微生物学通报 DEC 20, 2009, 36(12): 1871~1875 Microbiology tongbao@im.ac.cn 2009 by Institute of Microbiology, CAS 简报 正交实验优选光合细菌混合菌群产氢的最佳条件 * 崔宝臣 张国欣侯博邵姝媛 ( 163318) 摘要 : 对光合细菌混合菌群产氢影响因子进行了实验研究 通过单因素实验和正交实验, 系统考察了碳源 氮源 碳源浓度 氮源浓度 初始 ph 值 光照方式 接种量等因素对产氢量的影响, 实验得出最佳工艺条件为 : 采用 3 号菌群, 碳源为葡萄糖, 碳源浓度为 3 g/l, 氮源为尿素, 氮源浓度为 9 g/l, 接种量为 10%, ph 值为 8.5, 光照方式为 12 h 光照 -12 h 黑暗交替光照, 培养温度为 30 C 菌种 碳源 碳源浓度 氮源是影响产氢量的重要因素 关键词 : 光合细菌, 混合菌群, 产氢, 正交实验 Optimization of Hydrogen Production Conditions of Photosynthetic Bacteria Group by Orthogonal Test CUI Bao-Chen * ZHANG Guo-Xin HOU Bo SHAO Shu-Yuan (College of Chemistry and Chemical Engineering, Daqing Petroleum Institute, Daqing, Heilongjiang 163318, China) Abstract: The influence factors for hydrogen production using photosynthetic bacteria group were studied. The effects of carbon, nitrogen, carbon concentration, nitrogen concentration, initial ph value, illumination mode and inoculation amount on hydrogen production were investigated by experiment of single factor and orthogonal experiment. The optimum process conditions were as follows: the best bacteria group was number 3, the glucose was carbon source and concentration of carbon source was 3 g/l; the urea was nitrogen source and concentration of nitrogen source was 9 g/l; the inoculation amount was 10%; initial ph value was 8.5; the illumination mode was 12 h light and 12 h dark alternation; incubation temperature was 30 C. The main factors affecting hydrogen production were strain, carbon, carbon source concentration and nitrogen suorces. Keywords: Photosynthetic bacteria, Mixed bacteria group, Hydrogen production, Orthogonal test,,,, [1], (Rhodobacter sphaeroides)1.1737 [2] (Rhodopseudomonas)H, [3] C/N Barbosa [4] 基金项目 :(No. GZ08B205) * 通讯作者 :Tel: 86-459-6503502; : cuibaochen2005@163.com 收稿日期 :2009-09-15; 接受日期 :2009-10-16
1872 微生物学通报 2009, Vol.36, No.12 3, ph,, 1 材料与方法 1.1 菌种与培养基 123 : NH 4 Cl 0.1 g, NaHCO 3 0.1 g, K 2 HPO 4 0.02 g, CH 3 COONa 0.3 g, MgSO 4 7H 2 O 0.02 g, NaCl 0.15 g, 1 ml, 1 ml, 97 ml : (C 5 H 8 NO 4 Na) 0.1 g, NaHCO 3 0.1 g, K 2 HPO 4 0.02 g, CH 3 COONa 0.3 g, MgSO 4 7H 2 O 0.02 g, NaCl 0.15 g, 1 ml, 1 ml, 97 ml 1.2 试剂与仪器 ; SP3420 ; LRH-250 ; HWS26 ; PHS-25 ph 1.3 实验方法 10%, 50 C 30 min,,, 100 W, 30 C0.5 C 8 d,, = 1000/, : ml H 2 /(L-); =/( ), : ml H 2 /(L-h) 1 2, ph, 3 Wakayama [5], 4, 5 10%,, 1 Table 1 Effect of different carbon and nitrogen on hydrogen production Nutrient Carbon Nitrogen Category H 2 Hydrogen amount [mlh 2 /(L-culture)] The highest hydrogen rate [mlh 2 /(L-culture h)] 0.339 0.006 23.090 0.403 1.756 0.039 0 0 2.889 0.081 0 0 0 0 0.119 0.003 0.259 0.006 0 0 0.323 0.009 0.130 0.002 0.031 0.0007 0.057 0.001 0.889 0.031 2 结果与讨论 2.1 影响产氢量的主要参数的确定, ph 1 图 1 葡萄糖浓度对产氢影响 Fig. 1 Effect of glucose concentration on hydrogen production
: 1873 图 2 谷氨酸钠浓度对产氢影响 Fig. 2 Effect of nitrogen concentration on hydrogen production 图 3 不同初始 ph 值对产氢影响 Fig. 3 Effect of initial ph value on hydrogen production 图 5 接种量对产氢影响 Fig. 5 Effect of inoculation amount on hydrogen production 2.2 参数优化 正交实验结果, ph 9, 3, 2 L 27 (3 13 ) 9 3 3,,, ph 4, P 0.05,,, 3 结论 图 4 光照方式对产氢影响 Fig. 4 Effect of illumination mode on hydrogen production : 12/12: 12 h, 12 h ; 16/8: 16 h, 8 h ; 24 h:. Note: 12/12: 12 h light, 12 h dark alternation; 16/8: 16 h light, 8 h dark alternation; 24h: Continuous illumination. 1), ph, 2) 9, : 3 3 g/l 9 g/l 10%pH
