微生物学通报 APR 20, 2009, 36(4): 610~615 Microbiology 研究报告 2009 by Institute of Microbiology, CAS 嗜盐菌对高盐有机废水处理的强化作用 * 李维国徐仲张大伟王光玉柳锋马梅荣杨慧茹

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1 微生物学通报 APR 20, 2009, 36(4): 610~615 Microbiology 研究报告 2009 by Institute of Microbiology, CAS 嗜盐菌对高盐有机废水处理的强化作用 * 李维国徐仲张大伟王光玉柳锋马梅荣杨慧茹马放 ( ) 摘要 : 为解决高盐有机工业废水处理这一难题, 从山东省威海市路道口盐场晒盐池盐水中分离一株嗜盐菌株 YS-1, 通过对该菌株进行原子力显微镜观察生理生化测定全细胞脂肪酸分析和 16S rdna 序列同源性分析发现 YS-1 菌株 16S rdna 序列与 Halomonas sp. (AB167061) 的亲缘关系最为接近, 结合上述其它各项结果确定该菌株为盐单胞菌属 (Halomonas sp.), 属中度嗜盐菌 ; 在 SBR 反应器中对该菌株进行强化高盐有机废水处理试验, 结果表明, 含盐 12% CODcr= 1494 mg/l 的高盐模拟有机废水, 经 72 h CODcr 去除率为 90.0%, 120 h 的 COD Cr 去除率为 98.1%, 该菌株具有强化高盐有机废水处理的功能, 通过分离筛选嗜盐菌强化高盐有机工业废水处理具有可行性关键词 : 中度嗜盐菌, 分离鉴定, 高盐有机废水 The Invigoration Effect of Halophilic Bacterium and Its Bioaugmention in High-sality Organic Wastewater Treatment LI Wei-Guo * XU Zhong ZHANG Da-Wei WANG Guang-Yu LIU Feng MA Mei-Rong YANG Hui-Ru MA Fang (Harbin Institute of Technology (Weihai), Weihai, Shangdong , China) Abstract: Be subjected to study the application in the hypersaline organic wastewater treatment, A moderately halophilic bacterium YS-1 was isolated from the salty water of a Ludaokou salina of Weihai in China. The polyphasic taxonomy study was processed through identifying its morphological features by AFM, researched the features of culture and measured physiologic-biochemical index, analyzed 16S rdna sequence homology Phylogenetic analysis based on 16S rdna gene sequences showed that the closest relative of the strain was Halomonas sp. (AB167061). Experimented of intensifying hypersaline organic wastewater treatmented processing in the SBR demonstrate a significant result. The wastewater salt content of initial wastewater was 12% and initial CODcr was 1494 mg/l. After 72 h, the removal efficiency of CODcr achieved 90.0% and after 120 h, it achieved 98.1%. The experiments also showed the hypersaline organic wastewater treatment and presented the feasibility of acclimating of halophilic bacteria and running of processing system. Keywords: Moderately halophilic bacterium, Isolation and Identification, Hypersaline organic wastewater 基金项目 : () [No. HIT(WH)XB200804]; 973 (No. 2004CB185050) * 通讯作者 :Tel: ; : lwghm@tom.com 收稿日期 : ; 接受日期 :

