低溶氧适应型好氧反硝化细菌简单近芽孢杆菌NBNZ-3745的脱氮特性及在浅湖底泥生物修复中的应用

Aerobic denitrifying bacterium Peribacillus simplex NBNZ-3745 adaptive to low dissolved oxygen: nitrogen removal characteristics and application in shallow lake sediment bioremediation

摘要:

背景 异养硝化-好氧反硝化(heterotrophic nitrifying-aerobic denitrifying, HN-AD)细菌在浅湖底泥脱氮修复方面极具应用潜力。然而在重度污染的底泥环境中，较低的溶解氧浓度严重制约了HN-AD细菌的脱氮性能发挥。目的 筛选适应低溶解氧环境的HN-AD功能菌株，并探究其脱氮特性及在浅湖底泥中的实际修复效果。方法 通过菌株分离筛选获得菌株NBNZ-3745，采用细胞形态学观察及16S rRNA基因系统发育分析进行菌种鉴定；在30 ℃、150 r/min条件下测定其HN-AD脱氮性能，并在模拟浅水湖泊水-底泥界面低溶解氧环境下验证其适应性；结合基因组学分析与PCR验证鉴定氮代谢功能基因；通过原位接种实验评估菌株在浅湖底泥中的脱氮效能。结果 菌株NBNZ-3745被鉴定为简单近芽孢杆菌(Peribacillus simplex)。其表现出高水平的脱氮能力，在30 ℃、150 r/min条件的HN-AD过程中，对铵态氮(NH₄⁺-N)和硝态氮(NO₃^--N)的去除率分别达(96.8±2.6)%和(84.9±3.7)%，尤其是无有毒产物亚硝态氮(NO₂^--N)和羟胺氮(NH₂OH-N)积累。该结果对应的最大去除速率分别为(5.7±0.1) mg/(L·h)和(5.2±0.2) mg/(L·h)。在模拟低溶解氧环境下，对NH₄⁺-N和NO₃^--N的去除率仍可达(83.3±4.3)%和(78.1±2.8)%。基因组学分析结合PCR验证鉴定出nrt2A、nrt2B、narB、nasC、nirB、nasD、nasE、glnA、nirK等9个参与氮代谢的功能基因。原位底泥修复实验中，菌株在水下低溶解氧环境下60天内可消除(41.2±3.2)%的NH₄⁺-N，在富氧环境下则可去除(87.6±13.5)%的NH₄⁺-N。结论 菌株NBNZ-3745具有强低溶解氧环境适应性和高效脱氮能力，且无有毒中间产物积累，在脱氮修复领域尤其是浅湖底泥原位治理中极具应用前景。

Abstract:

Background Heterotrophic nitrifying-aerobic denitrifying (HN-AD) bacteria show great potential for use in denitrification and remediation of shallow lake sediments. Nevertheless, the low dissolved oxygen (LDO) in heavily polluted sediment environments greatly hampers the performance of HN-AD bacteria.Objective To screen functional HN-AD bacterial strains adapted to LDO environments, and to investigate their nitrogen removal characteristics as well as their practical bio-remediation effects in shallow lake sediments.Methods The strain NBNZ-3745 was obtained through screening and evaluation from the strain library, and identified via cellular morphological observation and 16S rRNA gene phylogenetic analysis. Its HN-AD nitrogen removal performance was determined at 30 ℃ and 150 r/min, and its adaptability was verified under a simulated LDO environment as the water-sediment interface of shallow lakes [25 ℃, (2.0±0.5) mg/L]. Functional genes involved in nitrogen metabolism were identified by combining genomics analysis and PCR verification. The nitrogen removal efficiency of the strain in shallow lake sediments was evaluated through in-situ inoculation experiments.Results Strain NBNZ-3745 was identified as Peribacillus simplex. The strain demonstrated high-level nitrogen removal capability, achieving NH₄⁺-N and NO₃^--N removal rates of (96.8±2.6)% and (84.9±3.7)%, respectively, under HN-AD conditions at 30 ℃ and 150 r/min, with no accumulation of toxic intermediates NO₂^--N and NH₂OH-N. The corresponding maximum removal rates were (5.7±0.1) mg/(L·h) and (5.2±0.2) mg/(L·h). Under simulated LDO conditions, the removal rates for NH₄⁺-N and NO₃^--N remained at (83.3±4.3)% and (78.1±2.8)%, respectively. A total of nine nitrogen metabolism functional genes, including nrt2A, nrt2B, narB, nasC, nirB, nasD, nasE, glnA, and nirK, were identified through genomic analysis combined with PCR verification. In the in-situ sediment remediation experiment, the strain eliminated (41.2±3.2)% of NH₄⁺-N under underwater LDO conditions and (87.6±13.5)% under oxygen-rich conditions within 60 days.Conclusion Strain NBNZ-3745 possesses strong adaptability to LDO environments and efficient nitrogen removal capability without accumulation of toxic intermediates, demonstrating substantial application prospects in the field of nitrogen removal remediation, particularly for in-situ treatment of shallow lake sediments.