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Photosynthetic hydrogen production by alginate immobilized bacterial consortium
Bioresource Technology 236 (2017) 44–48
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Photosynthetic hydrogen production by alginate immobilized bacterial consortium Huan Zhang a,b, Guanyi Chen a, Quanguo Zhang b, Duu-Jong Lee b,c,d,⇑, Zhiping Zhang b, Yameng Li b, Pengpeng Li b, Jianjun Hu a, Beibei Yan a a
School of Environmental Science & Engineering, Tianjin University, Tianjin 300350, PR China Collaborative Innovation Center of Biomass Energy, Henan Agricultural University, Zhengzhou 450002, PR China Department of Chemical Engineering, National Taiwan University, Taipei 10617, Taiwan d Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 10607, Taiwan b c
h i g h l i g h t s Photosynthetic H2 production from immobilized cells was studied. Immobilized cells produced more H2 than free cells. Optimal granule size, cell loadings, and cell ages for granules were reported. Minimum substrate concentration and maximum illumination intensity were reported. Alginate matrix can provide shield to embedded cells from external challenges.
a r t i c l e
i n f o
Article history: Received 27 February 2017 Received in revised form 26 March 2017 Accepted 27 March 2017 Available online 30 March 2017 Keywords: Photosynthetic bacteria Hydrogen production Immobilization Swine manure
a b s t r a c t Photosynthetic hydrogen production from organic wastewaters using immobilized mixed culture with photosynthetic bacteria (PSB) was studied. A PSB consortium was immobilized by alginate matrix to form granules. The so-yielded granules exhibited minimal diffusional resistances to substrates and to illumination penetration but still produced more hydrogen from synthetic wastewater than the free cells at identical experimental conditions. Optimal granule size, cell loadings, and cell ages for granules and the minimum substrate concentration and maximum illumination intensity requited to maximize hydrogen production were studied. The applied alginate matrix can provide shield to embedded cells from external challenges, likely the produced proton gradients from the surroundings. Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction Hydrogen (H2) is an energy carrier with high energy density (122 kJ/g), about 3.8 times to gasoline (Kumar et al., 2016). Biological hydrogen production is one of potentially feasible ways for supplying green hydrogen to sustainable society (Khan et al., 2016; Boboescu et al., 2016). Biological H2 production can be categorized into dark and photo-fermentative pathways (Kumar and Chowdhary, 2016), in which photo-H2 production by photosynthetic bacteria (PSB) is considered a promising technology which is driven by light energy (Guo et al., 2015; Lin et al., 2016) and
⇑ Corresponding author at: Department of Chemical Engineering, National Taiwan University, Taipei 10617, Taiwan. E-mail address: [email protected] (D.-J. Lee). http://dx.doi.org/10.1016/j.biortech.2017.03.171 0960-8524/Ó 2017 Elsevier Ltd. All rights reserved.
can couple with wastewater treatment processes at ambient temperature and pressure (Hosseini et al., 2015). Cell immobilization is widely used for enhanced fermentative hydrogen production (Kumar et al., 2016). Compared with free PSB cells, immobilized cells can effectively prolong H2 production time and improve H2 production rate. Guevara-Lopez and Buitron (2015) evaluated the different support materials for immobilizing Rhodopseudomonas palustris consortium for their capability to photofermentatively produce H2 from volatile fatty acids as substrates. Another effective immobilization technology is to immobilize functional substances in polymeric matrix (Lai et al., 2016). Zhu et al. (1999) studied hydrogen production from tofu wastewater by Rhodobacter sphaeroides immobilized in agar gels and found that the H2 production lasted up to 50 h and the yield of hydrogen was 1.9 mL/mL. Ishikawa et al. (2008) used agar gel immobilized Escherichia coli to yield 6.7 mL/(Lh) H2 from glucose
H. Zhang et al. / Bioresource Technology 236 (2017) 44–48
wastewater. Seon et al. (1993) optimized H2 production from glucose by immobilized Rhodospirillum rubum KS-301, maximizing H2 production rate at 91 mL/h from glucose wastewaters. Zhang et al. (2016) immobilized Rhodopsudomonas palustris as biofilm on optical fiber for producing H2 at 0.85 mmol/g-h with uniform lighting. Zagrodnik et al. (2015) immobilized Rhodobacter sphaeroides O. U.001 on porous glass plates or glass beads for photo-H2 production from malic acid. The average H2 production rate at 12.7 mL/ Lh was achieved. All the above-mentioned studies utilized pure culture cells for immobilized photo-fermentative tests. However, to produce H2 from waste materials cannot be realized in a sterilized environment; the pure culture would be continuously challenged by external strains. The mixed culture consortium always has a better adaption to environmental changes than pure culture systems (Lee et al., 2011). Sodium alginate is a carbohydrate with chemical formula (C6H7NaO6)x, which is commonly used as immobilized substrate for bacterial cells or other organic/inorganic compounds (Yang et al., 2014). This study used alginate to immobilized cultivated PSB consortium with mixed bacterial species for producing hydrogen from synthetic medium via photofermentative pathway. Effects of substrate concentration, biomass loading, granule size, cell age and illumination intensity on photo-H2 productivity were studied.
