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1 17 6 Vol.17 No Journal of Fishery Sciences of China Nov 熊焰, 刘力源, 罗睿, 朱欢 (,, ) 摘要 : 1, 16S rrna (Bacillus megaterium), SZ-3 SZ-3, : ph 7.0, 33, 0.01 mg/l, 24 h 69.58% 5 mg/l 100 L, ml CFU/mL SZ-3, 28, 96 h, SZ %[, 2010, 17(6): ] :; ; ; 中图分类号 : S94 文献标识码 : A 文章编号 : (2010)00,, [1],,, (Fe 2+ ) (Fe 3+ ),, [2 5],,,,,, MRS 37 [6 9],, [10-11],,,, 7% 77.7% [12], (Bacillus megaterium),,,, [13],,,,,,,,, 收稿日期 : ; 修订日期 : 基金项目 : (IRTO848). 作者简介 : (1955 ),,. :. xyan @yahoo.com.cn

2 2 17, 1 1.1, 6 8cm, () %, r/min, 5% 2 ml, 1, 1 ml 100 ml, r/min 48 h, -, [14] S rrna DNA, 16S rrna [15 16], 5-AGAGTTTGATCCTGGCT- CAG-3, 5-ACGGTTACCTTGTT- ACGACTT-3, 25 L PCR : 12.5μLTaq Mixture, 0.5μL(20μmol/L), DNA 2μL, 9.5μL : 94 3 min, s, min, 72 2 min 30 s, 35, min, GenBank BLAST clustralx 8.0, Mega 4.0, Neighbor-Joining 1000 Bootstrap, r/min 24 h 0.1 ml, 5%,, h, CFU/mL 10 L, r/min 10 min, 10 L, 10,, 15 d ph 24h ( CFU/ ml, ) 1%, 0.5 mg/l, ph , 28, 120 r/min 24 h, ph, 24 h 1%, 0.5 mg/l, , 120 r/min 24 h, ph, 1% 24 h, 4, r/min 10 min, 1mg/L 0.1mg/L 0.01mg/L 0.001mg/L, 10 L 4, 5 mg/l, 24 h, 7 d , 3, 5 mg/l 100 L, ph 7,

3 6 : 1 3 (28±1), 0.01 mg/l 6 8 cm 10 3 d, 1% 10 6 CFU/mL 10 7 CFU/ ml 10 8 CFU/ ml ml, r/min 10 min, 9 3% 3,, 24h ph, 7 d, 2 2.1, 42,, 42,, 7 ( 1), SZ % SZ SZ-3 : V-P,,, 7%, 2 Time Item 0 h OD Control 表 1 分离菌株对亚硝酸盐的降解率 Tab. 1 to nitrite of isolated strains SZ SZ SZ Strain SZ SZ SZ SZ /% h OD /% h OD c c b c a b bc /% a a 0.746b 0.395a 1.127c 0.684b 0.789ab : 3 ; (P<0.05) Note: The degradation rate is the mean value of three times duplicate tests data. Within a row, means with no common superscript letters are significantly different (P < 0.05). Tab. 2 表 2 SZ-3 菌株的形态及生化特性 Morphological and biochemical characteristics of SZ-3 strain Item Result Item Result Gram stain + Indol test Shape of spore R Sporangium distention Position of spore C Motility Manna sugar + Proteolysis + 7% 7%NaCl + Amylolysis + Acid-producing by glucose + Saccharose Gas-producing by glucose Gas-producing by nitrite V.P V.P test Nitrate reduction + Aerobism Facultative Catalase + : + ; - ; R ; C (). Note: + represents the positive; - represents the negative; E represents the ellipse or columnar; C represents the mesozoic gemma.

