一株高产胞外多糖巨大芽孢杆菌的筛选鉴定、发酵条件优化及其对土壤团聚体的影响

Screening,identification,fermentation condition optimization of an exopolysaccharide-producing strain and the influence on soil macro-aggregate formation

摘要:

【目的】筛选具有高产胞外多糖(exopolysaccharides,EPS)特性的微生物菌株，为功能性土壤改良菌剂的研制提供菌种资源。【方法】采用LB-苯胺蓝平板结合菌丝拉丝法定性筛选获得具有产EPS特性的植物根际促生细菌(plant growth promoting rhizobacteria,PGPR)功能菌株；采用低温醇沉分离、硫酸-蒽酮比色法测定PGPR功能菌株经4种培养基发酵后发酵液中EPS含量，获得具有高产EPS特性的PGPR功能菌株；以发酵液中EPS含量为衡量指标，采用正交试验对高产EPS菌株发酵条件进行优化；采用培养皿土壤培养试验，分析F1发酵液对砂壤土土壤大团聚体含量的影响。【结果】初选获得8株具有产EPS特性的PGPR功能菌株；经复选确定菌株F1具有高产EPS特性，PDA是F1产EPS的最优培养基，EPS含量为867.54 μg/mL。基于形态学、生理生化特性测定，以及16S rRNA基因序列系统发育分析、全基因组平均核苷酸相似度分析，确定F1为一株巨大芽孢杆菌(Bacillus megaterium)。F1产EPS的最优培养条件：28℃、180 r/min培养24 h，在该条件下EPS产量为1 123.39μg/mL。F1应用于砂壤土进行土壤培养40 d后，粒径>0.25 mm土壤水稳性大团聚体含量比对照提高了4.44倍。【结论】F1菌株具有较强的产EPS能力，能够促进砂壤土水稳性大团聚体的形成；F1产EPS最优培养基为PDA，最优发酵条件为28℃、180 r/min培养24 h。

Abstract:

[Objective] To screen microbial strains with high production of exopolysaccharides (EPS) and provide strain resources for the development of soil improvement agents.[Methods] The string test with a LB-aniline blue plate was employed to qualitatively screen the strains of plant growth-promoting rhizobacteria (PGPR) with EPS production.After fermentation with each strain in four media,the EPS content in the fermentation liquid was determined by low temperature alcohol precipitation and the sulfate-anthranone colorimetric method,on the basis of which the PGPR strain with high EPS production was screened out.The fermentation conditions of the strain screened out were optimized by orthogonal test with EPS content in fermentation liquid as the indicator.The influence of the fermentation liquid of strain F1 on the content of macro-aggregates in sandy loam soil was analyzed by the petri dish culture experiment.[Results] Eight EPS-producing PGPR strains were primarily screened out,among which strain F1 had the highest EPS production.PDA was the best medium for F1 to produce EPS,with the EPS content of 867.54 μg/mL.Based on morphological,physiological and biochemical characteristics,phylogenetic analysis based on 16S rRNA gene sequence,and also average nucleotide identity analysis,F1 was identified as a strain of Bacillus megaterium.The optimal culture conditions for F1 to produce EPS were 28℃ and 180 r/min for 24 h,under which the EPS yield reached1123.39 μg/mL.After F1 was incubated in sandy loam soil for 40 days,the content of water-stable macro-aggregates with the grain diameter>0.25 mm in the soil increased by4.44 times compared with that of the control.[Conclusion] Strain F1 with high EPS production can promote the formation of water-stable macro-aggregates in sandy loam soil.The optimal conditions for F1 to produce of EPS was incubation in PDA at 28℃ and 180 r/min for 24 h.