680 微生物学通报 2008, Vol.35, No.5,, [1 4],,,,, Paracoccus Pseudomonas [2,3] Bacillus [5] Rhodococcus [5],, [6],, [7], 4, 40 h, A1, 99.05% 16S rdna A1 ph A

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1 微生物学通报 MAY 20, 2008, 35(5): 679~684 Microbiology 2008 by Institute of Microbiology, CAS 研究报告生物滤塔体系好氧反硝化菌的分离鉴定与特性研究 * 范利荣黄少斌杨军张永清 ( ) 摘要 : 从实验室生物滤塔填料的生物膜上, 经选择性培养基筛选, 分离出 4 株好氧反硝化菌好氧状态下 4 种菌的 40 h 反硝化率均大于 80%, 其中菌种 A1 反硝化率可达到 99.05% 跟踪菌种反硝化过程中氮元素 24 h 变化过程, 发现 4 株菌除 A1 外都有亚硝酸根积累菌种 A1 为短杆菌, 革兰氏阴性生理生化特性研究与 16S rdna 序列测定 (GenBank 接受号 DQ ) 初步判定菌种 A1 为假单胞菌 Pseudomonas putida 适于菌 A1 生长的初始 ph 值是 7.0 左右, 温度 30 左右, 当 DO 大于 2.0 mg/l 时, DO 的变化对菌种 A1 的反硝化效果影响很小关键词 : 生物滤塔, 好氧反硝化菌, 菌种筛选, 菌种鉴定 Separating and Studying of the Aerobic Denitrifying Bacteria from Biofilter FAN Li-Rong HUANG Shao-Bin * YANG Jun ZHANG Yong-Qing (College of Environmental Science & Engineering, South China University of Technology, Guangzhou ) Abstract: Four strains of aerobic denitrifying bacteria were isolated from biomembrane of the laboratory biofilter by selective medium. The denitrifying rates of these four strains were found more than 80% under aerobic condition for 40 hours. The denitrifying rate of A1 was highest, which was 99.05%. When measuring the course of nitrogen element changing, it is found that they accumulated nitrite expect the strain A1. The strain A1 was gram positive and spherical. It is identified as Pseudomonas putida based on its biochemical and morphological characters and phylogenetic analysis of 16S rdna sequences(genbank accession NO.DQ ). For the strain A1, the optimum beginning ph was 7.0 around, and the optimum temperature was 30 around, The change of DO did not influence the effect of denitrification when it was more than 2.0 mg/l. Keywords: Biofilter, Aerobic denitrifying bacteria, Bacteria screening, Bacteria identification,,, 基金项目 : (No ); (No. 2006A ); (No ) * 通讯作者 : : [email protected] 收稿日期 : ; 接受日期 :

2 680 微生物学通报 2008, Vol.35, No.5,, [1 4],,,,, Paracoccus Pseudomonas [2,3] Bacillus [5] Rhodococcus [5],, [6],, [7], 4, 40 h, A1, 99.05% 16S rdna A1 ph A1, 1 材料与方法 1.1 培养基 DM(g/L) Na 2 HPO 4 7H 2 O 7.9; KH 2 PO 4 1.5; MgSO 4 7H 2 O 0.1; 9.4; 2 ml; ph 7~7.5; KNO (g/l) EDTA 50.0; ZnSO 4 2.2; CaCl 2 5.5; MnCl 2 4H 2 O 5.06; FeSO 4 7H 2 O 5.0; (NH 4 ) 6 Mo 7 O 2 4H 2 O 1.1; CuSO 4 5H 2 O 1.57; CoCl 2 6H 2 O 1.61; ph 菌种来源,,,,, 15 d, COD 50, DO 2.0 mg/l, 90 [9] 1.3 菌种分离 90 ml,,,,, ph, 24 h, DM 3, 100 ml DM, ( ), 9,, 40 h, 30, 160 r/min, 5.0 mg/l, 1 10 DM, 9,,, 160 r/min 3 h, ( - ), 1.4 A1 菌种鉴定菌形态观察生理生化反应 S rdna 的序列同源性分析 : 16S rdna PCR, f27(5 -AGAGTTTGATCATGGCTCAG-3 ), R1522 (5 -AAGGAGGTGATCCAGCCGC A-3 ) PCR (50 μl) 10 5 μl PCR, 5 μl MgCl 2 (25 mmol/l), 1 μl dntps, 1 μl f27 1 μl R1522, 1 μl DNA, 0.5 μl Taq (5 U/μL), 35.5 μl PCR 94, 5 min; 94, 1 min; 56, 1 min; 72, 2 min; 72, 7 min GenBank ( DQ ),,

