Hydrogen production using Rhodopseudomonas palustris WP 3-5 with hydrogen fermentation reactor effluent

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1 WHEC 16 / June 26 Lyon France Hydrogen production using Rhodopseudomonas palustris WP 3-5 with hydrogen fermentation reactor effluent Chi- Mei Lee, Kuo-Tsang Hung Department of Environmental Engineering, National Chung-Hsing University, 25 Kuo- Kuang Road, Taichung Taiwan,cmlee@dragon.nchu.edu.tw ABSTRACT: The possibility of utilizing the dark hydrogen fermentation stage effluents for photohydrogen production using purple nonsulfur bacteria should be elucidated. In the previous experiments, Rhodopseudomonas palustris WP3-5 was proven to efficiently produce hydrogen from the effluent of hydrogen fermentation reactors. The highest hydrogen production rate was obtained at a HRT value of 48 h when feeding a 5 fold effluent dilution from anaerobic hydrogen fermentation. Besides, hydrogen production occurred only when the NH 4 + concentration was below 17 mg- NH 4 + /l. Therefore, for successful fermentation effluent utilization, the most important things were to decrease the optimal HRT, increase the optimal substrate concentration and increase the tolerable ammonia concentration. In this study, a lab-scale serial photo-bioreactor was constructed. The reactor overall hydrogen production efficiency with synthetic wastewater exhibiting an organic acid profile identical to that of anaerobic hydrogen fermentation reactor effluent and with effluent from two anaerobic hydrogen fermentation reactors was evaluated. KEYWORDS : photohydrogen production, purple nonsulfur photosynthetic bacteria, ammonia, photobioreactor. 1. Introduction Energy requirements and consumption have dramatically increased with the development of technology. It is therefore necessary to search for an alternative energy resources. Hydrogen gas, a clean energy source that does not cause the greenhouse effect, serves well for this purpose. It is known that different taxonomic groups including some species of photosynthetic bacteria can produce hydrogen gas. Among these microorganisms, hydrogen gas production using purple nonsulfur bacteria can be characterized as: 1) High hydrogen gas production rate, 2) Hydrogen production is not inhibited by the produced oxygen because water is not used as the electron donor. 3) These bacteria can produce hydrogen gas in the dark or under light, 4) When used as the organic substrate source, wastewater can be treated because the wastes are used as the electron donors [1]. Hydrogen production from waste using photosynthetic bacteria is attractive because energy can be recovered from renewable resources. Photosynthetic bacteria can produce hydrogen from organic acids. The effluent from anaerobic hydrogen fermentation reactors contains high concentrations of organic acids. The possibility of utilizing these initial dark hydrogen fermentation stage effluents for photohydrogen production using purple nonsulfur bacteria should be elucidated. In the previous continuous flow experiments, Rhodopseudomonas palustris WP3-5 was proven to efficiently produce hydrogen from the effluent from hydrogen fermentation reactors [2]. The highest hydrogen production rate was obtained at a HRT value of 48 h when feeding a 5-fold effluent dilution from anaerobic + hydrogen fermentation as the substrate. Besides, hydrogen production occurred only when the NH 4 + concentration was below 17 mg- NH 4 /l [3]. Therefore, for successful hydrogen fermentation effluent utilization for photo-hydrogen production using Rhodopseudomonas palustris WP3-5, the most important things were to decrease the optimal HRT, increase the optimal substrate concentration and increase the tolerable ammonia concentration. In this study, a lab-scale serial photo-bioreactor was constructed. The reactor overall hydrogen production efficiency with synthetic wastewater exhibiting an organic acid profile identical to that of anaerobic hydrogen fermentation reactor effluent and with effluent from two anaerobic hydrogen fermentation reactors was evaluated. 2. Material and Methods 1/8

