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1 土壤 (Soils), 2014, 46(3): 两株抗铜细菌的筛选鉴定及对碳酸铜的溶解作用 * ( ) F16a Fw17a 16S rdna F16a (Enterobacter) Fw17a (Pseudomona) F16a Fw17a Fw17a ( 500 mg/l CuCO 3 ) 48 h F16a 300% Fw17a 60% Q939 [1] [2] [3-4] [5] [5] [6] [7 8] Chen [9] Cu Cu Cu Cu [9 10] Cu Cu Cu Cu Cu - Cu (30 45 N E ) (32 03 N E ) 1.2 (g/l) NaCl 0.5 g (NH 4 ) 2 SO g K 2 HPO 4 3H 2 O 2.0 g KH 2 PO g MgSO 4 7H 2 O 0.4 g ml ph 7.0 LB (g/l) 10.0 g NaCl 10.0 g 5.0 g ml ph 7.2(g/L) 10.0 g (NH 4 ) 2 SO g K 2 HPO g MgSO 4 7H 2 O 0.5 g (2012BAD15B00)(BE )(BK ) * (yxia@njau.edu.cn) (1963 ) gpwang@njau.edu.cn

2 3 499 NaCl 0.1 g 0.5 g ml ph 7.2 PCR DNA g [11] CuSO mg/l 3 Cu 100 mg/l 24 h Cu [12] Buchanan Gibbons [13] 1.5 LB mg/l 3 2% LB r/min 24 h 1.6 DNA CTAB [14 15] F16a Fw17a DNA 16S rdna 5'-AGAGTT- TGATCCTGGCTCAG-3' 5'-TACCTTGT- TACGACTT-3' 16S rdna (TianGen ) pmd19-t (TaKaRa ) DH5α X-gal(40 µg/ml) IPTG(40 µg/ml) Amp(100 µg/ml) LB GenBank( Bioedit Mega3.0 Kimura2-Parameter Distance Neighbor-Joining 1.7 CuCO 3 (500 mg/l CuCO 3 )30 C 60 h r/min 5 min Cu ph Cu 48 Cu(CuSO 4 ) 40 CuSO mg/l Cu 40 ( 500 mg/l CuCO 3 ) CuCO 3 13 Cu 6 Cu F16a Cu Fw17a 2.2 F16a Fw17a 1 2 F16a V.P. M.R. Fw17a F16a Fw17a F16a Fw17a Fw17a 2.4 F16a Fw17a DNA 16S rdna PCR F16a 16S rdna bp Fw17a16S rdna bp GenBank GQ GQ S rdna GenBank 16S rdna Blast F16a Fw17a 16S rdna NJ 1 2

3 Table 1 表 1 菌株 F16a 和 Fw17a 菌体和菌落形态 Colony and morphology characteristics of strains F16a and Fw17a F16a Fw17a (μm) 2.0 ~ ~ 1.7 (μm) 0.6 ~ ~ 0.9 G G 表 2 菌株 F16a 和 Fw17a 的生理生化特征 Table 2 Physiological and biochemical characteristics of Cu-resistant strains F16a and Fw17a F16a Fw17a F16a Fw17a (V.P.) (M.R.) W W 表 3 铜抗性菌株对不同抗生素的敏感性 Table 3 Antibiotic tolerance of Cu-resistant strains F16a and Fw17a F16a Fw17a F16a Fw17a W 2.5 CuCO 3 F16a CuCO 3 ( 3B) F16a Cu Fig. 1 图 1 基于菌株 F16a 16S rdna 序列的聚类分析 Phylogenetic analysis based on 16S rdna sequences of Cu-resistant strain F16a and related species

4 3 501 Fig. 2 图 2 基于菌株 Fw17a 16S rdna 序列的聚类分析 Phylogenetic analysis based on 16S rdna sequences of Cu-resistant strain Fw17a (B) and related species Fig. 3 ( P<0.05 ) 图 3 液体培养条件下菌株 F16a 和 Fw17a 对上清液中 ph(a) 及 Cu 含量 (B) 的影响 Effects of F16a and Fw17a on ph value (A) and concentration of water-soluble copper in supernatant (B) of liquid culture containing 500 mg/l CuCO 3 300% Fw17a Cu 60% Fw17a Cu 3A F16a ph F16a Fw17a ph 3 (Burkholderia)(Pseudomonas) (Xanthomonas) (Enterobacter) [16 20] Cu Cu F16a Fw17a 16S rrna Cu Cu F16a Fw17a Cu 500 mg/l