1874 微生物学通报 2009, Vol.36, No.12 Levels A Strains B Carbon C Nitrogen Table 2 表 2 正交实验水平因素 Levels and factors of orthogonal experiment D Carbon concentration (g/l) Factors E Nitrogen concentration (g/l) F Inoculation amount (%) G ph Initial ph value H Illumination mode I Incubation temperature ( C) 1 1 1 1 5 4.5 1* 25 2 2 3 6 10 6.5 2* 30 3 3 6 9 15 8.5 3* 35 : 1 * : 24 h ; 2 * : 16 h, 8 h ; 3 * : 12 h, 12 h. Note: 1 * : 24 h continuous illumination; 2 * : 16 h light, 8 h dark alternation; 3 * :12 h light,12 h dark alternation. No. 表 3 L 27 (3 13 ) 正交实验表与结果 Table 3 L 27 (3 13 ) orthogonal experiment table and results Factors A B C D E F G H L Hydrogen amount [mlh 2 /(L-culture)] 1 1 1 1 1 1 1 1 1 1 1 1 1 1 7.43 2 1 1 1 1 2 2 2 2 2 2 2 2 2 6.99 3 1 1 1 1 3 3 3 3 3 3 3 3 3 11.2 4 1 2 2 2 1 1 1 2 2 2 3 3 3 0.0562 5 1 2 2 2 2 2 2 3 3 3 3 1 1 0.540 6 1 2 2 2 3 3 3 1 1 1 2 2 2 0.339 7 1 3 3 3 1 1 1 3 3 3 2 2 2 0.211 8 1 3 3 3 2 2 2 1 1 1 3 3 3 0.143 9 1 3 3 3 3 3 3 2 2 2 1 1 1 0.852 10 2 1 2 3 1 2 3 1 2 3 1 2 3 3.58 11 2 1 2 3 2 3 1 2 3 1 2 3 1 0.0797 12 2 1 2 3 3 1 2 3 1 2 3 1 2 0.157 13 2 2 3 1 1 2 3 2 3 1 3 1 2 0.0916 14 2 2 3 1 2 3 1 3 1 2 1 2 3 0.152 15 2 2 3 1 3 1 2 1 2 3 2 3 1 0.0522 16 2 3 1 2 1 2 3 3 1 2 2 3 1 0.881 17 2 3 1 2 2 3 1 1 2 3 3 1 2 0.461 18 2 3 1 2 3 1 2 2 3 1 1 2 3 2.59 19 3 1 3 2 1 3 2 1 3 2 1 3 2 13.6 20 3 1 3 2 2 1 3 2 1 3 2 1 3 13.2 21 3 1 3 2 3 2 1 3 2 1 3 2 1 17.2 22 3 2 1 3 1 3 2 2 1 3 3 2 1 0.156 23 3 2 1 3 2 1 3 3 2 1 1 3 2 0.236 24 3 2 1 3 3 2 1 1 3 2 2 1 3 0.0208 25 3 3 2 1 1 3 2 3 2 1 2 1 3 0.111 26 3 3 2 1 2 1 3 1 3 2 3 2 1 0.743 27 3 3 2 1 3 2 1 2 1 3 1 2 3 0.0643 3.08 8.15 3.32 2.98 2.90 2.74 2.86 2.93 2.50 3.14 3.23 2.54 3.10 0.89 0.18 0.63 5.43 2.50 3.28 2.70 2.67 3.29 2.61 2.43 3.55 2.46 5.03 0.67 5.05 0.60 3.61 2.99 3.45 3.41 3.22 3.27 3.36 2.92 3.45 R 4.14 7.97 4.42 4.83 1.11 0.54 0.75 0.74 0.79 0.66 0.93 1.01 0.99
: 1875 表 4 方差分析 Table 4 Variance analysis Source SS DF MS F P A 25.7382 2 12.8691 19.3116 0.0492 B 119.6496 2 59.8248 89.7739 0.0110 C 29.7654 2 14.8827 22.3332 0.0429 D 34.9526 2 17.4763 26.2251 0.0367 E 1.8962 2 0.9481 1.4227 0.4128 F 0.4382 2 0.2191 0.3288 0.7526 G 0.9362 2 0.4681 0.7024 0.5874 H 0.8456 2 0.4228 0.6345 0.6118 I 1.1474 2 0.5737 0.8609 0.5374 Error 0.7334 2 0.3667 0.5503 -- 1.5218 2 0.7609 1.1418 -- 1.5614 2 0.7807 1.1715 -- 1.5146 2 0.7573 1.1364 -- 5.3312 8 0.6664 -- -- 8.5 12 h -12 h 30 C, 参考文献 [1],,,.., 2004, 55 (): 85 89. [2],,,. H., 1994, 21(5): 259 262. [3],,,.., 2007, 28( 2): 45 48. [4] Barbosa MJ, Rocha JMS, Tramper J, et al. Acetate as a carbon source for hydrogen production by photosynthetic bacteria. Journal of Biotechnology, 2001, 85(1): 25 33. [5] Wakayama T, Nakada E, Asada Y, et al. Effect of light/dark cycle on bacterial hydrogen production by Rhodobacter sphaeroides RV. Applied Biochemistry and Biotechnology, 2000, 1-9(84/86): 431 440. 2010 年中科院微生物所期刊联合编辑部联合征订全面启动! 征订启事 ( 4 ), 55.00, 660 ISSN 0001-6209; CODEN WSHPA82-504; BM67 ( 25 ), 65.00, 780 ISSN 1000-3061; CODEN SGXUED82-13; BM5608 ( 20 ), 48.00, 576 ISSN 0253-2654; CODEN WSWPDI2-817; BM413 ( 15 ), 80, 480 ISSN 1672-6472; CODEN JXUUAE2-499; Q723,. (100101) 1 3 B401 ; (010)64807521; E-mailbjb@im.ac.cn