4 SZ-3 16S rrna PCR 16S rrna PCR, bp bp ( 1), 1441bp, GeneBank, : HM BLAST, SZ-3, 99.5%, 2, 8 [14], 16S rrna, SZ-3 (Bacillus megaterium), SZ-3 1 SZ-3 Fig. 1 : SZ-3. Agarose gel electrophoreμsis map of SZ-3 strain Note: 1,2,3 and 4 represent the SZ-3 strain. Fig. 2 2 SZ-3 16S rrna Molecular phy logenetic tree of SZ-3 strain based on 16S rrna sequences %, h, SZ L SZ-3, 10 L, CFU/mL 15 d,,,, ph 0.5 mg/l, ph , SZ-3 ph ; ph , ph ph 7.0, 80.23% mg/l, ph 7.0, 18 33, SZ-3 ; 33 50, 28 33,, 88.94%, 表 3 Table 3 SZ-3 菌株在不同温度下对亚硝酸盐的降解率 to nitrite at different temperatures of SZ-3 stain / Incubation temperature /% b a a a a b c 3 ; (P < 0.05) Note: The degradation rate is the mean value of three times duplicate tests data. Within a column, means with no common superscript letters are significantly different(p < 0.05).

5 6 : ph , 7, 1 mg/l 0.1 mg/l 0.01 mg/l 79.64% 71.81% 69.58%, mg/l, 24.16% ph 3, SZ-3, ph, 7 ph 7.1, 7.5, ph 表 4 葡萄糖含量对 SZ-3 菌株亚硝酸盐降解率的影响 Tab.3 Influence of glucose content on degradability Glucose content Item 1 mg/l 0.1 mg/l 0.01 mg/l mg/l OD c b b a /% a b b c 3 ; (P < 0.05). Note: the degradation rate is the mean value of three times duplicate tests data. Within a row, means with no common superscript letters are significantly different(p < 0.05). 3 ph ph 3 ph Fig. 3 Fluctuation of ph value in the aquatic water The ph value in fig. 2 is the mean value of three different thallus concentrations in the treatment group., 7 ph , SZ CFU/ml, 91.78%, %, CFU/ml CFU/ml % %(P< 0.05), 0%(P<0.05) 5-7,, 100% Item OD Tab. 5 /% Degradation rate 表 5 添加菌量条件下的亚硝酸盐降解率 Relationship between degradation rate of nitrite and additive amount of bacteria /dtest date Additive bacterial amount ±0.006 b 0.091±0.014 b 0.070±0.015 b 0.059±0.011 b 0.045±0.008 b 0.040±0.010 b 0.041±0.010 b ±0.011b c 0.069±0.013 b 0.055±0.012 bc 0.046±0.007 bc 0.040±0.010 b 0.036±0.006 b 0.037±0.007 b ±0.013 c 0.030±0.009 c 0.017±0.003 c 0.012±0.004 c 0.011±0.003 c 0.012±0.004 b 0.010±0.002 c Control 0.146±0.021 a 0.143±0.025 a 0.130±0.045 a 0.162±0.037 a 0.162±0.015 a 0.170±0.044 a 0.168±0.012 a ±3.814 b 37.68± b 52.05± b 59.59±7.248 b 69.18±5.171 b 72.61±6.534 b 71.92±6.533 b ±7.409 a 52.74±9.515 b 62.34±7.899 b 68.49±4.939 b 72.61±6.746 b 75.34±3.813 b 74.66±4.745 b ±8.904 a 79.45±6.534 a 88.35±1.812 a 91.78±2.469 a 92.47±2.054 a 91.78±2.985 a 93.15± a Control (P < 0.05). Note: the degradation rate is the mean value of three times duplicate tests data. Within a column, means with no common superscript letters are significantly different (P < 0.05).

6 ,, [17],,,,, [6 9, 18], SZ-3,, 3.2 ph, 0.5 mg/l, ph7.0 28~33, 1% CFU/mL SZ-3, 24h, 88.94%, SZ-3 (28 33 ), ph , 1 mg/l 0.1 mg/l 0.01 mg/l mg/l, 1% CFU/ml SZ-3, 7 d, 79.64% 71.81% 69.58%, 24.16% SZ-3, 1mg/L 0.1mg/L, 2,,,,, 0.01mg/L,, 0.01 mg/l, SZ-3, 69.58% mg/l,, 24.16%, [6], 1 (10 9 CFU ml) 300 ml, 30 3d, , [7], () 37 36h 90,,,,,, 28-30,, mg/l, 0.5 mg/l,,, [19], 1 mmol/l(50 mg/l) [20 22] [23], 15 mg/l, SZ mg/l ph7.0 28, 100 L, ( 5) CFU/mL, 4d 5 mg/l mg/l,

