6 2. Continuous fermentative H2 production from a wheat starch coproduct by mixed microflora.075 13. 2004). Anal. M. Lin. 2002.. Microbiol.S..J.1 min 27.. R. Zhang. Int. 351–356. B.02 0. Iyer et al..2 8. J. G. New York. Smith. Appl. additional research is needed to prove the stability and usefulness of this technology for wastewater treatment. 619–626.6–2..2 6h 192 7.06 2..L. or a new type of microbial fuel process that produces electricity or H2 (Liu et al. (2000) 1. 1996. an anaerobic process such as Acknowledgment This research was funded by NSF grant BES 01-24674 and by NSF (IGERT) grant DGE-9972759. J. 74. Nat..H. Daigger. Chen.1 Iyer et al.. Rebers. Chang. as a result of larger size reactors.5 1. 2002.. M. Lee. Biotechnol. 14. These reactors will need to be much taller than the laboratory-scale reactor examined here in order to provide sufficient HRTs to fully remove the sugars and produce H2. Dubois. Chem. The main advantage of the unsaturated flow reactor compared to a stirred reactor is that the energy cost of stirring the system can be avoided. 2002.3 CSTR CSTR CSTR This study 8. 2004).T. H. Biotechnol... 1956.S. F. thus. Fang and Liu. H. It is envisioned that an unsaturated flow reactor of the type developed here would be most effectively used as a pretreatment method for wastewaters containing high concentrations of carbohydrates. R E F E R E N C E S Benemann. 383–388... Biotechnol. P. H. Implications of these results for H2 production using an unsaturated flow reactor The high H2 gas concentrations (70–79%) and H2 production rates (normalized by reactor volume) obtained here using unsaturated flow reactors demonstrate the feasibility of using this type of reactor for biological H2 production under mesophilic conditions. Liu.. S. J. Fang.R.9 0.. In addition.ARTICLE IN PRESS WAT E R R E S E A R C H 40 (20 06) 72 8 – 734 733 Table 3 – Comparison of H2 production rates in the trickle-bed reactor with those reported using a CSTR or fixed-bed reactor Comparison Items Tricklebed reactor Reference Glucose loading rate (g/L of reactor/h) Reactor retention time Volumetric H2 production rate (mL/h) Molar H2 production rate (mmol/h) Reactor working volume (L) Normalized H2 production rate (mmol H2/L h) H2 yield (mol/mol glucose) a Reactor configuration Saturated fixed-bed reactor Yokoi et al. H. Biohydrogen generation by mesophilic anaerobic fermentation of microcrystalline cellulose.9 Mizuno et al.L. but was lower than 1.. Colorimetric method for determination of sugars and related substances. influent glucose concentration of 10 g/L was comparable to 0. 2004. Wastewater leaving this reactor could therefore either be further treated with a conventional aerobic process such as activated sludge.9a Fang and Liu (2002) 1. 1167–1174. (1997) 5. 2003. Chou. Z.. Effect of pH on H2 production from glucose by a mixed culture. Hydrogen Energy 27.0 2h 25.P. 2002) (Table 3).57 3 2. 1997. .E. Wastewater treatment process trains optimized for H2 production have so far not been extensively tested at large scale. K. 84.6 No nitrogen sparging.2 1. 166–173. 28. Biohydrogen production with fixed-bed bioreactors.A. but scale-up of the process would require regular backwashing to control biofilm growth (Min et al..

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