耐盐碱秸秆降解微生物的定向筛选

Directional screening for straw-degrading microorganisms tolerant to saline-alkali soil

  • 摘要: 针对松嫩平原苏打盐碱地低丰度秸秆降解功能微生物难以分离的技术瓶颈,本研究采用“先富集-后筛选”策略,通过模拟盐碱胁迫与秸秆碳源限制双重选择压力,突破传统方法对耐盐碱秸秆降解菌的捕获效率限制,筛选耐盐碱秸秆降解微生物。研究从松嫩典型盐碱地的大安水稻田(DA)和洮北水稻田(TB)土壤中富集秸秆降解菌群,经12轮传代扩增获得目标菌群DA-12W和TB-12W。通过扩增子测序系统解析菌群结构与功能,结合苯胺蓝平板显色法筛选优势菌株,并通过形态学、生理生化及分子生物学手段进行多相分类鉴定。结果表明,从DA和TB土壤中共分离出165株初筛菌株,其中三株高效菌株在pH 9 ~ 11条件下保持高生长活性,分别表现出漆酶、木聚糖酶及纤维素酶活性优势,对水稻秸秆的降解率为11.8% ~ 16.2%。TB中菌株TB324A的初始相对丰度为0.49%,经12代连续富集后,其在TB-12菌群中的占比显著提升至2.19%,富集倍数达4.47倍。DA中的菌株524A展现出更强的富集潜力,其原始占比仅为0.02%,经同等代际培养后,在DA-12W菌群中占比激增至0.28%,实现14倍增幅,HY01菌株在相同培养条件下实现1.5倍增长(0.02%→0.03%)。“先富集-后筛选”策略可高效挖掘兼具耐盐碱与秸秆降解功能的微生物资源,筛选出的菌株具有良好的盐碱环境适应性及降解能力,为盐碱地微生物资源挖掘提供了技术范式。

     

    Abstract: To address the technical bottleneck of isolating low-abundance straw-degrading functional microorganisms in the soda saline-alkali soil of the Songnen Plain, this study adopted a "enrichment followed by screening" strategy. By simulating the dual selection pressure of salt-alkali stress and straw carbon source limitation, it broke through the limitation of the capture efficiency of salt-alkali-tolerant straw-degrading bacteria in traditional methods and screened salt-alkali-tolerant straw-degrading microorganisms. The study enriched the straw-degrading bacterial communities from the typical saline-alkali soil of the Songnen Plain, namely the Da'an rice field (DA) and the Taobei rice field (TB), and the target bacterial communities DA-12W and TB-12W after 12 rounds of serial dilution and amplification were obtained. The bacterial community structure and function were systematically analyzed through amplicon sequencing, and the dominant strains were screened by the aniline blue plate coloration method. The strains were then identified through morphological, physiological and biochemical, and molecular biological methods. A total of 165 initial screening strains were isolated from the DA and TB soils. Among them, three highly efficient strains maintain high growth activity under pH 9 - 11 with advantages in laccase, xylanase and cellulase activities. The degradation rate of rice straw by these three strains is 11.8% - 16.2%. The initial relative abundance of strain TB324A in TB soil is 0.49%; and after 12 generations of continuous enrichment, its proportion in the TB-12 community significantly increases to 2.19%, with an enrichment factor of 4.47 times. Strain 524A in DA exhibits stronger enrichment potential. Its original proportion is only 0.02%; and after the same number of generations of cultivation, its proportion in the DA-12W community increases sharply to 0.28%, achieving a 14-fold increase. Strain HY01 achieves a 1.5-fold increase (0.02% → 0.03%) under the same culture conditions. The "enrichment followed by screening" strategy can efficiently mine microbial resources with both salt-alkali tolerance and straw degradation functions. The screened strains have good adaptability to salt-alkali environments and degradation capabilities, providing a technical paradigm for the exploration of microbial resources in saline-alkali soils.

     

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