2 WHEC 16 / June 26 Lyon France 2.1 Organisms and culture medium The photosynthetic bacteria Rhodopseudomonas palustris WP3-5 used in this study was isolated from piggery wastewater. This strain was identified using the 16S rrna gene sequencing method. The microorganisms obtained from the liquid culture were used for this study. The liquid medium was an enrichment medium for Rhodospirillaceae[4]. The compositions of the Rhodospirillaceae medium were (in g/l) K 2 HPO 4,.5; KH 2 PO 4,.5; MgSO 4 7H 2 O,.2; NaCl,.4; CaCl 2 2H 2 O,.5; yeast extract,.2; Fe-citrate solution, 5 ml/l; vitamin B12, 1 ml/l. To the medium 1 ml of the trace element solution was added containing the following (in mg/l): ZnCl 2, 7; MnCl 2 4H 2 O, 1; H 3 BO 3, 6; CoCl 2 6H 2 O, 2; CuCl 2 2H 2 O, 2; NiCl 2 6H 2 O, 2; NaMoO 4 2H 2 O, 4; HCl (25%), 1 ml/l. The final Rhodospirillaceae medium ph value was Set-up and operation of a serial photo-bioreactor for H 2 production Schematic description of the lab-scale serial photo-bioreactor is shown in Fig.1. H 2 collection H 2 collection H 2 collection effluentt Influent Cell Recycle Fig 1 : Schematic diagram of a lab-scale serial photo-bioreactor The photo-bioreactor was composed of three main vertical columns. Each column was 68 cm in height, 8 cm in diameter and hat a working volume of 2.5 l volume and was equipped with a magnetic stirrer. The lamps were located outside the reactor. The light intensity measured at the reactor surface was varied between 4 klx and 7 klx. The effluent of the bioreactor was introduced into a sedimentation flask, where the cells were concentrated and returned to the photo-bioreactor. The temperature of the photo-bioreactor was controlled at Hydrogen production medium was prepared by diluting the organic acid composition of the synthetic wastewater with different folds of distilled water or prepared by real wastewater from two anaerobic hydrogen fermentation reactors. Initial ph was adjust to and then the medium was sterilized at 121 for 15 min by autoclaving. The synthetic wastewater consisted of 12 mg formic acid, 2 mg acetic acid, 3 mg butyric acid and 1 mg lactic acid, 2mg/l glutamate, 3mg K 2 HPO 4, and 4 mg FeCl 3 per liter. This wastewater exhibited an organic profile identical to that from the anaerobic hydrogen fermentation reactor effluent at Cheng-Kung University [5]. The real wastewater, that is the effluent from anaerobic hydrogen fermentation reactor was obtained from Cheng-Kung University and our laboratory. After centrifugation at 6 rpm for 15 minutes and adding.2mm FeCl 3, the supernatant was sterilized and used as the influent of the serial photo-bioreactor. During the experiments, the evolved gas was collected and measured volumetrically in a measuring cylinder. The composition of gas products and organic acid concentration in each column was monitored separately as a function of time. 2.3 Analytical methods After membrane filtration, the ammonia concentration was measured using the indophenol blue method [6]. Gas samples for hydrogen analysis were injected into a gas chromatograph (GC) equipped with a thermal conductivity detector (TCD). GC was performed with a HP 689 system equipped with a stainless-steel 2/8

3 WHEC 16 / June 26 Lyon France column containing carboxen-1. The oven, injector and detector temperatures were 8,15 and 2,respectively. Nitrogen gas was supplied as the carrier gas and the flow rate was 2 ml/min. The OD was measured using a spectrophotometer ( Beckman Du 53 ) at 66 nm. 3. Results and Discussion 3.1 Effect of cell recycle on hydrogen production The photo-bioreactor was fed with synthetic waste water, which organic acid concentration was diluted to.2 fold and 2 mg/l glutamate, 2 mg/l K 2 HPO 4,.2mM FeCl 3 was added as supplement of nitrogen sourse, buffer and cofactor, respectively. The