7 6 : % [12] [12], CFU/mL (MPF- 906),, 1 4, 5 7,,, SZ-3, ph, 7 d ph7.3 SZ-3 ph, SZ-3 [19], 0.5 mg/l,,,, 3.4, 0.5 mg/l, 24h 80.23% 88.94%( ), 10L, 0.01mg/L, %( 2.4.3), 100L, 0.01mg/L, %( 2.5.2),,,,,,,,, SZ-3, ph,,,, 参考文献 : [1],. [J]., 2000, 2(1): [2],. [J]., l989, l3(3): [3] AM Hilmy, NA EI-Domiaty, K Wershana. Acute and chronic toxicity of nitrite to Clarias Lazera[J]. Comp Biochem Physiol C, 1987, 86(2): [4],. [J]., 1991, 6(1): [5],,. Cl- NO 2 -N [J]., 1995, 25(6): 3 5 [6],,. [J]., 2008, 12(11): 59 6 [7],,. [J]., 2008, 9: [8],,. [J]. 2009, 17(4): [9],,. [J]. 2009, 2: 42 [10],. B11 5 [J]. 2006, 6: 9 20 [11],,. [J]. 2009, 6: [12],,,. MPF- 906 [J]., 2007, 28(3): 6 8 [13],,,. [J]., 2006, 37 (4): [14] R E, N E.: 8 [M]. :, 1984: [15],,,. 16S rdna 4 [J]., 2006, 5: [16] Weisburg W G, Barns S M, Pelletier D A, et al. 16S ribosomal DNA amplification for phylogenetic study[j]. J Bacteriol, 1991, 173(2):

8 8 17 [17]. [D].:, 2001, 5 [18],,. Ⅰ [J]., 2005, 24(6): [19]. [J], 2006, 11(2): 44 [20] Avnimelech Y, Weber B, Hepher B, et a1 Studies in circulated fish ponds: organic matter recycling and nitrogen trans formation[j] Aquca Fish Manag, 1986, 17(13): 23l 242 [21] Kamstra A, Span J A, Van Weerd J H. The acute toxicity and sublethal effects of nitrite on growth and feed utilization of European eel, Anguilla anguilla(l.)[j].aquac Res, 1996, 27(2): [22] Kroupova H, Machova J, Svobodova Z. Nitrite influence on fish: a review[j]. Vet.Med-Czech. 2005, 50(11): 46l 471. [23],,,. [J]., 2008, 4(4): Screening, identification, degradation condition and effects of a nitrite-degradating strain XIONG Yan, LIU Li-yuan, LUO Rui, ZHU Huan (Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Ya an , China) Abstract: An efficient, rapid multiplicative, stable and safe nitrite-degradating strain was selected from aquatic water. According to the conventional physiological, biochemical measurement and the 16S rrna sequence analysis, the strain was identified as Bacillus megaterium and named SZ-3. Based on the single-factor optimization tests to the strain, the optimum degradation conditions were found as follows, the optimal ph value is 7, the perfect temperature is 33 and the minimum glucose require of degradation is 0.01 mg/l. Under these circumstances, the degradation rate of SZ-3 strain to nitrite came up to 69.58% in 24 h. In breeding test of 100 L polluted water in which the concentration of nitrite is 5 mg/l, the degradation rate of SZ-3 strain on the nitrite achieved at 91.78% for 96 h in 28 via adding the pure thalli of SZ-3 which was obtained by centrifugated 1,000ml (1 108 CFU/mLl) culture fluid. All of these tests above indicated that the strain can degrade the nitrite in polluted water efficiently. [Journal of Fishery Sciences of China, 2010,17(6): ] Key words: screening of microorganisms, Bacillus megaterium, nitrite; pollution water

: 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