微生物学通报 APR 20, 2009, 36(4): 473~478 Microbiology 2009 by Institute of Microbiology, CAS 研究报告锰氧化菌 Bacillus sp. MK3-1 的 Mn(Ⅱ) 氧化特性和除锰能

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1 微生物学通报 APR 20, 2009, 36(4): 473~478 Microbiology 2009 by Institute of Microbiology, CAS 研究报告锰氧化菌 Bacillus sp. MK3-1 的 Mn(Ⅱ) 氧化特性和除锰能力研究 * 刘颜军周静晓王革娇 ( / ) 摘要 : 锰氧化微生物能够将可溶的 Mn(II) 氧化为不溶的锰氧化物沉淀, 因此在生物除锰研究上具有重要的应用价值本研究从锰污染土壤中分离到一株锰氧化菌 Bacillus sp. MK3-1, 该菌对 MnCl 2 有较高抗性, 其最低抑制浓度 (Minimal inhibitory concentration, MIC) 为 20 mmol/l 实验表明该菌在培养基中 Mn(Ⅱ) 的去除率高达 96%, 同时将其制成固体包埋菌剂应用于含 0.15 mmol/l 的 MnCl 2 水溶液实验, 结果表明其仍然具有稳定的除锰能力, 去除率为 87.12%, 使溶液的终锰浓度符合国家排放标准扫描电子显微镜观察和能谱分析实验表明, 实验产生的锰氧化物均匀地分布在 Bacillus sp. MK3-1 的细胞表面, 细胞表面含锰量为 19.60% (W/W) 用简并引物扩增目前被认为催化锰氧化的多铜氧化酶基因 mnxg, 获得了 903 bp 的基因片段, 其基因产物与已报道的多铜氧化酶具有 86% 的同源性关键词 : 锰氧化细菌, 芽胞杆菌, 锰氧化, 锰去除, 包埋, 扫描电子显微镜 Mn( ) Oxidation and Removal by a Manganeseoxidizing Bacterium Bacillus sp. MK3-1 LIU Yan-Jun ZHOU Jing-Xiao WANG Ge-Jiao * (State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, Hubei , China) Abstract: Manganese-oxidizing microorganisms are able to oxidize soluble Mn(II) into insoluble Mn oxides. Such microorganisms are very useful in treatment of Mn-contaminated water. In this research, a Mn(II)- oxidizing bacterium Bacillus sp. MK3-1 was isolated from Mn-contaminated soil. This bacterium has high MnCl 2 resistance with a MIC of 20 mmol/l. The results showed that it is able to oxidize and remove more than 96% of Mn(II) in the culture medium. The immobilized solid-embedding Bacillus sp. MK3-1can removed 87.12% of manganese contaminated water. The final concentration of MnCl 2 after the treatment reached the national discharge standard level. Scan electron microscope observation showed that the produced Mn oxides located on the cell surfaces of Bacillus sp. MK3-1. Energy dispersive spectrdmeter analysis indicated that the content of manganese of cell surfaces of Bacillus sp. MK3-1 was 19.60% (W/W). At last we amplified a 903 bp multicopper oxidase gene mnxg encoding the putative Mn(II)-oxidizing protein. 基金项目 :(No ); (SRF) * 通讯作者 :Tel: ; : gejiao@mail.hzau.edu.cn 收稿日期 : ; 接受日期 :

2 474 微生物学通报 2009, Vol.36, No.4 The product of mnxg showed 86% identity to the reported multicopper oxidase. Keywords: Manganese-oxidizing bacteria, Bacillus, Mn(II)-oxidation, Mn removal, Embedding, Scan electron microscope 15,, 0.1 mg/l, 2 mg/l, Mn( ) [1], Mn( ),, [2] (MOCS) MOCS,, MnxG,,, mnxg 16S rdna [3],,,,,,, 1 材料和方法 1.1 样品采集与分析 cm, 2 mm ph [4] [5] [6] 1.2 锰氧化菌的分离鉴定及其系统进化分析 100 gmncl mg/kg, 28 C 1 10 g 90 ml, 28 C 30 min, 20 mmol/l HEPES(pH 7.5) 0.15 mmol/l MnCl 2 K [7], 28 C 1,, 16S rdna, PCR 27F(5'-AGAGTTTGATCMTGGCTCAG- 3') 1492R(5'-GGYTACCTTGTTACGACTT-3') [8] PCR 94 C 5 min; 94 C 1 min, 49 C 1 min, 72 C 2 min, 35 ; 72 C 10 min PCR 16S rdna Blastn GenBank ( nih.gov/blast), Clustal W, MEGA 3.1 ( 1.3 锰氧化菌锰抗性及生理生化分析 MnCl 2 K (MIC) MnCl 2 0~ 30.0 mmol/l, 0.5 mmol/l, 28 C 150 r/min, 72 h (OD 600 ) [9] 1.4 锰氧化菌除锰能力测定 MnCl mmol/l K 3, 28 C 150 r/min 24 h 10 ml, r/min 10 min, 0.2 μm, Mn(II) ; 40 mmol/l CuCl 2 10 h, Cu 2+ Mn 2+, 10 ml, r/min 10 min, 0.2 μm, Mn(II) ; 20 mmol/l 10 h, Mn 2+, 10 ml r/min 10 min, 0.2 μm, Mn( );

: Bacillus sp. MK3-1 Mn( ) 475 1.5 包埋菌剂除锰能力测定 1) 1:1(W/V), ; 2) 10%( 10 g 1 ml, 1 OD ), 28 C 24 h, 50 C ; 3) (PP) 10 cm 20 cm,, ; 4) 20 cm 25 cm 30 cm, 10 L 0.15 mmol/l MnCl 2, 28 C ; 5) 24 h 1, ph 1.

