海水养殖生境中柠檬酸杆菌降解半胱氨酸产生硫化物的机制研究

Mechanism of sulfide production from cysteine degradation by Citrobacter in mariculture habitats

摘要:

【目的】含硫氨基酸的生物降解是造成海水养殖环境中毒性硫化物上升的重要因素，而微生物降解含硫氨基酸的机制和影响因素解析是控制该系统中硫化物浓度的关键环节。【方法】本研究利用稀释涂布-叠皿夹法自本实验室海水养殖环境的沉积物中分离得到一株产生硫化物的厌氧菌株，并通过代谢组学研究其以半胱氨酸为底物产生硫化物的机制和途径。【结果】经鉴定，该菌株为弗氏柠檬酸杆菌(Citrobacter freundii)，能够在厌氧条件下还原硫酸盐，能够降解半胱氨酸产生硫化物，投加l-半胱氨酸可提高其还原硫酸盐的能力。该菌株以1 g/L半胱氨酸为底物，在35 ℃、盐度为10、pH 8.0的条件下，硫化物的最高积累量可达302.4 mg/L。对菌株中硫化物产生具有重要贡献的半胱氨酸脱巯基酶研究表明，该酶最适温度为35 ℃，在pH 6.0-8.0有较高的活性，能够快速降解半胱氨酸产生硫化物。结合代谢组学研究发现，该菌株中含有的3-巯基丙酮酸硫酸转移酶、胱硫醚-γ-裂解酶、半胱氨酸脱巯基酶催化半胱氨酸降解是产生硫化氢的主要途径；亚硫酸盐还原酶还原硫酸盐、亚硫酸盐是其产生硫化氢的次要途径。【结论】通过揭示柠檬酸杆菌降解半胱氨酸产生硫化物的机理，可为海水养殖环境中毒性硫化物的防控提供理论基础。

Abstract:

[Objective] The biodegradation of sulfur-containing amino acids is an important factor in the rise of toxic sulfides in the mariculture environment, and the analysis of the mechanisms and influencing factors of microbial degradation of sulfur-containing amino acids is a key link to control the sulfide concentration in this system. [Methods] This study used the dilution coating-stacked dish clamp method to isolate a sulfide-producing anaerobic strain from the sediment of the mariculture environment in this laboratory, and the mechanism and pathway of sulfide production with cysteine as substrate were investigated by metabolomics. [Results] The strain was identified as Citrobacter freundii, which reduced sulfate under anaerobic conditions and degraded cysteine to produce sulfide. The addition of l-cysteine improved its ability to reduce sulfate. The maximum accumulation of sulfide in this strain was 302.4 mg/L at 35 ℃, salinity 10, and pH 8.0 with 1 g/L l-cysteine as substrate. The study of cysteine desulfhydrase, the important contributor to sulfide production in the strain, revealed that the optimum temperature of this enzyme was 35 ℃, and it had high activity at pH 6.0-8.0, which could rapidly degrade cysteine to produce sulfide. Combined with the metabolomics study, it was found that the strain contained 3-mercaptopyruvate sulfotransferase, cystathionine gamma-lyase, and cysteine desulfhydrase catalyzing the degradation of cysteine was the main pathway to produce hydrogen sulfide, and sulfite reductase reducing sulfate and sulfite was its secondary pathway to produce hydrogen sulfide.[Conclusion] By revealing the mechanism of sulfide production from cysteine degradation by Citrobacter, this paper provides a theoretical basis for the prevention and control of sulfide in mariculture.