reactor was kept at HRT=16h. The photo-bioreactor was started without cellular recycle. After start-up for 11 days, it was operated with cellular recycle with recycle ratio of 1:3. As shown in Fig 2a, the optical density of all three column steadily descresed from 1 to 11 days of operation, especially in the first column. On day 11 the optical density of.18 was observed in the first column. The optical density decreasing of the second and the third column was less than that of the first column, on day 11 was.39 and. 61, respectively. O. D a 時 Time 間 (day) ( ) gas ml l //L l /. d a y 1 9 b H 2 % 時 Time 間 (day) ( ) c 時 Time 間 (day) ( ) ph d 時 Time 間 (day) ( ) Fig 2 : Effect of cell recycle on hydrogen production ~11 d : without cellular recycle 11~24 d : with cellular recycle (a) optical density (b) gas production rate (c) H 2 content (d) ph The optical density of all three column incerased and then maintain a value of.58,.71 and.75, respectively for more than 12 days, when the photo-bioreactor was operated with cellular recycle (Fig 2a). This clearly indicated that the cells are washed out from the system faster than they can reproduce when the photo-bioreactor was operated without cellular recycle. When the photo-bioreactor was operated without cellular recycle, the H 2 production rate in the first column was in average 65.6 ml H 2 /l/d lower than that of the following two column, this could be due to the low cell concentration in the first column. When the experiment was shifted to operation with cellular recycle, there was a sudden increase of hydrogen production. However, the enhancement in H 2 production only lasted for a few days, before the average H 2 production performance in the three column dropped to 323.8, 283.4, and 21.3 ml H 2 /l/d, respectively. The average H 2 production obtained was ml H 2 /l/d higher than the result obtained without cellular recycle (Fig 2b). 3/8

4 WHEC 16 / June 26 Lyon France The H 2 content in biogas was 8% after start-up for 7 days and lasted until the end of experiment (Fig 2c), while it was unstable during the first 7 days. Relatively stable H 2 content seemed to suggest that the H2- producing culture had high operation stability. 3.2 Effect of HRT on hydrogen production In this experiment, the same operation conditions were conducted as 3.1. except the reactor was kept at HRT=8h. At the first stage of reaction, the optical density increased to a value of 1.8 (Fig 3a), meanwhile the H2 production decreased obviously (Fig 3b). It could be observed that all the columns were coated with cells. The possible reason was that biofilm was a shelter from the light, and this interfered with the hydrogen production. After removing the coated cells, the photo-bioreactor was started up. As shown in Fig 3a, the optical density of cells in the three columns remained at.33,.46,.58, respectively. This result clearly indicated that the cell density was lower at a HRT of 8h than at a HRT of 16h. In contrast, the average hydrogen production rate was 361, 262, 277 ml H 2 /l/d, respectively, and the total hydrogen production rate was 846 ml H 2 /l/d, that was slightly higher than the result obtained at a HRT of 16hr. This indicated that the photo-bioreactor could be operated at lower HRT and still hat the excellent H 2 producing performance. O.D a (day) ( ) gas 產氣量 (m ml / l/l.day) l / d H 2 % b (day) ( ) c (day) ( ) Fig 3 : Variation of optical density (a) gas production rate (b) H 2 content (c) during the experiment in the serial photo-bioreactor at a HRT of 8h, on day 12, coated cells were removed 3.3 Effect of organic acid concentration Four organic acid concentrations were examined, that was.2 fold,.4 fold,.6 fold and 1 fold. The result was shown in Table 1. When the organic acid concentration was diluted to.2 fold, the cell density was lower that of.4 fold dilution. When the organic acid concentration was diluted to.6 fold, the cell density was nearly equal to that of.4 fold dilution. It indicated that, when the organic acid concentration was higher than.4 fold dilution, the concentration of nitrogen source glutamate might limit cell growth. Therefore in the experiment of 1 fold organic acid concentration, 3 mg/l NH 4 Cl was used as nitrogen source. 4/8

