McCully Alexandra L, Loop Yao McKenna, Brower Kara K, Fordyce Polly M, Spormann Alfred M
Department of Civil and Environmental Engineering, Stanford University, Stanford, CA, USA.
Department of Chemical Engineering, Stanford University, Stanford, CA, USA.
ISME Commun. 2023 May 9;3(1):47. doi: 10.1038/s43705-023-00241-9.
Our understanding of in situ microbial physiology is primarily based on physiological characterization of fast-growing and readily-isolatable microbes. Microbial enrichments to obtain novel isolates with slower growth rates or physiologies adapted to low nutrient environments are plagued by intrinsic biases for fastest-growing species when using standard laboratory isolation protocols. New cultivation tools to minimize these biases and enrich for less well-studied taxa are needed. In this study, we developed a high-throughput bacterial enrichment platform based on single cell encapsulation and growth within double emulsions (GrowMiDE). We showed that GrowMiDE can cultivate many different microorganisms and enrich for underrepresented taxa that are never observed in traditional batch enrichments. For example, preventing dominance of the enrichment by fast-growing microbes due to nutrient privatization within the double emulsion droplets allowed cultivation of slower-growing Negativicutes and Methanobacteria from stool samples in rich media enrichment cultures. In competition experiments between growth rate and growth yield specialist strains, GrowMiDE enrichments prevented competition for shared nutrient pools and enriched for slower-growing but more efficient strains. Finally, we demonstrated the compatibility of GrowMiDE with commercial fluorescence-activated cell sorting (FACS) to obtain isolates from GrowMiDE enrichments. Together, GrowMiDE + DE-FACS is a promising new high-throughput enrichment platform that can be easily applied to diverse microbial enrichments or screens.
我们对原位微生物生理学的理解主要基于对快速生长且易于分离的微生物的生理学特征描述。当使用标准实验室分离方案时，通过微生物富集来获得生长速率较慢或适应低营养环境生理学特征的新分离株，会受到生长最快物种的内在偏差的困扰。因此需要新的培养工具来尽量减少这些偏差，并富集研究较少的分类群。在本研究中，我们基于单细胞封装和在双重乳液中生长开发了一个高通量细菌富集平台（GrowMiDE）。我们表明，GrowMiDE可以培养许多不同的微生物，并富集传统批次富集中从未观察到的代表性不足的分类群。例如，通过防止双重乳液液滴内营养物质私有化导致快速生长的微生物在富集中占主导地位，使得在丰富培养基富集培养中能够从粪便样本中培养出生长较慢的厌氧革兰氏阴性菌和甲烷杆菌。在生长速率和生长产量专家菌株之间的竞争实验中，GrowMiDE富集防止了对共享营养池的竞争，并富集了生长较慢但效率更高的菌株。最后，我们证明了GrowMiDE与商业荧光激活细胞分选（FACS）的兼容性，以从GrowMiDE富集中获得分离株。总之，GrowMiDE + DE-FACS是一个很有前景的新型高通量富集平台，可轻松应用于各种微生物富集或筛选。
