土壤 (Soils), 2013, 45(4): 683 690 1 一株根际好氧反硝化菌的筛选及其反硝化条件研究 1,2 1,2 1* (1 ( ) 210008; 2 100049) RWX31 NO 3 -N 140 mg/l 24 h 82% N 2 O RWX31 1% Mg 2+ 0.05 g/l NO 2 -N 0 28 ~ 32 ph 7.0 ~ 7.5 C/N 8 ~ 12 DO 6.5 ~ 7.0 mg/l NO 3 -N 90% RWX31 NO 3 -N X52 [1] [2-4] 1983 Robertson [5] Thiosphaera pantotropha [6-9] ph [10] RWX31 1 1.1 1.1.1 (EM g/l) 5 KNO 3 2 KH 2 PO 4 1 K 2 HPO 4 0.5 Mg 2 SO 4 7 H 2 O 0.2 1 ml ph 7.2 1.5 % EM 1.1.2 (g/l) EDTA 50.0 ZnSO 4 7H 2 O 2.2 CaCl 2 2H 2 O 5.5 MnCl 2 4H 2 O 5.06 FeSO 4 7H 2 O 5.0 (NH 4 ) 6 Mo 7 O 24 4H 2 O 1.1 CuSO 4 5H 2 O 1.57 CoCl 2 6H 2 O 1.61 ph 6.0 1.1.3 (DM g/l) 5 KNO 3 ( ) (201003014-1) (30821140542) * (wmshi@mail.issas.ac.cn) (1985 ) E-mail: zhouyingru1985@163.com
684 45 1.011 KH 2 PO 4 1 K 2 HPO 4 0.5 Mg 2 SO 4 7H 2 O 0.2 ph 7.2 1.1.4 Luria-Bertani (LB g/l) 10 5 NaCl 5 ph 7.0 1.5% LB 1.1.5 Steinberg NH 4 Cl 12.5 KH 2 PO 4 1.756 Mg 2 SO 4 7H 2 O 100 Ca(NO 3 ) 2 4H 2 O 98.9 Na 2 EDTA 2H 2 O 1.5 ZnSO 4 7H 2 O 0.18 MnCl 2 4H 2 O 0.18 H 3 BO 3 0.12 NaMoO 4 2H 2 O 0.04 FeCl 3 6H 2 O 0.76 ph 6.8 1.2 1.2.1 10 g ( 5 cm) 10 ml 250 ml EM 160 r/min 28 1 h 1 ml 9 ml EM 28 3 1 ml EM 1.2.2 Lemna minor 3 Steinberg 15 mg/l KNO 3 23 65% / 16 h/8 h 3 10 1.5 ml 1 ml 5 mg/l (Na 5 P 3 O 10 ) 150 W 6 5 s EM [11] 1.2.3 N 2 O 250 ml 100 ml DM 1 ml RWX31 (OD 600 = 0.5) 704 0.22 μm (80% He + 20% O 2 )( 15 min 3 ) 704 160 r/min 30 24 h NO 3 -N TN N 2 O 1.3 RWX31 LB ( 12 h) 1% 100 ml 30 160 r/min OD 600 NO 3 -N NO 2 -N TN LB 1.4 RWX31 1 ml (OD 600 = 0.5) 100 ml 30 160 r/min 24 h OD 600 NO 3 -N NO 2 -N 1.4.1 DM 1.4.2 0.2 0.5 1.0 1.5 2.0 2.5 ml 1.4.3 Mg 2+ DM Mg 2+ 0 0.05 0.10 0.15 0.20 0.25 g/l 1.4.4 NO 2 -N DM TN 140 mg/l NO 2 -N 0 20 40 60 80 100 NO 2 -N 1.5 RWX31 1.5.1 20 25 28 30 32 35 40 1.5.2 ph ph 5.5 6.0 6.5 7.0 7.5 8.0 1.5.3 C/N C/N 2 4 6 8 8.7 10 12 1.5.4 (DO) DO 0 50 100 150 200 250 r/min 1.6 (Bio-Rad 3000 USA) 600 nm NO 3 -N NO 2 -N N-(1- )- TN - DO JPB-607 N 2 O ( Porapak Q 3 m 3 mm) 3 Excel 2003 SPSS 17.0 Duncan (P 0.05
4 685 2 2.1 128 69 140 mg/l NO 3 -N 24 h 6 NO 3 -N 70% 1 1 25 10 34 75% 4 3 [12] [13] [14] 1987 50% ~ 70% (34) 50% RWX31 RWX31 16S rrna RWX31 Pseudomonas [15] 表 1 环境样品中分离反硝化菌株 Table 1 Denitrifying bacteria isolated from different samples (%) ( >75%) (%) 25 3.1 ~ 56.3 0 0 10 32.9 ~ 79.7 1 10 34 15.3 ~ 81.3 3 9 69 3.1 ~ 81.3 4 6 RWX31 DM 80% He + 20% O 2 24 h DO 3.8 ~ 5.9 mg/l O 2 [16] RWX31 N 2 O NO 3 -N 140 25 mg/l TN 144 58 mg/l N 2 O 0 197 mg/kg 24% NO 3 -N 18% NO 3 -N 58% N 2 O-N 1% N 2 RWX31 2.2 RWX31 RWX31 1% DM OD 600 NO 3 -N TN 4 1 RWX31 9 h 21 h 18 ~ 21 h NO 3 -N RWX31 12 h NO 3 -N 12 h 21 h 24 h NO 3 -N 25 mg/l [17] X31 36 h NO 3 -N N 2 O 24 h NO 3 -N 82% 24 h TN NO X -N TN [18] 1 36 h NO 3 -N 80% TN 76% [19]