45
pig manure was exposed to air in 120 rpm oscillator at 25 °C for four days. And then the suspension was screened with 40-mesh sieve and the filtrate was sterilized at 121 °C for 30 min. 1 g/L glucose was added to enhance growth of H2-producing bacteria. 250-mL conical flasks sealed with rubber stoppers were applied in batch tests for photo-fermentation of the pre-prepared swine wastewater. The flasks were placed in thermostat at 30 °C with two incandescent lamps being placed at opposite sides of each flask to assure uniform incident light distributions. The PSB granules formed in Section 2.2 were cultured with cultivation medium for 4 h before being fed into the photobioreactor. The control tests were conducted with the same ingredients (40 mL sodium alginate solution +60 mg cell (dry basis) +40 mL cell-free fermentation medium) at pH 7.0. Effects of cell age (cultivation time before immobilization) (36–108 h), biomass quantity in sodium alginate matrix (1–3 mg cells/mL), substrate concentration (1200–8000 mg COD/L), granule diameter (0.5–2.5 mm), and illumination intensity (2000–10000 lx) on H2 production were studied (Table 1). All runs were done in triplicate to assure the data reproducibility. Using glucose as model compounds, the COD decrease equivalent is assumed as follows: 8 g COD reduced = 1 g H2 produced. 2.4. Analytical methods
2. Materials and methods 2.1. Microorganisms and medium The PSB HAU-M1 is a consortium composing of Rhodospirillum rubrum, Rhodobacter capsulatus and Rhodopseudomonas palustris (Lu et al., 2016). The cultivation medium had the following compositions (g/L): NH4Cl, 1; NaHCO3, 2; K2HPO4, 0.2; CH3COONa, 3; MgSO47H2O, 0.2; NaCl, 2; yeast extract, 1; and micronutrient solution (1 mL/L) with FeCl36H2O (5 mg/L), ZnSO47H2O (1 mg/L), CuSO45H2O (0.05 mg/L), H3BO4 (1 mg/L), MnCl24H2O (0.05 mg/L), and Co(NO3)26H2O (0.5 mg/L). Argon gas purged the solutions to expel oxygen to form anaerobic conditions. The consortium was cultivated anaerobically at 30 °C at illumination of incandescent lamp (3000 lx). The solution pH was adjusted to 7.0 by adding 50% (w/w) KOH solution. The C, N, P and micro-nutrient of the fermentation medium were supplied at (g/L): NH4Cl (0.4), MgCI2 (0.2), yeast extract (0.1), K2HPO4 (0.5), NaC1 (2), and sodium glutamate (3.5). 2.2. Immobilization of bacterial cells After PSB HAU-M1 consortium was cultivated in batch reactor to its exponential growth phase with cell concentration of 1.25 ± 0.02 g/L, the cells were washed with sterilized deionized water and were rapidly mixed with sodium alginate solution (at final sodium alginate concentration of 3%) at 30 °C. A peristaltic pump extruded the mixed solution as drops into 5% CaCl2 solution for forming 6.5 ± 0.1 mm PSB granules. The formed granules were washed thoroughly with physiological brine and were ready for use. The entire immobilization protocol was under sterile condition. 2.3. Bio-H2 production tests Fresh swine manure was collected from farms in the eastern suburbs of Zhengzhou City, China. The physical and chemical characteristics of swine manure were analyzed following Standard Methods for chemical oxygen demand (COD), nitrogen, and total phosphorus (APHA, 2012), water 79% (w/w), COD 179 mg/g, nitrogen 8.4 mg/g, phosphorus 2.9 mg/g, pH 7–8. Prior to tests, the fresh
The compositions of generated gas from the flasks were measured every 12 h using a gas chromatography (6820GC-14B, Agilent, USA). Nitrogen at a flow rate of 45 mL/min was the carrier gas; the temperatures of the injector, detector and column were 100, 80 and 150 °C, respectively. The solution pH value was measured by a pH meter (PHS-3C, Shanghai, China). The optical density of cell biomass was determined at 660 nm using a spectrophotometer (HP8453 Ultraviolet Spectrophotometer, Agilent, USA) (Pattanamanee, 2012). 3. Results and discussion 3.1. Immobilized cells on photo-H2 production In this section the photo-H2 production using immobilized granules at 30 °C, 5500 mg COD/L, pH 7, 2.0 mm granules with 72 h cell age and the control test with free suspended cells (with identical quantity of alginate and other ingredients) is compared (Table 2). As this table shows, the immobilized cells produced 1.37–1.43 times H2 at identical conditions with free cells. For instance, at 120 h, the granules produced 152.1 mL H2 (=reduction of 1120 mg COD/L) while free cells yielded 977 mL H2 (=reduction of 977 mg COD/L). 3.2. Single-factor photo-hydrogen tests 3.2.1. Effects of immobilized biomass on H2 production The hydrogen production rate at 30 °C, pH 7, 8000 lx, 2 mm granule size, 72 h cell age, 5500 mg COD/L is increased with immobilized biomass quantity, but not in proportionality (Fig. 1a). For instance, at 144 h, the H2 production quantity was 142 mL for 1 mg/mL granules and was 186 mL for 2 mg/mL. At 2.5 mg/mL, the H2 production peaked to 193 mL at 144 h; but at 3 mg/mL, the H2 production reduced to 164 mL. Restated, there is an optimal biomass loading in immobilized matrix, 2.5 mg/mL in the present case. During the tests, the solution COD was declined with time, while the pH was reduced from 7 to 5.5 in the first 20-h testing. The solution pH was declined owing to the production of volatile fatty acids in substrate hydrolysis. The COD decreasing rate was
