高通量培养方法在苏打盐碱土细菌资源发掘中的应用

Application of high-throughput cultivation methods for exploring bacterial resources in soda saline-alkaline soils

摘要:

目的 评价培养基浓度、土壤悬液稀释度及土壤类型对细菌高通量培养结果的影响，为苏打盐碱土微生物资源的高效挖掘提供理论依据和技术支撑。方法 以盐碱荒地与玉米地表层土壤为研究对象，设置1×TSB、1/5×TSB、1/10×TSB 3种培养基浓度，结合最适和2×最适土壤悬液稀释度，开展细菌的高通量分离培养与分子鉴定，系统分析不同处理条件下细菌的培养偏好性。结果 2种土壤中原位细菌群落的优势门类为假单胞菌门(Pseudomonadota)、放线菌门(Actinomycetota)、酸杆菌门(Acidobacteriota)、芽孢杆菌门(Bacillota)和绿屈挠菌门(Chloroflexota)；前10个优势属中仅芽孢杆菌属(Bacillus)和红色杆菌属(Rubrobacter)为可培养类群。高通量培养共获得2 256个阳性体系，纯菌孔占比达79.3%；从中鉴定出153个扩增子序列变体(amplicon sequence variants, ASVs)，隶属于4门52属。平均每100个纯菌孔可获得6.8个ASVs或2.3个属。1/10×TSB培养基处理的纯菌孔占比最高，而1×TSB培养基的ASVs分离效率最优；2×最适稀释度在纯菌比例和分离效率方面均优于最适稀释度。盐碱荒地土壤在纯菌比例、ASV及属水平分离效率方面均高于玉米地土壤，且其特有ASVs数量更多。高通量培养获得的高频属包括假单胞菌属(Pseudomonas)、嗜氢菌属(Hydrogenophaga)、芽孢杆菌属(Bacillus)、类芽孢杆菌属(Paenibacillus)、食酸菌属(Acidovorax)和节杆菌属(Arthrobacter)，其中仅Bacillus为原位土壤中的优势属。结论 高通量培养技术是快速获取苏打盐碱土细菌纯培养菌株的有效手段。相较于旱地土壤，盐碱荒地中可获得更高多样性的可培养细菌；适度降低培养基浓度和提高土壤悬液稀释度有助于提升纯菌分离效率和物种多样性。然而，多数原位优势类群难以通过单一类型培养基获得，未来需进一步拓展培养条件多样性以提高土壤优势细菌的可培养性。

Abstract:

Objective To evaluate the effects of medium concentration, soil suspension dilution, and soil type on bacterial high-throughput cultivation outcomes, providing a reference for the exploration of bacterial resources in soda saline-alkali soils.Methods High-throughput cultivation and identification of bacteria from soda saline-alkali wildland and maize field soils were conducted. Three medium concentrations (1×TSB, 1/5×TSB, and 1/10×TSB), two soil suspension dilutions (optimal dilution and 2× optimal dilution), and two soil types (wildland and maize field) were set as experimental factors to analyze bacterial cultivation preferences under different treatments.Results The dominant bacterial phyla in both soils were Pseudomonadota, Actinomycetota, Acidobacteriota, Bacillota, and Chloroflexota. Among the top 10 dominant genera, only Bacillus and Rubrobacter were cultivable. A total of 2 256 positive cultures were obtained through high-throughput cultivation, with pure cultures accounting for 79.3%. A total of 153 amplicon sequence variants (ASVs) were identified, belonging to 52 genera of 4 phyla. On average, every 100 pure cultures yielded 6.8 ASVs or 2.3 genera. The 1/10×TSB medium resulted in the highest proportion of pure culture wells, while the 1×TSB medium showed the highest ASVs isolation efficiency. Cultivation with 2× optimal soil suspension dilution achieved higher pure culture ratios and isolation efficiency than the optimal dilution. The proportion of pure cultures, ASVs isolation efficiency, and genus isolation efficiency were all higher in wildland soil than in maize field soil, with more unique ASVs detected in wildland. The most frequently isolated genera via high-throughput cultivation were Pseudomonas, Hydrogenophaga, Bacillus, Paenibacillus, Acidovorax, and Arthrobacter, among which only Bacillus was a dominant genus in the soda saline-alkali soils.Conclusion High-throughput cultivation is an efficient method for obtaining numerous pure bacterial strains from soda saline-alkali soils within a short period. Natural wildland soil yielded more diverse cultivable bacteria than dryland soil. Moderately reducing medium concentration and soil suspension dilution improved pure culture isolation efficiency and diversity. However, most dominant soil taxa could not be cultivated via a single medium type, underscoring the need to diversify cultivation conditions to enhance the cultivability of dominant soil bacteria.