3 : Bacillus sp. MK3-1 Mn( ) 包埋菌剂除锰能力测定 1) 1:1(W/V), ; 2) 10%( 10 g 1 ml, 1 OD ), 28 C 24 h, 50 C ; 3) (PP) 10 cm 20 cm,, ; 4) 20 cm 25 cm 30 cm, 10 L 0.15 mmol/l MnCl 2, 28 C ; 5) 24 h 1, ph 1.6 扫描电子显微镜观察和能谱分析 MnCl 2 0 mmol/l 0.15 mmol/l K, 28 C 150 r/min 4 d,, 1), ph 7.2 ; 2), 2.5% 4 C 24 h; 3), (30%-50%-70%-85%-95%-100%), 2, 15 min; 4), 1:1 10 min 10 min, ; 5), ; 6) JSM-6390/LV ( 20 kv, 1 na) 1.7 锰氧化菌多铜氧化酶基因 mnxg 的扩增 DNA, [10], DNA PCR MnxG : mnxgif (5'-ACGCATGTCTTTCACTATCATGTTCA T-3') mnxgir(5'-aaataagtggtcatggaaga ACCATGC-3') [11], PCR 94 C 5 min; 94 C 30 s, 53 C 30 s, 60 C 1 min, 30 PCR pgem-t (Promega, USA), E. coli DH5α, 100 μg/ml Amp, 50 μg/ml X-Gal 100 μg/ml IPTG LB, 37 C 16 h mnxg 16S rdna 2 结果 2.1 锰氧化菌 MK3-1 的分离鉴定及锰抗性, 0.89 g/kg, 30.23%, ph 7.19 MK3-1, MnCl 2 K,,,,, 0.15 mmol/l MnCl 2 K,, 1 图 1 MK3-1 的菌落形态 Fig. 1 The colony morphology of strain MK3-1: a K ; b 0.15 mmol/l MnCl 2 的 K. Note: a: On K medium; b: On K medium containing 0.15 mmol/l MnCl 2. 16S rrna, MK3-1 Bacillus sp. PL-26(AF326369) 98%, 16S rdna, 2, 9, ( 1) 16S rrna, Bacillus sp. MK3-1, GenBank FJ389509, MK3-1 MIC 20 mmol/l, 2.2 Bacillus sp. MK3-1 除锰能力 3 Bacillus sp. MK3-1 1, 67.53%±2.04% 4, 50.51%±1.67% 7,, 96.80%±2.64%, 77.56%±2.64%, 0.48 mg/l±0.02 mg/l,,, 4 Bacillus sp. MK3-1 PP, 0.15 mmol/l MnCl 2, 4, 84.80%±0.31% 0.87 mg/l±0.02 mg/l,

4 476 微生物学通报 2009, Vol.36, No.4 图 2 菌株 Bacillus sp. MK3-1 16S rdna 系统发育树 ( 括号内为序列 GenBank 登录号 ) Fig. 2 16S rdna phylogenetic tree of Bacillus sp. MK3-1 (Numbers in parentheses represent the sequences accession number in GenBank) bootstrap 1000 (%); 2%. Note: Numbers near the branches are bootstrap probability values (%) with 1000 replicates; Scale bar represents 2% difference in nucleotide sequences. Table 1 表 1 菌株 MK3-1 的生理生化特性 Biophysical and biochemical characteristics of strain MK3-1 Test items Results Test items Results Test items Maltose Pectinose L- L-tyrosine D- D-galactopyranoside Results Raffinose L- L-arginine Sorbose Glycerol Sodium nitrite Sucrose Inositol Potassium nitrate D- D-fructose Methanol Ammonium chloride Rhamnose Mannitol V.P. V-P test Trehalose Ethanol Methyl red D (+) - D-xylose Sorbitol Starch hydrolysis Glucose Gluconate Gelatin liquefaction Lactose Creatine Ammonia producing test Melezitose Citric acid Nitrate reduction Arabinose Glycine Hippuric acid + ;. Note: +: Positive reaction; : Negative reaction. 2.3 Bacillus sp. MK3-1 的电镜分析及能普扫描 Bacillus sp. MK a Bacillus sp. MK3-1 0 mmol/l MnCl 2 K, 1.94 μm, 1.29 μm 5 b, Bacillus sp. MK3-1,, 19.60%± 2.97% (W/W) 2.4 多铜氧化酶基因的扩增 mnxg 903 bp, GenBankFJ389508, Francis [11] Bacillus sp. GB02-16 MnxG 86% mnxg, 9, 6 MnxG 16S rdna, Bacillus sp. MK3-1,