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H. Zhang et al. / Bioresource Technology 236 (2017) 44–48
Table 1 Variable design of single factor tests. Variable
Unit
Level
Biomass Substrate concentration Size particles Bacteria age Light intensity
mg cell (dry weight)/mL in COD, mg/L mm h Lx
1 1200 0.5 24 2000
1.5 3500 1 48 4000
2 5500 1.5 72 6000
2.5 6700 2 96 8000
3 8000 2.5 120 10,000
Table 2 Photo-hydrogen production by granules and by free cells. 30 °C, pH 7, 8000 lx, 5500 mg COD/L, 72 h cell age. F: free cells; G: granules. Time (h)
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Bacterial state
F
G
F
72 G
96 F
G
120 F
G
H2 produced (mL) H2 productivity (mL/hL) Specific H2 productivity (mL/gh)
25.6 2.37 21.3
35.8 4.14 29.8
56.3 3.48 31.3
79.5 6.13 44.2
132.4 6.13 55.2
151.1 8.74 62.9
152.1 5.64 50.7
174.6 8.08 58.2
Fig. 1. Effects of immobilized biomass quantity on hydrogen production (a) and on solution pH and COD (b). 30 °C, pH 7, 8000 lx, 2 mm granule size, 72 h cell age, 5500 mg COD/L.
Fig. 2. Effects of substrate concentration on hydrogen production (a) and on solution pH and COD (b). 30 °C, pH 7, 8000 lx, 2 mg/mL loading rate, 2 mm granule size, 72 h cell age.
increased with immobilized biomass quantity, neither in proportionality (Fig. 1b). Although less H2 was produced by 3 mg/mL granules than 2.5 mg/mL (Fig. 1a), the former degraded more COD than the latter (Fig. 1b). 3.2.2. Effects of substrate concentration on H2 production The H2 production at 30 °C, pH 7, 8000 lx, 2 mg/mL loading rate, 2 mm granule size, 72 h cell age were shown in Fig. 2a. The hydrogen production rate was increased with substrate concentration
from 1200 to 2500 mg COD/mL, and reach plateau at and above 5500 mg COD/mL. This observation reveals that the 5500 mg COD/mL can be regarded as reaching the maximum growth rate of cells and there showed no substrate inhibition over the studied concentration range. As in Section 3.2.1, the solution pH dropped from 7 to around 5.5 in the first phase of testing (Fig. 2b). Correspondingly, the COD was degraded by biomass and was declined in concentration over testing time. The COD decrease ranged 2100–2800 mg COD/mL.
H. Zhang et al. / Bioresource Technology 236 (2017) 44–48
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Fig. 3. Effects of granule size on hydrogen production (a) and on solution pH and COD (b). 30 °C, pH 7, 8000 lx, 2 mg/mL loading rate, 72 h cell age, 5500 mg COD/L.
Fig. 4. Effects of cell age on hydrogen production (a) and on solution pH and COD (b). 30 °C, pH 7, 8000 lx, 2 mg/mL loading rate, 2 mm granule size, 5500 mg COD/L.
3.2.3. Effects of granule sizes on H2 production Fig. 3a shows the effects of granule size on photo-H2 production at 30 °C, pH 7, 8000 lx, 2 mg/mL loading rate, 72 h cell age and 5500 mg COD/L. The corresponding pH and COD changes are demonstrated in Fig. 3b. Interestingly, the H2 productivity followed 1 mm = 1.5 mm > 0.5 mm > 2 mm > 2.5 mm. Restated, there is an optimal granule size with sufficient embedded biomass for reaction but with insignificant limitations by the large matrix size. However, since the difference was not large, at most 20% amongst different granule size, the present granules exhibit low mass transfer resistances for substrates and low shielding effects for incipient lights for the embedded cells.
3.2.4. Effects of cell age on H2 production Fig. 4a and b shows respectively the cell age and pH and COD changes during photo-H2 production tests at 30 °C, pH 7.0, 8000 lx, 5500 mg COD/L and 2 mm granules. There was an optimal cell age of 72–96 h with maximum H2 productivity: younger PSB cells are not ready for effective operation while older cells have passed over their best production age. 3.2.5. Effects of illumination intensity on H2 production Fig. 5a reveals the effects of illumination intensity on photo-H2 production at 30 °C, pH 7, 2 mg/mL loading rate, 2 mm granules, 72 h cell age and 5500 mg COD/L. The corresponding pH and
Fig. 5. Effects of light intensity on hydrogen production (a) and on solution pH and COD (b). 30 °C, pH 7, 2 mg/mL loading rate, 2 mm granule size, 72 h cell age, 5500 mg COD/L.