686 45 CW 24 h 108 mg/l NO 3 -N 75% [4] YL-1 24 h 115 mg/l NO 3 -N 50% [20] 2-8 NO 3 -N 140 mg/l 48 h 92% RWX31 140 mg/l NO 3 -N 24 h 82% RWX31 图 1 菌株 RWX31 生长与 - NO 3 -N 和 TN 去除曲线 Fig.1 Growth curve and -N and TN remove performance of strain RWX31 2.3 2.3.1 RWX31 2 RWX31 24 h 91.5% P. stutzeri D6 [21] D5 [22] 2 RWX31 NO 3 -N 2.3.2 RWX31 RWX31 DM 24 h OD 600 NO 3 -N 表 2 菌株 RWX31 在不同碳源中的反硝化活性 Table 2 Denitrification activity of strain RWX31 in different carbon sources OD 600 NO2 -N (%) 0.013 138.4 0 13.80 0.563 92.8 4.89 30.43 1.534 11.3 5.50 91.54 0.021 138.8 0 4.13 0.025 134.9 0 1.15 0.874 67.6 5.41 49.33 2 0.5% OD 600 1.0% NO 3 -N OD 600 [23] AM-4 2% 图 2 初始接种量对 RWX31 生长和反硝化效率的影响 Fig. 2 Effects of inoculation size on growth and denitrification performance of strain RWX31 2.3.3 Mg 2+ RWX31 Mg 2+ [24] Mg 2+ 3 Mg 2+ 0 NO 3 -N 6.8% Mg 2+ 0.05 g/l Mg 2+ Mg 2+ 0.05 g/l Mg 2+ NO 2 -N [24] Mg 2+
4 687 表 3 Mg 2+ 浓度对 RWX31 反硝化作用的影响 Table 3 Effect of Mg 2+ concentration on denitrification of strain RWX31 Mg 2+ (g/l) OD 600 NO2 -N (%) 0 0.03 0.33 155.71 6.81 0.05 1.58 13.56 22.42 85.60 0.10 1.51 16.36 48.08 69.12 0.15 1.45 0 38.69 75.15 0.20 1.38 8.55 31.15 79.99 0.25 1.38 15.58 42.72 72.56 2.3.4 NO 2 -N RWX31 NO 2 -N 4 NO 2 -N 0 79.9% NO 2 -N NO 2 -N NO 2 -N NO 2 -N 50% RWX31 NO 2 -N NO 2 -N NO 2 -N 20 ~ 30 mg/l NO 2 -N [25] NO X -N NO 2 -N ph NO 2 -N NO 2 -N NO X -N 20 mg/l 140 mg/l NO 2 -N [26] AQ-3 NO 2 -N 20 mg/l 2.4 2.4.1 RWX31 25 ~ 35 [27] ( 3a) 30 - 表 4 NO2 -N 在初始氮源中所占比例对 RWX31 反硝化作用的影响 Table 4 Effect of nitrite proportion on denitrification of strain RWX31 NO2 -N (%) OD 600 NO2 -N -N (%) 0 1.47 0.56 29.23 79.90 20 1.42 28.26 44.84 69.18 40 1.38 21.71 69.17 52.45 60 1.44 28.47 44.44 69.45 80 1.44 34.73 47.15 67.58 100 1.37 21.77 62.03 57.36 24 h 92.7% NO 3 -N 30 [28] F1 NO 3 -N 30 ~ 35 2.4.2 ph RWX31 ph [29] 3b RWX31 ph 7.2 90.3% (ph < 6) (ph > 7.5) RWX31 2.4.3 C/N RWX31 C/N C/N [30] 3c RWX31 C/N C/N 10 NO 3 -N 91.2% C/N RWX31 Chiu [31] C/N Stouthamer [32] C/N 2.4.4 DO RWX31 DO DO [4,33-34] 0 50 r/min (DO 3.5 ~ 5.8 mg/l) NO 3 -N 63% 31.2%
