以亚硝氮为唯一氮源生长的海洋紫色硫细菌去除无机三态氮

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1 微生物学通报 Microbiology China 研究报告 May 20, 2014, 41(5): by Institute of Microbiology, CAS DOI: /j.microbiol.china 以亚硝氮为唯一氮源生长的海洋紫色硫细菌去除无机三态氮 蒋鹏 * 赵春贵 贾雅琼 * 杨素萍 ( ) 摘要 目的 揭示以亚硝氮为唯一氮源生长的海洋紫色硫细菌去除水体中无机三态氮的特征和规律 方法 在光照厌氧环境下, 以乙酸盐为唯一有机物, 在分别以氨氮 亚硝态氮 硝态氮为唯一氮源和三氮共存的模拟水体中, 采用 Nessler s 试剂分光光度法 N-(1- 萘基 )- 乙二胺分光光度法和紫外分光光度法分别测定水体中氨氮 亚硝态氮和硝态氮的含量, 比浊法测定菌体生物量 结果 随着时间的延长, 海洋紫色硫细菌 Marichromatium gracile YL28 分别在氨氮 亚硝态氮和硝态氮为唯一氮源的水体中对三氮的去除量增加, 生物量增大, 水体 ph 升高, 并逐渐趋于平衡 ;YL28 对氨氮的最大去除量和最大耐受浓度分别为 9.64 mmol/l 和 mmol/l, 当氨氮浓度低于 3.21 mmol/l 时, 去除率可达 97.61% 以上 ; 与氨氮相比, 以亚硝态氮和硝态氮为唯一氮源, 菌体的生长速率 生物量和水体最终 ph 较低, 但对亚硝态氮和硝态氮的去除速率和去除量仍然很高, 当亚硝态氮和硝态氮浓度分别达 mmol/l 和 mmol/l 时,YL28 仍能够完全去除 在三氮共存的水体中,YL28 也能良好的去除无机三态氮, 对亚硝态氮和硝态氮去除能力更强 结论 在模拟水体中, 海洋紫色硫细菌 YL28 能够分别以氨氮 亚硝态氮和硝态氮为唯一氮源生长, 具有良好的耐受和去除无机三态氮的能力, 尤其对亚硝态氮具有良好的去除能力 本研究为进一步开发高效脱氮, 尤其是去除亚硝态氮的不产氧光合细菌水质调节剂奠定了基础, 也为微生物制剂的合理应用提供参考 关键词 紫色硫细菌, 氨氮, 硝态氮, 亚硝态氮 Inorganic nitrogen removal by a marine purple sulfur bacterium capable of growth on nitrite as sole nitrogen source JIANG Peng ZHAO Chun-Gui * JIA Ya-Qiong YANG Su-Ping * (Department of Bioengineering and Biotechnology, Huaqiao University, Xiamen, Fujian , China) Abstract: [Objective] To clarify the removal of ammonium, nitrate and nitrite by a marine purple sulfur bacterium capable of growing on nitrite as sole nitrogen source. [Methods] Ammonium, nitrate and nitrite concentration in simulated wastewater were determined using the Nessler s reagent spectrophotometry, N-(1-naphthyl)-1,2-diaminoethane dihydrochloride spectrophotometry, UV 基金项目 : (No ) (No. 2012J01136) (No. JB11275) * 通讯作者 :Tel chungui@hqu.edu.cn yangsuping@hqu.edu.cn 收稿日期 : 接受日期 : 优先数字出版日期 (

2 : 825 spectrophotometry, respectively. [Results] The removal of ammonium, nitrate and nitrite as well as bacterial biomass and ph increased with time, and then tended to equilibrium. Strain YL28 exhibited effective ammonium removal ability, with a maximal removal and tolerance of 9.64 mmol/l and mmol/l, respectively. The removal rate exceeded 97.61% when the concentration of ammonium was less than 3.21 mmol/l. Compared to ammonium, the cell growth rate, biomass and ph of wastewater enhanced slowly while using nitrate and nitrite as sole nitrogen source. However, the removal of nitrate and nitrite were higher than that of ammonium. Nitrate and nitrite in wastewater could be completely removed when their concentrations were up to mmol/l and mmol/l, respectively. When the three inorganic nitrogen existed simultaneously in wastewater, ammonium, nitrate and nitrate could be removed by strain YL28, the removal of nitrate and nitrite was higher than that of ammonium. [Conclusion] Strain YL28 would be a promising candidate for bioremediation of polluted aquaculture wastewater, especially for nitrite-polluted marine culture wastewater. Keywords: Purple sulfur bacterium, Ammonium, Nitrite, Nitrate [1] [2-4] (Anoxygenic phototrophic bacteria APB) APB ( ) H 2 S [5] [6-8] ( ) APB [9] APB Rhodobacter (Rba.) sphaeroides Rhodopseudomonas (Rps.) palustris Rubrivivax gelatinosa Phaeospirillum fulvum Rba. capsulata [2] ( ) Rba. sphaeroides P [8] 4 Rhodopseudomonas sp. wps [7] Rhodobacter sp. S [10] Rps. palustris PS1 [11] APB Rhodospirillum rubrum S1 [12] APB [13] 2007 Benjamin [14] Science Thiocapsa (Tca.) roseopersicina (98% identical) Thiocapsa sp. KS1 [15] YL28 Marichromatium gracile [16]