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H. Zhang et al. / Bioresource Technology 236 (2017) 44–48
COD changes are also shown in Fig. 5b. The hydrogen production at 144 h was increased from 43 to 178 mL with illumination intensity from 2000 to 8000 lx. Further increase in illumination intensity to 12,000 lx decreased the hydrogen productivity to 143 mL. The illumination intensity above 8000 lx would exhibit light inhibition to the present embedded PSB cells, correlating to the finding in previous report (Hu et al., 2016). 3.3. Use of immobilized cells on photo-hydrogen production The alginate immobilized HAU-M1 PSB granules can produced more photo-H2 than the free cells at identical production conditions at 30 °C and pH 7. The adopted granule size (0.5–2.5 mm), the loaded cell quantity (1–3 mg cells/mL), cell age (36–108 h) and the light intensity (2000–10,000 lx) ranged studied revealed no inhibition to PSB cell activities for H2 production. Owing to hydrolysis reaction the solution pH dropped to around 5.5 in all tests (Figs. 1–5b); likely the alginate matrix can resist proton diffusion to minimize photo-H2 production under < 5500 mg COD/mL and <8000 lx conditions. Use of mixed culture in inoculum leads to competition of substrates by different bacterial groups. In the present study, the COD decrease corresponding to photo-H2 production at optimal condition ranged 1100–1200 mg COD/mL, while the overall COD decrease ranged 2000–2800 mg COD/mL. Restated, the fraction of COD decrease by photo-fermentative pathway ranged 42–57%. Therefore, the present alginate matrix provided a shield for photofermentative bacteria to compete with heterotrophic bacteria to promote photo-H2 production. 4. Conclusions The sodium alginate immobilized HAU-M1 consortium produced 1.37–1.43 times quantity of H2 to identical quantity of free cells from synthetic wastewaters at the identical composition. In single-factor tests, the HAU-M1 granules with 2 mm size, 72–96 cell age and 2.5 mg/mL cell loading can produce photo-H2 at maximum productivity from wastewater at 8000 lx and >5500 mg COD/L. The alginate matrix leads to minimal diffusional resistance to substrates or to illumination penetration at granule size less than 2.5 mm, however, still provides shield to embedded cells from external challenges, likely the produced proton gradients from the surroundings. Acknowledgements Financial supports from National Natural Science Foundation of China (51676065) and Doctoral Scientific Fund Project of the Ministry of Education of China (20134105130001).
References APHA, 2012. Standard Methods for the Examination of Water and Wastewater. APHA, Washington DC, USA. Boboescu, I.Z., Cherman, V.D., Lakatos, G., Pap, B., Biro, T., Maroti, G., 2016. Surpassing the current limitations of biohydrogen production systems: The case for a novel hybrid approach. Bioresour. Technol. 204, 192–201. Guevara-Lopez, E., Buitron, G., 2015. Evaluation of different support materials used with a photo-fermentative consortium for hydrogen production. Int. J. Hydrogen Energy 40, 17231–17238. Guo, C.L., Cao, H.X., Pei, H.S., Guo, F.Q., Liu, D.M., 2015. A multiphase mixture model for substrate concentration distribution characteristics and photo-hydrogen production performance of the entrapped-cell photobioreactor. Bioresour. Technol. 181, 40–46. Hosseini, S.S., Aghbashlo, M., Tabatabaei, M., Younesi, H., Najafpour, G., 2015. Exergy analysis of biohydrogen production from various carbon sources via anaerobic photosynthetic bacteria (Rhodospirium rubrum). Energy 93, 730–739. Hu, J.J., Zhang, Q.G., Jing, Y.Y., Lee, D.J., 2016. Photosynthetic hydrogen production from enzyme-hydrolyzed micro-grinded maize straws. Int. J. Hydrogen Energy 41, 21665–21669. Ishikawa, M., Yamamura, S., Ikeda, R., Takamura, Y., Sode, K., Tamiya, E., Tomiyama, M., 2008. Development of a compact stacked flatbed reactor with immobilized high-density bacteria for hydrogen production. Int. J. Hydrogen Energy 33, 1593–1597. Khan, M.A., Ngo, H.H., Guo, W.S., Liu, Y., Nghiem, L.D., Hai, F.I., Deng, L.J., Wang, J., Wu, Y., 2016. Optimization of process parameters for production of volatile fatty acid, biohydrogen and methane from anaerobic digestion. Bioresour. Technol. 