2 : 611 (Moderately halophilic bacteria) [1,2],,, [3] 0~32% NaCl, 3%~15% [4] 3.5%(TDS),, 10%~25%,,,, 3.5%,,, [5], Woolard [6] (Great Salt Lake) YS-1, 16S rdna,, 1 材料和方法 1.1 菌种的来源实验材料 YS-1 5 g, 12 g, 6 g, 3 g, KCl 3 g, MgSO 4 7H 2 O 18 g, CaCl g, FeSO g, NaCl 70 g, 1000 ml, ph 7.5, Pa 20 min 1 ml 100 ml 300 ml, 35 C 120 r/min 5 d, (30 mm 200 mm), 5 35 C 2 d~4 d,, ; [7] ( ) [8] [9 11] 1.2 甘油二醚衍生物测定 YS-1 30 ml, 3500 r/min,, 3 ml, 3 ml, 0.1 ml, 50 C 15 h,, 1.7 g 10 ml 0.5 %,, 110 C 1 h, R f [12] 1.3 细菌全细胞脂肪酸组分分析 MIDI [13,14] S rdna 的扩增与测序 DNA DNA PCR, 16S rdna Eu-bac27F: 5'-AGAGTT TGATCCTGGCTCACG-3'; 1492R: 5'-GGTTACCTTG TTACGACTT-3' PCR PTC-200, 20 μl 40 ng, rtaq DNA 0.3 U, dntps 0.3 mmol/l, 0.1 μmol/l; 94 C 5 min; 94 C 30 s, 58 C 45 s, 72 C 90 s, 30 ; 72 C 10 min 1.0% 16S rdna GenBank Blast, BioEdit v 5.06, MEGA 3.1 (Neighbor-joining, NJ) 1.5 YS-1 在高盐有机废水中降解有机物性能试验, CODcr:N:P 100:5:1(V/V/V), (1250 mg/l), (128 mg/l) (72 mg/l)(30 mg/l), NaCl 12%, ph 7.5 CODcr=1494 mg/l 32 C~35 C 150 r/min 2 d (40 ml) SBR1 # (+ + +); 2 # (+ +);

3 612 微生物学通报 2009, Vol.36, No.4 3 # (+); 4 # () 1 # 2 #, (2500 ml) 4, 3000 ml, YS-1 ( 5%), 4 (2800 ml) 4, 4 mg/l~ 5 mg/l(ysi MODEL 58 ), (32 C), ph 7.5, 12 h 1 h, [15] COD OH KI, K CODcr, CODcr, 4 3, h, (MLSS) 5000 mg/l, 0.3 kg(bod 5 )/kg(mlss) d 2 结果与讨论 2.1 菌株的形态和生理生化 YS-1,,,,,,,,, 4 d 1 mm, (1.3~2.1) µm (0.6~0.8) µm, ( 1) 图 1 原子力显微镜下菌体的形态 Fig. 1 Bacterial morphology scanned by AFM YS-1 0~25%, 10%,, ; YS-1 10 C~55 C, 35 C; ph 5~10, ph 7.5;, ;,,,,, H 2 S,,, ;, 1.1, 35 C(150 r/min), 10 h, 5000 r/min 10 min,, OD 480, ( 2) YS-1 20 h, 20 h 50 h, 50 h 110 h, 110 h G=(t 2 t 1 )/[3.322 (lgod 2 lgod 1 )](G, t 1 OD 1, t 2 OD 2 ) YS-1 (35 C) 12.9 h 2.2 甘油二醚衍生物测定,

4 : 613 图 2 生长曲线 Fig. 2 Growth curve,, R f 0.6, 0.2, 2.3 全细胞脂肪酸组分分析 3, MIS, 15:0 iso (17.86%), 15:0 anteiso (34.10%), 17:0 anteiso (6.58%), 58.54%, S rdna 测序及同源性比较 16S rdna 1458 bp, GenBank, YS-1 4 Halomonas sp. (AB167061) Halomonas sp. (HSA295145) Halomonas sp. (HSA243448) Halomonas sp. (HSA243447) 99%, Halomonas sp. (AB167061) (Neighbor-joining, NJ) 3, YS-1 (Halomonas sp.) [16,17], Fig. 3 图 3 菌株脂肪酸组成和含量色谱图 The Chromatogram about the fatty acid component and content Fig. 4 图 4 YS-1 菌株的系统发育树 Phylogenetic tree of the YS-1 bacterium