5 Altimira [21] 5 Cu 5 O12 A32 A55 C21 O4 Cu 550 ~ 750 mg/l Chen [9] 12 Cu Cu 2+ Cu F16a Fw17a Cu 30 5 O12 A32 A55 C21 O4 Cu Cu 379 ~ 784 mg/kg [21] Cu Cu [9] Cu M1 ~ M12 M2 11 Cu [9] F16a CuCO 3 Cu 4.4 F16a ph F16a [6] Cu Cu HQN2 Cu JYC17 2 Cu CuCO 3 Cu CuCO [22] F16a CuCO 3 Cu F16a Fw17a Cu 40% ph Fw17a Cu - Płociniczak [23] MH8b ACC 3- Cu 2+ Cd 2+ Zn 2+ Zhang [20] Y1-3-9 ACC IAA Cu [10] 1 Cu F16a Fw17a IAA( 2) ACC 4 2 Cu Cu F16a Fw17a 16S rdna (Enterobacter sp. F16a)(Pseudomonas sp. Fw17a) F16a Cu - Cu [1]. [M]. :, 2002 [2] Magidin M, Pittman JK, Hirschi KD, Bartel B. ILR2, a novel gene regulating IAA conjugate sensitivity and metal transport in Arabidopsis thaliana[j]. Plant Journal, 2003, 35: [3] Chen YH, Shen ZG, Li XD. The use of vetiver grass (Vetiveria zizanioides) in the phytoremediation of soils contaminated with heavy metals[j]. Applied Geochemistry, 2004, 19: [4] Chen YH, Wang CC, Wang GP, Luo CL, Mao Y, Shen ZG, Li XD. Heating treatment schemes for enhancing chelant-assisted phytoextraction of heavy metals from contaminated soils[j]. Environmental Toxicology and Chemistry, 2008, 27: [5] Sheng XF, Xia JJ. Improvement of rape (Brassicanapus) plant growth and cadmium uptake by cadmium-resistant bacteria[j]. Chemosphere, 2006, 64: [6] Yang XE, Feng Y, He ZL, Stoffella PJ. Molecular mechanisms of heavy metal hyperaccumulation and phytoremediation[j]. Journal of Trace Elements in Medicine and Biology, 2005, 18: [7] Miransari M. Hyperaccumulators, arbuscular mycorrhizal fungi and stress of heavy metals[j]. Biotechnology Advances, 2011, 29:

6 3 503 [8] Vamerali T, Bandiera M, Mosca G. Field crops for phytoremediation of metal contaminated land: A review[j]. Environmental Chemistry Letters, 2010, 8: 1 17 [9] Chen YX, Wang YP, Lin Q, Luo YM. Effect of coppertolerant rhizosphere bacteria on mobility of copper in soil and copper accumulation by Elsholtzia splendens[j]. Environment International, 2005, l 31: [10]. [D]. :, 2005 [11],. [M]. 3 :, 1999 [12],. [M]. :, 2001 [13] Buchanan RE, Gibbons NE. Bergey s manual of determinative bacteriology[m]. 8th ed. USA The Willianm & wilkins Company, 1986 [14] Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells[j]. Nucleic Acids Research, 1988, 16(3): [15]. [M]. :, 1999 [16],,,,,. WS34 [J]., 2008, 28: [17] Poirier I, Hammann P, Kuhn L, Bertrand M. Strategies developed by the marine bacterium Pseudomonas fluorescens BA3SM1 to resist metals: A proteome analysis[j]. Aquatic Toxicology, 2013, 128/129: [18] Pernezny K, Nagata R, Havranek N, Sanchez J. Comparison of two culture media for determination of the copper resistance of Xanthomonas strains and their usefulness for prediction of control with copper bactericides[j]. Crop Protection, 2008, 27: [19],,. 16S rdna [J]., 2006, 32: [20] Zhang YF, He LY, Chen ZJ, Wang QY, Qian M, Sheng XF. Characterization of ACC deaminase-producing endophytic bacteria isolated from copper-tolerant plants and their potential in promoting the growth and copper accumulation of Brassica napus[j]. Chemosphere, 2011, 83:57 62 [21] Altimira F, Yáñez C, Bravo G, González M, Rojas L A, Seeger M. Characterization of copper-resistant bacteria and bacterial communities from copper-polluted agricultural soils of central Chile[J]. BMC Microbiology, 2012, 12: 193 [22],,,,. [J]., 2009, 41(6): [23] Płociniczak T, Sinkkonen A, Romantschuk M, Piotrowska- Seget Z. Characterization of Enterobacter intermedius MH8b and its use for the enhancement of heavy metals uptake by Sinapis alba L[J]. Applied Soil Ecology, 2013, 63: 1 7 Isolation and Biological Characterization of Two Copper-resistant Bacteria and Their Solubilization of Copper Carbonate WANG Gui-ping, GUO Ming-zhi, CHEN Ya-hua, SHEN Zhen-guo, XIA Yan * (College of Life Sciences, Nanjing Agricultural University, Nanjing , China) Abstract: In this study, Cu-resistant bacteria strains F16a and Fw17a were isolated from Cu-contaminated soil in copper mining wasteland, the strains F16a and Fw17a were identified preliminarily as Enterobacter sp. and Pseudomona sp., respectively, based on their morphological, physiological and biochemical characteristics and 16S rdna sequences. The strains F16a and Fw17a exhibited the resistance to ampicillin and ceftazidime, and moreover, Fw17a was also resistant to chloramphenicol, tetracyclin and lower concentration kanamycin. F16a significantly increased the concentration of water-soluble copper in supernatant of liquid N-containing medium with 500 mg/l CuCO 3, the concentration of water-soluble copper in supernatant was increased by 300% of water-soluble copper concentration in control. However, Fw17a decreased the concentration of water-soluble copper in supernatant by close to 60% compared with the control under the same conditions. Key words: Cu-resistant bacteria, Biological characteristics, Solubilization

13期

13期中国农学通报 2012,28(13):271-275 Chinese Agricultural Science Bulletin 磷石膏改良基质中 Cd Zn 在内的富集和迁移特征王运长 1, 李金娟 1, 解田 2, 杨丽萍 2 1, 郭兴强 ( 1 贵州大学资源与环境工程学院, 贵阳 550025; 2 瓮