219, 738–748. Kumar, G.R., Chowdhary, N., 2016. Biotechnological and bioinformatics approaches for augmentation of biohydrogen production: a review. Renew. Sustain. Energy 56, 1194–1206. Kumar, G., Mudhoo, A., Sivagurunathan, P., Nagarajan, D., Ghimire, A., Lay, C.H., Lin, C.Y., Lee, D.J., Chang, J.S., 2016. Recent insights into the cell immobilization technology applied for dark fermentative hydrogen production. Bioresour. Technol. 219, 725–737. Lai, Y.C., Chang, Y.R., Chen, M.L., Lo, Y.K., Lai, J.Y., Lee, D.J., 2016. Poly(vinyl alcohol) and alginate cross-linked matrix with immobilized Prussian blue and ion exchange resin for cesium removal from waters. Bioresour. Technol. 214, 192– 198. Lee, D.J., Show, K.Y., Su, A., 2011. Dark fermentation on biohydrogen production: pure culture. Bioresour. Technol. 102, 8393–8402. Lin, R.C., Cheng, J., Yang, Z.B., Ding, L.K., Zhang, J.B., Zhou, J.H., Chen, K.F., 2016. Enhanced energy recovery from cassava ethanol wastewater through sequential dark hydrogen, photo hydrogen and methane fermentation combined with ammonium removal. Bioresour. Technol. 213, 686–691. Lu, C.Y., Zhang, Z.P., Ge, X.M., Wang, Y., Zhou, X.H., You, X.F., Liu, H.L., Zhang, Q.G., 2016. Bio-hydrogen production from apple waste by photosynthetic bacteria HAU-M1. Int. J. Hydrogen Energy 41, 13399–13407. Seon, Y.H., Lee, C.G., Park, D.H., Hwang, K.Y., Joe, Y.I., 1993. Hydrogen production by immobilized cells in the nozzle loop bioreactor. Biotechnol. Lett. 15, 1275– 1280. Yang, Y.C., Liu, X., Wang, C.L., Sun, S.P., Lee, D.J., 2014. Accelerated aerobic granulation using alternating feed loadings: algniate-like exopolysaccharides. Bioresour. Technol. 171, 360–366. Zagrodnik, R., Seifert, K., Stodolny, M., Laniecki, M., 2015. Continuous photofermentative production by immobilized Rhodobacter sphaeroides OU001. Int. J. Hydrogen Energy 40, 5062–5073. Zhang, C., Fu, J., Wang, B.W., Liu, X.Y., Cheng, M., Zhang, Q.G., 2016. Enhancing continuous photo-H2 production using optical fiber for biofilm formation. Environ. Progr. Sustain. Energy 35, 455–460. Zhu, H., Suzuki, T., Tsygankov, A.A., Asada, Y., Miyake, J., 1999. Hydrogen production from tofu wastewater by Rhodobacter sphaeroides immobilized in agar gels. Int. J. Hydrogen Energy 24, 305–310.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Photosynthetic hydrogen production by alginate immobilized bacterial consortium Huan Zhang a,b, Guanyi Chen a, Quanguo Zhang b, Duu-Jong Lee b,c,d,⇑, Zhiping Zhang b, Yameng Li b, Pengpeng Li b, Jianjun Hu a, Beibei Yan a a
School of Environmental Science & Engineering, Tianjin University, Tianjin 300350, PR China Collaborative Innovation Center of Biomass Energy, Henan Agricultural University, Zhengzhou 450002, PR China Department of Chemical Engineering, National Taiwan University, Taipei 10617, Taiwan d Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei 10607, Taiwan b c
h i g h l i g h t s Photosynthetic H2 production from immobilized cells was studied. Immobilized cells produced more H2 than free cells. Optimal granule size, cell loadings, and cell ages for granules were reported. Minimum substrate concentration and maximum illumination intensity were reported. Alginate matrix can provide shield to embedded cells from external challenges.
a r t i c l e
i n f o
Article history: Received 27 February 2017 Received in revised form 26 March 2017 Accepted 27 March 2017 Available online 30 March 2017 Keywords: Photosynthetic bacteria Hydrogen production Immobilization Swine manure
a b s t r a c t Photosynthetic hydrogen production from organic wastewaters using immobilized mixed culture with photosynthetic bacteria (PSB) was studied. A PSB consortium was immobilized by alginate matrix to form granules. The so-yielded granules exhibited minimal diffusional resistances to substrates and to illumination penetration but still produced more hydrogen from synthetic wastewater than the free cells at identical experimental conditions. Optimal granule size, cell loadings, and cell ages for granules and the minimum substrate concentration and maximum illumination intensity requited to maximize hydrogen production were studied. The applied alginate matrix can provide shield to embedded cells from external challenges, likely the produced proton gradients from the surroundings. Ó 2017 Elsevier Ltd. All rights reserved.