5 : Bacillus sp. MK3-1 Mn( ) 477 图 3 Bacillus sp. MK3-1 在 K 培养基中对 Mn(Ⅱ) 的氧化 / 去除曲线 Fig. 3 Mn(II) oxidation and removal curve of Bacillus sp. MK3-1 in K medium Mn( ) ; Mn(II) ; Mn( ) ; ( 0.15 mmol/l MnCl 2 K ). Note: : Remained MnCl 2 in the K medium; : Adsorption of MnCl 2 in the K medium; : Manganese oxids of Mn(II); : Blank (K medium containing 0.15 mmol/l MnCl 2 without bacterial inoculation). 图 4 制成包埋菌剂的 Bacillus sp. MK3-1 在 0.15 mmol/l 的 MnCl 2 水溶液中 Mn(Ⅱ) 的氧化 / 去除曲线 Fig. 4 Oxidation and removal curve of the immobilized solid-embeding Bacillus sp. MK3-1 in manganese containing water MK3-1 ; 0.15 mmol/l MnCl 2 Mn(II) ; ph; ( 0.15 mmol/l MnCl 2 ). Note: : Concentration of MK3-1; : Remained MnCl 2 in manganese containing water; : ph; : Blank (0.15 mmol/l MnCl 2 solution without bacterial inoculation). 3 讨论 K Bacillus sp. MK3-1,, 图 5 扫描电镜照片 Fig. 5 Scanning electron microscope images of cells of Bacillus sp. MK3-1 a: Bacillus sp. MK3-1 ; b: Bacillus sp. MK3-1. Note: a: Cells of Bacillus sp. MK3-1; b: Cells of MK3-1 with the surounded biogenic Mn oxides locating on the cell surfaces. Mn( )MK3-1, PP,,,,,, PP,,,, [12] MK3-1 16S rrna,,,, ( Zn Ni Cu Co Cd Cr ), As( ) As(V) [ As( ), As(V) ] [13], MK3-1

6 478 微生物学通报 2009, Vol.36, No.4 图 6 菌株 Bacillus sp. MK3-1 MnxG 系统发育树 ( 括号内为序列 GenBank 登录号 ) Fig. 6 MnxG phylogenetic tree of Bacillus sp. MK3-1 (Numbers in parentheses represent the sequences accession number in GenBank) bootstrap 1000 (%); 2%. Note: Numbers near the branches are bootstrap probability values (%) with 1000 replicates; Scale bar represents 2% difference in nucleotide sequence. 参考文献 [1].., 1989, 6(4): [2] Tebo BM, Johnson HA, McCarthy JK, et al. Geomicrobiology of manganese( ) oxidation. Trends Microbiol, 2005, 13(9): [3] Francis CA, Tebo BM. CumA multicopper oxidase genes from diverse Mn( )-oxidizing and non-mn(ii)-oxidizing Pseudomonas strains. Appl Environ Microbiol, 2001, 67(9): [4],,. ISO10390: 2005 ph., 2006, 1: [5].. :, 2000, pp [6],,.., 2005, 4(2): [7] van Waasbergen LG, Hoch JA, Tebo BM. Genetic analysis of the marine manganese-oxidizing Bacillus sp. strain SG-1: protoplast transformation, Tn917 mutagenesis, and identification of chromosomal loci involved in manganese oxidation. J Bacteriol, 1993, 175(23): [8],,,.., 35(5): [9],.. :, 1999, pp [10] Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual. 3rd ed. New York: Cold Spring Harbor Laboratory Press, [11] Francis CA, Tebo BM. Enzymatic manganese( ) oxidation by metabolically dormant spores of diverse Bacillus species. Appl Environ Microbiol, 2002, 68(2): [12] Rosson RA, Nealson KH. Manganese binding and oxidation by spores of a marine Bacillus. J Bacteriol, 1982, 151(2): [13] Tebo BM, Bargar JR, Clement BG, et al. Biogenic manganese oxides: properties and mechanisms of formation. Annu Rev Earth Planet Sci, 2004, 32: 栏目介绍显微世界,,, ( )

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Microsoft Word - 631-645 tb20120353吴慧昊.doc 微生物学通报 Microbiology China tongbao@im.ac.cn Apr. 20, 2013, 40(4): 631645 2013 by Institute of Microbiology, CAS 简报乳酸菌低温菌株的复合诱变选育吴慧昊 1 牛锋 (1. 西北民族大学实验中心甘肃兰州 730030) (2.

微生物学通报 APR 20, 2009, 36(4): 473~478 Microbiology 2009 by Institute of Microbiology, CAS 研究报告 锰氧化菌 Bacillus sp. MK3-1 的 Mn(Ⅱ) 氧化特性和除锰能

微生物学通报 APR 20, 2009, 36(4): 473~478 Microbiology 2009 by Institute of Microbiology, CAS 研究报告锰氧化菌 Bacillus sp. MK3-1 的 Mn(Ⅱ) 氧化特性和除锰能