5 WHEC 16 / June 26 Lyon France Table 1 showed that with.4 fold organic acid concentration, each of the three column showed ca. 2 ml H2/l/d higher hydrogen production than that of.2 fold organic acid concentration. The hydrogen production rate with.6 fold dilution was lower in the first column. The reason could be that the hydrogen production was inhibited by high concentration of organic acid, while the cell growth was not affected. The hydrogen production rate with 1 fold dilution was much lower. Apparently, high organic acid concentration inhibited cell growth and hydrogen production. As shown in Table1, the best hydrogen production rate was obtained when the organic acid concentration was diluted to.4 fold. Table 1:Hydrogen production with different mixed organic acid concentration Reactor 1 st column 2 nd column 3 rd column.2x.4x.6x 1X ml ml ml ml OD66 OD H 66 OD H 66 OD H 66 2/l/d 2/l/d 2/l/d H 2/l/d total Effect of ammonia concentration This study was fed with synthetic waste water, which organic acid concentration was not diluted (1 fold), the reactor was kept a HRT of 8h with cellular recycle. Three ammonia concentrations : 3, 5 and 7 mg/l were examined. As show in Fig 4a, the average cell density increased with increasing ammonia concentration. It indicated that high ammonia concentration was helpful for cell growth. Fig 4b showed that the higher the ammonia concentration, the inhibition of hydrogen production rate in the first column was more obvious. The first column had the function of decreasing ammonia concentration to the concentration below inhibition concentration (Fig 4c), therefore the ammonia concentration in the next two column remained below 1 mg/l and hydrogen production rate could keep constant despite the high ammonia concentration in the influent. O.D a NH 4 Cl 3 mg/l NH 4 Cl 5 mg/l NH 4 Cl 7 mg/l (day) gas 產氣量 ml (m l / /L l / d day) 8 b (day) mg NH 4 Cl/L c 1 5 Time 時間 (day) Fig 4 : Effect of ammonium concentration on hydrogen produ ction (a) optical density (b) gas production rate (c) NH 4 Cl concentration 5/8

6 WHEC 16 / June 26 Lyon France 3.5 Hydrogen production using real wastewater In this study effluent from two different anaerobic hydrogen fermentation reactors was used as influent for photo hydrogen production. The ammonia and organic acid composition of these two effluent was shown in Table 2. The reactor was fed with effluent 1 at the first stage. Because of the high organic acid concentration in the influent, the optical density could maintain the value of (Fig 5a). The ammonia content of effluent 1 was as high as 1 mg/l (Table 2). As indicated by Fig 5c, ammonia could be consumed in the first Table 2:Composition of effluent from hydrogen fermentation reactors NH 4 Cl (mg/l) Acetic acid Organic acid (mg/l) Propionic acid Butyric acid Effluent Effluent column of th e reactor, and in the second and third column only low ammonia concentration could be detected. Fig 5b shown that the hydrogen production in the first column was inhibited. Compare this result with Fig 5c, it could be explained that high ammonia concentration inhibit hydrogen production. As the ammonia concentration in the second and third reactor was low, they produced as high as 44 ml H 2 /l/d in the begining. At the second stage, effluent 2 was used as influent of photo-bioreactor. The optical density 1.13 was lower than that of the first stage (Fig 5a). The reson might be the lower organic acid concentration in the influent. At this stage, the hydrogen production rate in the first column is not inhibited (Fig 5b) because of ammonia concentration was nearly undetectable (Fig 5c). The average hydrogen production rate in each column was 232.8, 149., ml H 2 /l/d, respectively (Table 3). O.D 66 2 Effluent 1 Effluent 2 a Time 時間 (day) gas 產氣量 ml (m l / /L l /.day) d b Time 時間 (day) mg NH 4 Cl/L 1 9 c Time 時間 (day) Fig 5 : Variation of optical density(a) gas production rate(b) NH4Cl concentration(c) during the hydrogen production using real wastewater 6/8