1. Introduction Hydrogen (H2) is an energy carrier with high energy density (122 kJ/g), about 3.8 times to gasoline (Kumar et al., 2016). Biological hydrogen production is one of potentially feasible ways for supplying green hydrogen to sustainable society (Khan et al., 2016; Boboescu et al., 2016). Biological H2 production can be categorized into dark and photo-fermentative pathways (Kumar and Chowdhary, 2016), in which photo-H2 production by photosynthetic bacteria (PSB) is considered a promising technology which is driven by light energy (Guo et al., 2015; Lin et al., 2016) and
⇑ Corresponding author at: Department of Chemical Engineering, National Taiwan University, Taipei 10617, Taiwan. E-mail address: [email protected] (D.-J. Lee). http://dx.doi.org/10.1016/j.biortech.2017.03.171 0960-8524/Ó 2017 Elsevier Ltd. All rights reserved.
can couple with wastewater treatment processes at ambient temperature and pressure (Hosseini et al., 2015). Cell immobilization is widely used for enhanced fermentative hydrogen production (Kumar et al., 2016). Compared with free PSB cells, immobilized cells can effectively prolong H2 production time and improve H2 production rate. Guevara-Lopez and Buitron (2015) evaluated the different support materials for immobilizing Rhodopseudomonas palustris consortium for their capability to photofermentatively produce H2 from volatile fatty acids as substrates. Another effective immobilization technology is to immobilize functional substances in polymeric matrix (Lai et al., 2016). Zhu et al. (1999) studied hydrogen production from tofu wastewater by Rhodobacter sphaeroides immobilized in agar gels and found that the H2 production lasted up to 50 h and the yield of hydrogen was 1.9 mL/mL. Ishikawa et al. (2008) used agar gel immobilized Escherichia coli to yield 6.7 mL/(Lh) H2 from glucose
H. Zhang et al. / Bioresource Technology 236 (2017) 44–48
wastewater. Seon et al. (1993) optimized H2 production from glucose by immobilized Rhodospirillum rubum KS-301, maximizing H2 production rate at 91 mL/h from glucose wastewaters. Zhang et al. (2016) immobilized Rhodopsudomonas palustris as biofilm on optical fiber for producing H2 at 0.85 mmol/g-h with uniform lighting. Zagrodnik et al. (2015) immobilized Rhodobacter sphaeroides O. U.001 on porous glass plates or glass beads for photo-H2 production from malic acid. The average H2 production rate at 12.7 mL/ Lh was achieved. All the above-mentioned studies utilized pure culture cells for immobilized photo-fermentative tests. However, to produce H2 from waste materials cannot be realized in a sterilized environment; the pure culture would be continuously challenged by external strains. The mixed culture consortium always has a better adaption to environmental changes than pure culture systems (Lee et al., 2011). Sodium alginate is a carbohydrate with chemical formula (C6H7NaO6)x, which is commonly used as immobilized substrate for bacterial cells or other organic/inorganic compounds (Yang et al., 2014). This study used alginate to immobilized cultivated PSB consortium with mixed bacterial species for producing hydrogen from synthetic medium via photofermentative pathway. Effects of substrate concentration, biomass loading, granule size, cell age and illumination intensity on photo-H2 productivity were studied.
45
pig manure was exposed to air in 120 rpm oscillator at 25 °C for four days. And then the suspension was screened with 40-mesh sieve and the filtrate was sterilized at 121 °C for 30 min. 1 g/L glucose was added to enhance growth of H2-producing bacteria. 250-mL conical flasks sealed with rubber stoppers were applied in batch tests for photo-fermentation of the pre-prepared swine wastewater. The flasks were placed in thermostat at 30 °C with two incandescent lamps being placed at opposite sides of each flask to assure uniform incident light distributions. The PSB granules formed in Section 2.2 were cultured with cultivation medium for 4 h before being fed into the photobioreactor. The control tests were conducted with the same ingredients (40 mL sodium alginate solution +60 mg cell (dry basis) +40 mL cell-free fermentation medium) at pH 7.0. Effects of cell age (cultivation time before immobilization) (36–108 h), biomass quantity in sodium alginate matrix (1–3 mg cells/mL), substrate concentration (1200–8000 mg COD/L), granule diameter (0.5–2.5 mm), and illumination intensity (2000–10000 lx) on H2 production were studied (Table 1). All runs were done in triplicate to assure the data reproducibility. Using glucose as model compounds, the COD decrease equivalent is assumed as follows: 8 g COD reduced = 1 g H2 produced. 2.4. Analytical methods