3 826 微生物学通报 Microbiol. China 2014, Vol.41, No.5 YL28 ( ) APB 1 材料与方法 1.1 材料 菌株 : (Marichromatium gracile) YL28 16S rrna GenBank JF [16] 主要仪器和试剂 :UV-3200PCS MAPADA 5417R Eppendorf DELTA 320 ph METTLER-TOLED 1.2 方法 培养基及菌种的制备 : Pfennig [17] 24.3 mmol/l 3% NaCl 0.75 mmol/l Na 2 S 2 O mmol/l Na 2 S 9H 2 O ph 7.0 5% ( ) 28±1 C lx 5 d 溶液的配制 : 30.0 g/l 模拟污染水体样品的制备 : ph 无机三态氮去除体系和条件 : 3% NaCl ml 28±1 C lx (CK) ph 生物量测定 : 1 cm UV-3200PCS 660 nm (OD 660 ) ph 值测定 : ph ph 无机三态氮的测定和计算 : 3 Nessler s [18] N-(1- )- [19] [20] r=c t C 0 R=(C t C 0 )/C 0 100% (r) (R) C 0 C t 2 结果与分析 2.1 菌株 YL28 对氨氮的去除特性 YL28 8 d (r) (R) (OD 660 ) ph mmol/l mmol/l 9.64 mmol/l 3.21 mmol/l 97.61% mmol/l 50.37% 62.55% ( 1A) 6.77 mmol/l 6.77 mmol/l ph ph ( 1B) YL28 ph

4 : 827 Figure 1 图 1 氨氮初始浓度对氨氮去除量 (r) 去除率 (R) 菌体生物量 (OD 660 ) 和水体 ph 的影响 Effects of initial concentrations of ammonium-nitrogen on removal amount (r) and removal rate (R) of NH 4 + -N, bacteria biomass (OD 660 ) and ph in simulated wastewater 30 mmol/l ph YL28 ph 2 ph 2 d 2 4 d ph 6 d ph mmol/l ph YL28 ph 2.2 菌株 YL28 对亚硝氮的去除特性 YL28 5 d (r) (R) (OD 660 ) ph 3 ( mmol/l) mmol/l mmol/l 100% ph mmol/l 19.65% ph YL28 YL mmol/l YL28 ph 4 ph ph mmol/l ph YL 菌株 YL28 对硝氮的去除特性 YL28 6 d (r) (R) (OD 660 ) ph mmol/l ph ( 4.46 mmol/l)

5 828 微生物学通报 Microbiol. China 2014, Vol.41, No.5 图 2 模拟污染水体中 NH 4 + -N 的去除率 (A) 菌体生物量 (B) 及水体 ph (C) 随时间变化曲线 Figure 2 Change of NH + 4 -N removal rate (A), bacteria growth (B) and ph (C) in the simulated water with increasing time NH + 4 -N C (mmol/l) CK Note: NH + 4 -N initial concentration C (mmol/l). : 3.21; ; 12.49; : 36.64; : CK Figure 3 图 3 亚硝氮初始浓度对亚硝氮去除量 (r) 去除率 (R) 菌体生物量 (OD 660 ) 和 ph 的影响 Effects of initial concentrations of nitrite-nitrogen on removal amount (r) and removal rate (R) of NO 2 -N, bacterial biomass (OD 660 ) and ph in stimulated water 图 4 模拟污染水体中 NO 2 -N 的去除率 (A) 菌体生物量(B) 及水体 ph (C) 随时间变化曲线 Figure 4 Change of removal rate of NO 2 -N (A), cell growth (B) and ph (C) in the simulated water with increased time NO 2 -N C (mmol/l) CK Note: NO 2 -N initial concentration C (mmol/l). : 0; : 9.37; : 13.50; : 20.37; : CK 4.27.