7 WHEC 16 / June 26 Lyon France Table 3:Hydrogen production with effluent from two hydrogen fermentation reactors Acetic acid Organic acid removal (%) Propionic acid Hydrogen production rate Total (ml H 2/l/d) hydrogen Butyric 1 st 2 nd 3 rd production acid column column column ra te (ml H 2 /l/d) Effluent Effluent Compare the result of this study and other stud ies (Table 4), the hydrogen production rate.19 l/l/h for synthetic wastewater and.8 l/l/h for real wastewater were higher than that of lactic acid fermentation wastewater[7], sewage[9] and 2%OMW medium[1]. Table 4 : Example studies on the use of wastewater for hydrogen production Substrate.4 X synthetic wastewater+ glutamate Effluent from a hydrogen fermentation reactor Hydrogen production rate (l/l/h) references Suspended Immobilized cell cell.19 - This study.8 - This study Lactic acid fermentation wastewater.5 - [7] Toufu wastewater -.59 [8] Sewage.3.3 [9] 1% OMW medium -.9 [1] 2% OMW medium -.4 [1] 4. Conclusion A combination of purple nonsulfur photosynthetic bacteria and anaerobic bacteria could be used for efficient wastewater conversion into hydrogen. Using a serial photobioreactor with cellular recycle, the impact of a high flow rate and high organic acid and ammonia concentrations could be solved. Even for a HRT at a value of 8 h for the reactor, the biomass could still be retained and the total hydrogen production rate was 846 ml/l/d. 5. Acknowlegement This research was supported by the National Science Council, Taiwan (NSC E-5-25 ), Thanks Prof. Sheng-Shung Cheng, NCKU for technical assistance of the construction of a hydrogen fermentation reactor and for providing wastewater. References: 1. Zuerrer. H. and Bachhofen. R. Aspects of the growth and hydrogen production of the photosynthetic bacterium Rhodospirillum rubrum in continuous culture. Biomass 2: , Lee C.-M. Chen P.-C.,Wang C.-C. and Tung Y.- C. Tung. Photohydrogen production using purple nonsulfur bacteria with hydrogen fermentation reactor effluent, Int. J. Hydrogen Energy 27, , 22. 7/8

8 WHEC 16 / June 26 Lyon France 3. Lee C.-M., Yu K.-M., and Chen P.-C. Limiting factors of photo-hydrogen production by Rhodopseudornoncos palustris WP 3-5, International Conference on Environmental, Industrial and Applied Microbiology, Badajoz, Spain. March Pfenning, N., Rhodocyclus purpureus gen. nov. and sp. nov., a ring-shaped vitamin B 12 -requiring member of the family Rhodospirillaceae. International Journal of Systematic Bacteriology. 28: , Cheng S.S., Chen S.T., Bai M.D., Chang S.M., and Wu K.L., Anaerobic hydrogen production in mesophilic and thermophilic fermenting processes. Proceeding of 9th World Congress on Anaerobic Digestion 21. Antwerpen. Belgium. 2, , Keeney D.R., Nelson D.W. Indophenol-blue method. In: Page A.L., Miller R.H., Keeney D.R., (editors). Methods of soil analysis, part 2: chemical and microbiological properties, ed. p Sasikala K. Ramana C.V.. Photoproduction of hydrogen from waste water of a lactic acid fermentation plant by a purple non-sulfur photosynthetic bacterium, Rhodobacter sphaeroides O.U. 1. Indian J Experimental Biol, 29: 74-75,1991b. 8. 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, 24: 35-1, Sunita M., Mitra C.K., Photoproduction of hydrogen by photosynthetic bacteria from sewage and wastewater. J. Bioscience 18: 155-6, Eroğlu E, Eroğlu İ, Gündüz U, Yücel M, Türker L. Biological Hydrogen production from olive mill wastewater by Rhodobacter sphaeroides O.U. 1. In: Gökcekus H, editor. International Conference on Environmental Problems of Mediterranean Region (EPMR-22) in Near East University, Nicosa- Northern Cyprus, Book of Abstracts, p. 62, 22. 8/8