2. Materials and methods 2.1. Microorganisms and medium The PSB HAU-M1 is a consortium composing of Rhodospirillum rubrum, Rhodobacter capsulatus and Rhodopseudomonas palustris (Lu et al., 2016). The cultivation medium had the following compositions (g/L): NH4Cl, 1; NaHCO3, 2; K2HPO4, 0.2; CH3COONa, 3; MgSO47H2O, 0.2; NaCl, 2; yeast extract, 1; and micronutrient solution (1 mL/L) with FeCl36H2O (5 mg/L), ZnSO47H2O (1 mg/L), CuSO45H2O (0.05 mg/L), H3BO4 (1 mg/L), MnCl24H2O (0.05 mg/L), and Co(NO3)26H2O (0.5 mg/L). Argon gas purged the solutions to expel oxygen to form anaerobic conditions. The consortium was cultivated anaerobically at 30 °C at illumination of incandescent lamp (3000 lx). The solution pH was adjusted to 7.0 by adding 50% (w/w) KOH solution. The C, N, P and micro-nutrient of the fermentation medium were supplied at (g/L): NH4Cl (0.4), MgCI2 (0.2), yeast extract (0.1), K2HPO4 (0.5), NaC1 (2), and sodium glutamate (3.5). 2.2. Immobilization of bacterial cells After PSB HAU-M1 consortium was cultivated in batch reactor to its exponential growth phase with cell concentration of 1.25 ± 0.02 g/L, the cells were washed with sterilized deionized water and were rapidly mixed with sodium alginate solution (at final sodium alginate concentration of 3%) at 30 °C. A peristaltic pump extruded the mixed solution as drops into 5% CaCl2 solution for forming 6.5 ± 0.1 mm PSB granules. The formed granules were washed thoroughly with physiological brine and were ready for use. The entire immobilization protocol was under sterile condition. 2.3. Bio-H2 production tests Fresh swine manure was collected from farms in the eastern suburbs of Zhengzhou City, China. The physical and chemical characteristics of swine manure were analyzed following Standard Methods for chemical oxygen demand (COD), nitrogen, and total phosphorus (APHA, 2012), water 79% (w/w), COD 179 mg/g, nitrogen 8.4 mg/g, phosphorus 2.9 mg/g, pH 7–8. Prior to tests, the fresh
The compositions of generated gas from the flasks were measured every 12 h using a gas chromatography (6820GC-14B, Agilent, USA). Nitrogen at a flow rate of 45 mL/min was the carrier gas; the temperatures of the injector, detector and column were 100, 80 and 150 °C, respectively. The solution pH value was measured by a pH meter (PHS-3C, Shanghai, China). The optical density of cell biomass was determined at 660 nm using a spectrophotometer (HP8453 Ultraviolet Spectrophotometer, Agilent, USA) (Pattanamanee, 2012). 3. Results and discussion 3.1. Immobilized cells on photo-H2 production In this section the photo-H2 production using immobilized granules at 30 °C, 5500 mg COD/L, pH 7, 2.0 mm granules with 72 h cell age and the control test with free suspended cells (with identical quantity of alginate and other ingredients) is compared (Table 2). As this table shows, the immobilized cells produced 1.37–1.43 times H2 at identical conditions with free cells. For instance, at 120 h, the granules produced 152.1 mL H2 (=reduction of 1120 mg COD/L) while free cells yielded 977 mL H2 (=reduction of 977 mg COD/L). 3.2. Single-factor photo-hydrogen tests 3.2.1. Effects of immobilized biomass on H2 production The hydrogen production rate at 30 °C, pH 7, 8000 lx, 2 mm granule size, 72 h cell age, 5500 mg COD/L is increased with immobilized biomass quantity, but not in proportionality (Fig. 1a). For instance, at 144 h, the H2 production quantity was 142 mL for 1 mg/mL granules and was 186 mL for 2 mg/mL. At 2.5 mg/mL, the H2 production peaked to 193 mL at 144 h; but at 3 mg/mL, the H2 production reduced to 164 mL. Restated, there is an optimal biomass loading in immobilized matrix, 2.5 mg/mL in the present case. During the tests, the solution COD was declined with time, while the pH was reduced from 7 to 5.5 in the first 20-h testing. The solution pH was declined owing to the production of volatile fatty acids in substrate hydrolysis. The COD decreasing rate was
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Table 1 Variable design of single factor tests. Variable
Unit
Level
Biomass Substrate concentration Size particles Bacteria age Light intensity
mg cell (dry weight)/mL in COD, mg/L mm h Lx
1 1200 0.5 24 2000
1.5 3500 1 48 4000
2 5500 1.5 72 6000
2.5 6700 2 96 8000
3 8000 2.5 120 10,000
Table 2 Photo-hydrogen production by granules and by free cells. 30 °C, pH 7, 8000 lx, 5500 mg COD/L, 72 h cell age. F: free cells; G: granules. Time (h)
46
Bacterial state
F
G
F
72 G
96 F
G
120 F
G
H2 produced (mL) H2 productivity (mL/hL) Specific H2 productivity (mL/gh)
25.6 2.37 21.3
35.8 4.14 29.8
56.3 3.48 31.3
79.5 6.13 44.2
132.4 6.13 55.2
151.1 8.74 62.9
152.1 5.64 50.7
174.6 8.08 58.2
Fig. 1. Effects of immobilized biomass quantity on hydrogen production (a) and on solution pH and COD (b). 30 °C, pH 7, 8000 lx, 2 mm granule size, 72 h cell age, 5500 mg COD/L.