6 : 829 YL28 ph YL28 ph 6 ph ph 3 4 d YL28 ph 2.4 菌株 YL28 对无机三态氮共存的去除特性 (R) ph 7 2 d 4 d (73%) 6 d ph ph mmol/l YL28 Figure 5 图 5 硝氮起始浓度对硝氮去除量 (r) 去除率 (R) 菌体生物量 (OD 660 ) 和 ph 的影响 Effects of initial concentrations of nitrate-nitrogen on removal amount (r) and removal rate (R) of NO 3 -N, bacteria biomass (OD 660 ) and ph in simulated water 图 6 模拟污染水体中 NO 3 -N 的去除率 (A) 菌体生物量(B) 及水体 ph (C) 随时间变化曲线 Figure 6 Change of removal rate of NO 3 -N (A), cell growth (B) and ph (C) in the simulated water with increased time NO 3 -N C (mmol/l) CK Note: NO 3 -N initial concentration C (mmol/l). : 0; : 4.46; : 9.00; : 22.90; : CK 5.06.

7 830 微生物学通报 Microbiol. China 2014, Vol.41, No.5 图 7 模拟污染水体中无机三态氮的去除率 (A) 菌体生物量和 ph (B) 随时间变化曲线 Figure 7 Change of removal rate of inorganic nitrogen (A), cell growth and ph (B) in simulated water with increasing time C (mmol/l). Ammonium 6.53 Nitrite 4.00 Nitrate Note: Initial inorganic nitrogen concentration C (mmol/l). : Ammonium 6.53; : Nitrite 4.00; : Nitrate 讨论 APB ( ph ) 95% [21-22] 18 mg/l 38.9% [23] APB YL mmol/l 97% mmol/l 50% mmol/l 20% YL28 (50 mg/l) Rba. sphaeroide P [8] 4 Rps. palustris wps [7] APB YL mmol/l mmol/l [24] Rba. sp. S [10] Rba. capsulata [23] Rps. palustris HZ-5 [25] 5 mmol/l 6 mg/l mg/l 80% 94.8% 97.79% APB [13] Benjamin [14] Tca. roseopersicina KS YL mmol/l [11] APB

8 : 831 APB YL mmol/l YL28 ph YL28 ph YL28 YL28 参考文献 [1] Crab R, Avnimelech Y, Defoirdt T, et al. Nitrogen removal techniques in aquaculture for a sustainable production[j]. Aquaculture, 2007, 270(1/4): [2] Qi Z, Zhang XH, Boon N, et al. Probiotics in aquaculture of China current state, problems and prospect[j]. Aquaculture, 2009, 290(1/2): [3] Zhou Q, Li K, Jun X, et al. Role and functions of beneficial microorganisms in sustainable aquaculture[j]. Bioresource Technology, 2009, 100(16): [4]. [J]., 2012, 39(1): 1. [5] Lu H, Zhang G, Dai X, et al. Photosynthetic bacteria treatment of synthetic soybean wastewater: direct degradation of macromolecules[j]. Bioresource Technology, 2010, 101(19): [6] Zumft WG. Cell biology and molecular basis of denitrification[j]. Microbiology and Molecular Biology Reviews, 1997, 61(4): [7],,,. [J]., 2011, 51(2): [8],,. P4 [J]., 2000, 34(11): [9] Iribarren D, Dagá P, Moreira MT, et al. Potential environmental effects of probiotics used in aquaculture[j]. Aquaculture International, 2012, 20(4): [10],,,. [J]., 1995, 16(6): [11],,,. PS1 [J]., 2009, 21(1): 4-6. [12] Katoh T. Nitrate reductase in photosynthetic bacterium, Rhodospirillum rubrum. Purification and properties of nitrate reductase in nitrate-adapted cells[j]. Plant and Cell Physiology, 1963, 4(1): [13] Madigan MT, Jung DO. An overview of purple bacteria: systematics, physiology and habitats[a]//the Purple Phototrophic Bacteria[M]. Netherlands: Springer, 2008: [14] Griffin BM, Schott J, Schink B. Nitrite, an electron donor for anoxygenic photosynthesis[j]. Science, 2007, 316(5833): [15] Schott J, Griffin BM, Schink B. Anaerobic phototrophic nitrite oxidation by Thiocapsa sp. strain KS1 and Rhodopseudomonas sp. strain LQ17[J]. Microbiology, 2010, 156(8): [16],,,. [J]., 2011, 51(10): [17] Pfennig N, Trüper H. The prokaryotes[m]. Edition 2. New York: Springer, 1992: [18]. [J]., 2009, 27(1): [19] Mahmood Q, Zheng P, Hayat Y, et al. Effects of nitrite to sulfide ratios on the performance of anoxic sulfide oxidizing reactor[j]. Arabian Journal for Science and Engineering, 2009, 34(1): 45. [20],,. [M]. :, 2002: 266. [21],,,. [J]., 2000, 19(6): [22],,,. RPD-1 [J]., 2010(007): [23],,,. NH 4, NO2 [J]., 1999, 23(S1): [24] Jensen FB. Uptake, elimination and effects of nitrite and nitrate in freshwater crayfish (Astacus astacus)[j]. Aquatic Toxicology, 1996, 34(2): [25],,,. [J]., 2008, 28(8):