5 614 微生物学通报 2009, Vol.36, No YS-1 在高盐有机废水中降解有机物的性能 5, 4, 1 # (+ ++),, 72 h CODcr 90%( 2 # 78.53%, 3 # 40.31%) 1 # (120 h) CODcr 98.11% YS-1 2 # ( + +), YS-1, ; 3 # YS-1 3 # YS-1 1 # 2 #,, 72 h CODcr 40.31%, CODcr 81.23%, 1 # 3 # YS-1, 4 # (),, 3 结论图 5 四个反应器对比试验 Fig. 5 Comparison Test of four sequencing batch reactors 1 # 2 #, 2 # YS-1, 1 #, 1 #,,,, CODcr %, 3 # (+) YS-1, 1 #, 1 # 是由多种微生物构成的一个生态系统, YS-1,, 3 # YS-1, 3 # 2 #, YS-1, YS-1,,, ; 15:0 iso (17.86%) 15:0 anteiso (34.10%) 17:0 anteiso (6.58%); 16S rdna Halomonas sp. (AB167061) ; 0~25%, 10%;, ; YS-1, SBR YS-1, 12% CODcr=1494 mg/l, 72 h CODcr 90%, 120 h CODcr 98.1%,, 参考文献 [1] Kushner DJ. Life in high salt and solute concentrations: halophilic bacteria. Microbial Life in Extreme Environments. London: Academic Press, 1978, 6(2): [2],.., 2003, 43(3): [3] Galinski EA. Osmoadaption in bacteria. Adv Micro Physiol, 1995, 37(4): [4] Binder use of SBR S to treat pesticide wastewater. Presented at the no tre danemile haza rdous waste conference University of Notre DameSouth Bend, [5] LeFevre E, Round LA. A preliminary report upon some halophilic bacteria. Bacteriol, 1919, 4: [6] Woolard CR, Irvine RL. Treatment of hypersaline waste-

6 : 615 water in the sequencing batch reactor. Wat Res, 1995, 29(4): [7],.. :, [8] Hot GJ, Krieg RN, Sneath HAP, et al. Bergey's Manual of Determinative Bacteriology. 9th ed. Baltimore: Williams & Wilkins Company, [9] Nielsen P, Fritze D, Priest FG. Phenetic diversity of alkaliphilic Bacillus strains: proposal for nine new species. Microbiology, 1995, 141(3): [10] Yumoto L, Yamaga S, Sogabe Y, et al. Bacillus krulwichiae sp. nov., a halotolerant obligate alkaliphile that utilizes benzoate and m-hydroxybenzoate. Int J Syst Envol Microbiol, 2003, 53(5): [11] Fritze D, Flossdorf J, Claus D. Taxonomy of alkaliphilic Bacillus trains. Int J Syst Bacteriol, 1990, 40(5): [12] Ross HN, Collins MD, Tindall BJ, et al. A rapid procedure for the detection of archae bacterial lipids in halopilic bacterial. J Gen Microl, 1981, 123(1): [13],,,.. ( ), 2006, 27(7): [14],,,. 16S rdna PCR., 1991, 34(1): 6 8. [15],,.., 2005, 15(2): [16],,,. 16S rdna DNA., 2002, 42(2): [17],,,.. ( ), 1995, 34(4): ( p.580) 2009 年中国微生物学会及各专业委员会学术活动计划表 (2-2)

: 307, [], [2],,,, [3] (Response Surface Methodology, RSA),,, [4,5] Design-Expert 6.0,,,, [6] VPJ33 ph 3,, ph, OD, Design-Expert 6.0 Box-Behnken, VPJ3

: 307, [], [2],,,, [3] (Response Surface Methodology, RSA),,, [4,5] Design-Expert 6.0,,,, [6] VPJ33 ph 3,, ph, OD, Design-Expert 6.0 Box-Behnken, VPJ3 微生物学通报 FEB 20, 2008, 35(2) 306~30 Microbiology 2008 by Institute of Microbiology, CAS tongbao@im.ac.cn 生物实验室响应面分析法优化副溶血性弧菌生长条件刘代新宁喜斌 * 张继伦 2 (. 200090) (2. 20202) 摘要

微生物学通报 APR 20, 2009, 36(4): 610~615 Microbiology 研究报告 2009 by Institute of Microbiology, CAS 嗜盐菌对高盐有机废水处理的强化作用 * 李维国 徐仲张大伟王光玉柳锋马梅荣杨慧茹

微生物学通报 APR 20, 2009, 36(4): 610~615 Microbiology 研究报告 2009 by Institute of Microbiology, CAS 嗜盐菌对高盐有机废水处理的强化作用 * 李维国徐仲张大伟王光玉柳锋马梅荣杨慧茹