Fig. 2. Effects of substrate concentration on hydrogen production (a) and on solution pH and COD (b). 30 °C, pH 7, 8000 lx, 2 mg/mL loading rate, 2 mm granule size, 72 h cell age.
increased with immobilized biomass quantity, neither in proportionality (Fig. 1b). Although less H2 was produced by 3 mg/mL granules than 2.5 mg/mL (Fig. 1a), the former degraded more COD than the latter (Fig. 1b). 3.2.2. Effects of substrate concentration on H2 production The H2 production at 30 °C, pH 7, 8000 lx, 2 mg/mL loading rate, 2 mm granule size, 72 h cell age were shown in Fig. 2a. The hydrogen production rate was increased with substrate concentration
from 1200 to 2500 mg COD/mL, and reach plateau at and above 5500 mg COD/mL. This observation reveals that the 5500 mg COD/mL can be regarded as reaching the maximum growth rate of cells and there showed no substrate inhibition over the studied concentration range. As in Section 3.2.1, the solution pH dropped from 7 to around 5.5 in the first phase of testing (Fig. 2b). Correspondingly, the COD was degraded by biomass and was declined in concentration over testing time. The COD decrease ranged 2100–2800 mg COD/mL.
H. Zhang et al. / Bioresource Technology 236 (2017) 44–48
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Fig. 3. Effects of granule size on hydrogen production (a) and on solution pH and COD (b). 30 °C, pH 7, 8000 lx, 2 mg/mL loading rate, 72 h cell age, 5500 mg COD/L.
Fig. 4. Effects of cell age on hydrogen production (a) and on solution pH and COD (b). 30 °C, pH 7, 8000 lx, 2 mg/mL loading rate, 2 mm granule size, 5500 mg COD/L.
3.2.3. Effects of granule sizes on H2 production Fig. 3a shows the effects of granule size on photo-H2 production at 30 °C, pH 7, 8000 lx, 2 mg/mL loading rate, 72 h cell age and 5500 mg COD/L. The corresponding pH and COD changes are demonstrated in Fig. 3b. Interestingly, the H2 productivity followed 1 mm = 1.5 mm > 0.5 mm > 2 mm > 2.5 mm. Restated, there is an optimal granule size with sufficient embedded biomass for reaction but with insignificant limitations by the large matrix size. However, since the difference was not large, at most 20% amongst different granule size, the present granules exhibit low mass transfer resistances for substrates and low shielding effects for incipient lights for the embedded cells.
3.2.4. Effects of cell age on H2 production Fig. 4a and b shows respectively the cell age and pH and COD changes during photo-H2 production tests at 30 °C, pH 7.0, 8000 lx, 5500 mg COD/L and 2 mm granules. There was an optimal cell age of 72–96 h with maximum H2 productivity: younger PSB cells are not ready for effective operation while older cells have passed over their best production age. 3.2.5. Effects of illumination intensity on H2 production Fig. 5a reveals the effects of illumination intensity on photo-H2 production at 30 °C, pH 7, 2 mg/mL loading rate, 2 mm granules, 72 h cell age and 5500 mg COD/L. The corresponding pH and
Fig. 5. Effects of light intensity on hydrogen production (a) and on solution pH and COD (b). 30 °C, pH 7, 2 mg/mL loading rate, 2 mm granule size, 72 h cell age, 5500 mg COD/L.
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COD changes are also shown in Fig. 5b. The hydrogen production at 144 h was increased from 43 to 178 mL with illumination intensity from 2000 to 8000 lx. Further increase in illumination intensity to 12,000 lx decreased the hydrogen productivity to 143 mL. The illumination intensity above 8000 lx would exhibit light inhibition to the present embedded PSB cells, correlating to the finding in previous report (Hu et al., 2016). 3.3. Use of immobilized cells on photo-hydrogen production The alginate immobilized HAU-M1 PSB granules can produced more photo-H2 than the free cells at identical production conditions at 30 °C and pH 7. The adopted granule size (0.5–2.5 mm), the loaded cell quantity (1–3 mg cells/mL), cell age (36–108 h) and the light intensity (2000–10,000 lx) ranged studied revealed no inhibition to PSB cell activities for H2 production. Owing to hydrolysis reaction the solution pH dropped to around 5.5 in all tests (Figs. 1–5b); likely the alginate matrix can resist proton diffusion to minimize photo-H2 production under < 5500 mg COD/mL and <8000 lx conditions. Use of mixed culture in inoculum leads to competition of substrates by different bacterial groups. In the present study, the COD decrease corresponding to photo-H2 production at optimal condition ranged 1100–1200 mg COD/mL, while the overall COD decrease ranged 2000–2800 mg COD/mL. Restated, the fraction of COD decrease by photo-fermentative pathway ranged 42–57%. Therefore, the present alginate matrix provided a shield for photofermentative bacteria to compete with heterotrophic bacteria to promote photo-H2 production. 4. Conclusions The sodium alginate immobilized HAU-M1 consortium produced 1.37–1.43 times quantity of H2 to identical quantity of free cells from synthetic wastewaters at the identical composition. In single-factor tests, the HAU-M1 granules with 2 mm size, 72–96 cell age and 2.5 mg/mL cell loading can produce photo-H2 at maximum productivity from wastewater at 8000 lx and >5500 mg COD/L. The alginate matrix leads to minimal diffusional resistance to substrates or to illumination penetration at granule size less than 2.5 mm, however, still provides shield to embedded cells from external challenges, likely the produced proton gradients from the surroundings. Acknowledgements Financial supports from National Natural Science Foundation of China (51676065) and Doctoral Scientific Fund Project of the Ministry of Education of China (20134105130001).
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