688 45 100 ~ 250 r/min (DO 6.5 ~ 7.0 mg/l) NO 3 -N 90% Robertson Kuenen [16] 3 O 2 ( ) O 2 ( 1% ~ 2% DO 0.1 ~ 0.2 mg/l) O 2 3 100 ~ 250 r/min (DO 6.5 ~ 7.0 mg/l) 100 r/min RWX31 RWX31 Patureau [35] NO X O 2 DO DO RWX31 NO 3 -N DO 100 ~ 250 r/min (DO 6.5 ~ 7.0 mg/l) Fig.3 图 3 培养温度 ph 碳氮比和摇床转速对 RWX31 反硝化作用的影响 Effects of temperature, ph, carbon nitrogen ration and shaking speed on denitrification of strain RWX31 3 RWX31 140 mg/l NO 3 -N 24 h NO 3 -N 82% N 2 O RWX31 RWX31 1% Mg 2+ 0.05 g/l NO 2 -N 28 ~ 32 ph 7.0 ~ 7.5 C/N 8 ~ 12, DO 6.5 ~ 7.0 mg/l RWX31 NO 3 -N 140 mg/l 24 h 90% RWX31 [1],,,,,,,,. [J]., 2012, 44(2): 213-217 [2] Borges MT, Sousa A, De Marco P, Matos A, Honigova P, Castro PML. Aerobic and anoxic growth and nitrate removal capacity of a marine denitrifying bacterium isolated from a recirculation aquaculture system[j]. Microbial Ecology, 2008, 55(1): 107-118 [3] Kim M, Jeong SY, Yoon SJ, Cho SJ, Kim YH, Kim MJ, Ryu EY, Lee SJ. Aerobic denitrification of Pseudomonas putida AD-21 at different C/N ratios[j]. Journal of Bioscience and Bioengineering, 2008, 106(5): 498-502 [4],,,,,. [J]., 2010, 16(3): 394-398
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690 45 Isolation and Denitrification Conditions of Rhizosphere Aerobic Denitrifying Bacterium RWX31 ZHOU Ying-ru 1,2, LU Yu-fang 1,2, SHI Wei-ming 1 (1 State Key Laboratory of Soil and Sustainable Agriculture(Institute of Soil Science, Chinese Academy of Sciences), Nanjing 210008,China; 2 University of Chinese Academy of Sciences, Beijing 100049, China) Abstract: The aim of this study was to screen aerobic denitrifying strain from various environmental sites and to investigate the denitrification conditions for the isolated strain. Enrichment culture technique was used in screening and acclimatization of strain, a rhizosphere aerobic denitrifying bacterium RWX31 was isolated and single-factor experiments were used to investigate the denitrification conditions and characteristics. The nitrate remove efficiency of aerobic denitrifying strain RWX31 was 82% when cultivated in 140 mg/l nitrate for 24 hours, N 2 O could be produced aerobically through aerobic denitrification of strain RWX31. The optimum medium was: sodium citrate as carbon source, 1% inoculation volume, 0.05 g/l Mg 2+ concentration and nitrite proportion of 0 in the initial denitrification nitrogen source. The optimum condition was: temperature 28-32, ph 7.0-7.5, C/N ratio 8-12 and DO concentration 6.5-7.0 mg/l. The highest nitrate removal efficiency was higher than 90% under the above conditions. Rhizosphere bacterium RWX31 was an efficient aerobic denitrifying strain with higher nitrate removal efficiency than reported previously and has application potential. Key words: Rhizosphere, Aerobic denitrifying, Nitrate nitrogen removal efficiency, Condition investigation