3 : 环境条件对 A1 菌好氧反硝化效果的影响研究 1 10 DM, 9,, 24 h ph,, DO, 1.6 分析项目与检测方法 NO 3 -N ; NO 2 -N N-(1- )- ; (DO) YSI5000 DO ; (A), 721 OD 480 ; Philips XL30 ESEM 2 结果与分析 125 mg/l 4,, ; A2 A4 A5 NO 2 -N,, NO 3 -N NO 2 -N, NO 2 -N [10],, A2 A4 A5 3 ; A1 NO 2 -N,,, NO 3 -N A1 2.1 分离菌种及其反硝化率 8, mg/l, 40 h 1, 4, 80, 4 A1 A2 A4 A5 A 表 1 8 株反硝化菌的脱氮率 Table 1 Denitrifying rates of 8 strains of denitrifying bacteria (Strain) (Denitrifying rates, %) A A A A A A A A 图 1 Fig.1 菌种 A1 反硝化过程 The denitrification process of strain A1 2.2 菌种的好氧反硝化过程研究 1~ h 4 图 2 Fig.2 菌种 A2 反硝化过程 The denitrification process of strain A2

682 微生物学通报 2008, Vol.35, No.5 图 3 Fig.3 图 4 Fig.

denitrification process of strain A5, 94.5, 2.3

3.3 A1 菌株 16S rdna 序列同源性分析 : GenBank, ATCC DSM, A1 Pseudomonas( ), Pseudomonas

4 初始 ph 值对反硝化效果的影响 ph, ph,, ph,, 6, ph ph, A1, ph 4 5 ph,, ph 7 94.1 ph, 图 5 Fig.

4 682 微生物学通报 2008, Vol.35, No.5 图 3 Fig.3 图 4 Fig.4 菌种 A4 反硝化过程 The denitrification process of strain A4 菌种 A5 反硝化过程 The denitrification process of strain A5, 94.5, 2.3 菌种 A1 鉴定形态观察 A 生理生化鉴定 A1,,,,, ;,,,, D-, V-P,, (MR), A1 Pseudomonas putida A1 菌株 16S rdna 序列同源性分析 : GenBank, ATCC DSM, A1 Pseudomonas( ), Pseudomonas putida Pseudomonas plecoqlossicida 99.8, A1 Pseudomonas putida 2.4 初始 ph 值对反硝化效果的影响 ph, ph,, ph,, 6, ph ph, A1, ph 4 5 ph,, ph ph, 图 5 Fig.5 菌种 A1 的电镜照片 The microscopy photograph of strain A1 图 6 不同初始 ph 值对脱氮率和亚硝酸根生成率的影响 Fig.6 The effect of beginning ph on denitrifying rate and the production rate of nitrite

5 : 683, ph 9.0, 50.20,, A1,, N 2 6 ph A1, ph, ph, A1 ph 温度对反硝化效果的影响 7,, , 18.3 ; ~35, 30, A1 30, A1, 25 35,, 25, A1 30 图 7 温度对脱氮率和亚硝酸根生成率的影响 Fig.7 The effect of temperature on denitrifying rate and the production rate of nitrite 2.6 溶解氧对反硝化效果的影响 8, DO 2.0 mg/l 11.2 mg/l, 24 h 90 DO 2.0 mg/l 11.2 mg/l A1 [4 7], A1, 图 8 不同 DO 下的脱氮率和亚硝酸根生成率 Fig.8 The effect of DO on denitrifying rate and the production rate of nitrite 3 结论 1),,, 8, 4 40 h 80, A1, ),, 4, A1, 3 3) A1, 16S rdna, A1 Pseudomonas putida Pseudomonas plecoqlossicida 99.8,, A1 Pseudomonas putida 4) A1,, A1 ph 7.0

6 684 微生物学通报 2008, Vol.35, No.5 A1, A1 30 A1, DO 2.0 mg/l 11.2 mg/l, A1 24 h 90, DO 参考文献 [1] Hung SJ, Mitsuyo H, Makoto S. Characteristics of Ammonium Removal by Heterotrophic Nitrification-Aerobic Denitrification. Bioscience and Bioengineering, 2005, 100(2): [2] Chen F, Xia Q, Ju LK. Aerobic denitrification of Pseudomonas aeruginosa monitored by online NAD(p) H fluorescence. Applied & Environmental Microbiology, 2003, 69(11): [3] Kesser ǚ P, Kiss I, Bihari Z, et al. Biological denitrification in a continuous flow pilot bioreactor containing immobilizes Pseudomonas butannovora cells. Bioresource technology, 2003, 87(9): [4] Joong KK, Kyoung JP, Kyoung SC. Aerobic nitrification denitrification by heterotrophic Bacillus strains. Bioresource Technology, 2005, 96(17): [5].., 2004, 25(1): [6],,,.., 2004, 44(6): [7],. SBR-BAF., 2006, 24(5): [8],,,.., 2006, 8(161) [9],,,.., 2006, 26(11): [10],,.., 2005, 25(1): 编辑部公告关于微生物学通报 2008 年度开始专题刊申请的通知,,,,,,,,, 2008,,,,,,,, 1., ; 2. () ;, [email protected], ; 3.,, [email protected] 微生物学通报编辑部 2007 年 8 月 29 日

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc

Microsoft Word - 793-797 tb20150504赵宏宇s-高校教改纵横.doc 微生物学通报 Microbiology China [email protected] Apr. 20, 2016, 43(4): 793 797 http://journals.im.ac.cn/wswxtbcn DOI: 10.13344/j.microbiol.china.150504 高校教改纵横生物工程专业发酵课程群建设探索 * 赵宏宇