基于电子分配的化能自养细菌培养策略

Culture strategy of chemoautotrophic bacteria based on electron distribution

摘要:

目的 针对化能自养细菌培养困难的问题，本研究基于电子分配理论分析它们难培养的原因，并基于纯培养探究电子分配策略对提高化能自养细菌生物量的可行性。方法 从维持细胞内pH平衡和最适能量代谢的角度，计算得出硫氧化细菌盐硫小杆菌(Halothiobacillus) DCM-3、亚硝酸盐氧化细菌硝化杆菌(Nitrobacter) N1及氨氧化细菌亚硝化单胞菌(Nitrosomonas) SCUT-1氧化相应底物产生的电子最终分配给O₂与CO₂的最优比例，并以相应菌株为对象，分别设置不同的O₂与HCO₃^- (CO₂)摩尔量比例以形成不同的电子分配比例进行纯培养验证，用离子色谱仪和紫外分光光度计检测底物和产物浓度，用稀释涂布法测定细胞密度。结果 DCM-3、N1和SCUT-1菌株的最优分配比例分别为0.733:0.267、0.867:0.133和0.6:0.4。若基于最优电子分配比例的条件，DCM-3、N1和SCUT-1菌株分别可合成3.967 ATP/S₂O₃^2-、0.433 ATP/NO₂^-和1.35 ATP/NH₃。根据计算结果，其难培养的原因主要为单位底物提供的能量合成的ATP数量少，且要求适当控制低氧气浓度及补充适量的无机碳。纯培养验证中，DCM-3菌株在最优比例下的生物量为6.5×10⁷ CFU/mL，是不控制比例的对照组的2.2倍。N1菌株在最优比例下的生物量为7×10⁶ CFU/mL，与对照组无显著差异，但最优比例的HCO₃^-浓度(0.4 mmol/L)明显低于对照组(2.5 mmol/L)，该菌株表现出倾向于较高氧气但对CO₂需求低的生长特性，与计算得到的最优比例相符。SCUT-1菌株在控制O₂和CO₂量的实验组中的单位NH₄⁺浓度生物量积累达1.3×10⁶ CFU/(mL·(mmol/L))以上，比充足O₂和CO₂量条件的高25%-40%。结论 基于电子分配的化能自养细菌培养策略，通过限制O₂和CO₂摩尔量并形成一定比例从而限制电子分配的培养条件，有助于提高同一底物条件下的生物量积累，为化能自养细菌的培养提供一定的策略参考。

Abstract:

Objective In view of the difficulty in the culture of chemoautotrophic bacteria, this study analyzed the reasons for the difficulty based on the theory of electron distribution and explored the feasibility of using the electron distribution strategy for increasing the biomass of chemoautotrophic bacteria based on pure culture.Methods From the perspective of maintaining intracellular pH balance and optimal energy metabolism, we calculated the optimal distribution ratios of electrons produced by the sulfur-oxidizing bacterial strain Halothiobacillus sp. DCM-3, nitrite-oxidizing bacterial strain Nitrobacter sp. N1, and ammonia-oxidizing bacterial strain Nitrosomonas sp. SCUT-1 to O₂ and CO₂ by oxidizing corresponding substrates. Furthermore, different molar ratios of O₂ to HCO₃^- (CO₂) were set respectively to form different electron distribution ratios for pure culture verification of the strains. Substrate and product concentrations were measured by ion chromatography and ultraviolet spectrophotometry, and cell density was measured by the dilution coating method.Results The optimal electron distribution ratios of strains DCM-3, N1, and SCUT-1 were 0.733:0.267, 0.867:0.133, and 0.6:0.4, respectively. Based on the optimal electron distribution ratios, strains DCM-3, N1, and SCUT-1 could synthesize 3.967 ATP/S₂O₃^2-, 0.433 ATP/NO₂^-, and 1.35 ATP/NH₃, respectively. According to the calculation results, the main reasons for the difficulty in culture were the small amount of ATP synthesized with the energy provided by per unit substrate and the need to control a low oxygen concentration and supplement an appropriate amount of inorganic carbon. The results of pure culture verification showed that the biomass of DCM-3 under the optimal ratio was 6.5×10⁷ CFU/mL, which was 2.2 times that of the control group. The biomass of N1 under the optimal ratio was 7×10⁶ CFU/mL, which was not significantly different from that of the control group. However, the HCO₃^- concentration (0.4 mmol/L) of the optimal ratio of strain N1 was significantly lower than that (2.5 mmol/L) of the control group, which meant that the strain showed a growth characteristic of tending to higher oxygen concentration but lower CO₂ demand, which was consistent with the calculated optimal ratio. The biomass accumulation per unit NH₄⁺ concentration of SCUT-1 strain in the group with controlled O₂ and CO₂ was more than 1.3×10⁶ CFU/(mL·(mmol/L)), which was 25%-40% higher than that obtained under sufficient O₂ and CO₂.Conclusion The culture strategy of chemoautotrophic bacteria based on electron distribution restricts the culture conditions of electron distribution by limiting the molar amounts and forming a certain ratio of O₂ and CO₂, which helps to improve the biomass accumulation under the same substrate condition and provides certain strategic reference for the culture of chemoautotrophic bacteria.