Table_1_A Simple Culture Method Enhances the Recovery of Culturable Actinobacteria From Coastal Sediments.XLSX

Molecular methods revealed that the majority of microbes in natural environments remains uncultivated. To fully understand the physiological and metabolic characteristics of microbes, however, culturing is still critical for microbial studies. Here, we used bacterial community analysis and four culture media, namely, traditional marine broth 2216 (MB), water extracted matter (WEM), methanol extracted matter (MEM), and starch casein agar (SCA), to investigate the diversity of cultivated bacteria in coastal sediments. A total of 1,036 isolates were obtained in pure culture, and they were classified into five groups, namely, Alphaproteobacteria (52.51%), Gammaproteobacteria (23.26%), Actinobacteria (13.32%), Firmicutes, and Bacteroidetes. Compared to other three media, WEM recovered a high diversity of actinobacteria (42 of 63 genotypes), with Micromonospora and Streptomyces as the most cultivated genera. Amplicon sequencing of the bacterial 16S ribosomal RNA (rRNA) gene V3–V4 fragment revealed eight dominant groups, Alphaproteobacteria (12.81%), Gammaproteobacteria (20.07%), Deltaproteobacteria (12.95%), Chloroflexi (13.09%), Bacteroidetes (8.28%), Actinobacteria (7.34%), Cyanobacteria (6.20%), and Acidobacteria (5.71%). The dominant members affiliated to Actinobacteria belonged to “Candidatus Actinomarinales,” “Candidatus Microtrichales,” and Nitriliruptorales. The cultivated actinobacteria accounted for a small proportion (<5%) compared to the actinobacterial community, which supported that the majority of actinobacteria are still waiting for cultivation. Our study concluded that WEM could be a useful and simple culture medium that enhanced the recovery of culturable actinobacteria from coastal sediments.

分子生物学方法已证实，自然环境中的绝大多数微生物仍未实现人工培养。然而，若要全面解析微生物的生理与代谢特征，纯培养技术仍是微生物学研究的关键手段。本研究依托细菌群落分析方法，结合四种培养基——即传统海洋肉汤2216（marine broth 2216, MB）、水提取物培养基（water extracted matter, WEM）、甲醇提取物培养基（methanol extracted matter, MEM）以及淀粉酪素琼脂（starch casein agar, SCA）——对沿海沉积物中可培养细菌的多样性展开探究。本研究共获得1036株纯培养菌株，可划分为5个类群：α-变形菌纲（Alphaproteobacteria，占比52.51%）、γ-变形菌纲（Gammaproteobacteria，占比23.26%）、放线菌门（Actinobacteria，占比13.32%）、厚壁菌门（Firmicutes）以及拟杆菌门（Bacteroidetes）。相较于其余三种培养基，水提取物培养基（WEM）可分离得到多样性更高的放线菌（63个基因型中的42个），其中小单孢菌属（Micromonospora）与链霉菌属（Streptomyces）为最易培养的优势菌属。通过对细菌16S核糖体RNA（ribosomal RNA, rRNA）基因V3-V4可变区进行扩增子测序，本研究共鉴定出8个优势类群：α-变形菌纲（12.81%）、γ-变形菌纲（20.07%）、δ-变形菌纲（Deltaproteobacteria，12.95%）、绿弯菌门（Chloroflexi，13.09%）、拟杆菌门（8.28%）、放线菌门（7.34%）、蓝细菌门（Cyanobacteria，6.20%）以及酸杆菌门（Acidobacteria，5.71%）。隶属于放线菌门的优势类群包括候选放线菌目（Candidatus Actinomarinales）、候选微毛菌目（Candidatus Microtrichales）以及硝化嗜热菌目（Nitriliruptorales）。相较于沉积物中原生放线菌群落，本研究获得的可培养放线菌占比极低（<5%），这进一步证实绝大多数放线菌仍未实现人工培养。本研究结果表明，水提取物培养基（WEM）是一种简便高效的培养基，可有效提升沿海沉积物中可培养放线菌的分离效率。