Partipilo Gina, Bowman Emily K, Palmer Emma J, Gao Yang, Ridley Rodney S, Alper Hal S, Keitz Benjamin K
McKetta Department of Chemical Engineering, The University of Texas at Austin, Austin, Texas, USA.
Interdisciplinary Life Sciences Graduate Program, The University of Texas at Austin, Austin, Texas, USA.
Appl Environ Microbiol. 2025 Jan 31;91(1):e0246524. doi: 10.1128/aem.02465-24. Epub 2025 Jan 14.
Electroactive organisms contribute to metal cycling, pollutant removal, and other redox-driven environmental processes via extracellular electron transfer (EET). Unfortunately, developing genotype-phenotype relationships for electroactive organisms is challenging because EET is necessarily removed from the cell of origin. Microdroplet emulsions, which encapsulate individual cells in aqueous droplets, have been used to study a variety of extracellular phenotypes but have not been applied to investigate EET. Here, we describe the development of a microdroplet emulsion system to sort and enrich EET-capable organisms from complex populations. We validated our system using the model electrogen and described the tooling of a benchtop microfluidic system for oxygen-limited conditions. We demonstrated the enrichment of strains exhibiting electroactive phenotypes from mixed wild-type and EET-deficient populations. As a proof-of-concept application, we collected samples from iron sedimentation in Town Lake (Austin, TX) and subjected them to microdroplet enrichment. We measured an increase in electroactive organisms in the sorted population that was distinct compared to a population growing in bulk culture with Fe(III) as the sole electron acceptor. Finally, two bacterial species not previously shown to be EET-capable, and , were further cultured and characterized for electroactivity. Our results demonstrate the utility of microdroplet emulsions for isolating and identifying EET-capable bacteria.IMPORTANCEThis work outlines a new high-throughput method for identifying electroactive bacteria from mixed populations. Electroactive bacteria play key roles in iron trafficking, soil remediation, and pollutant degradation. Many existing methods for identifying electroactive bacteria are coupled to microbial growth and fitness-as a result, the contributions from weak or poor-growing electrogens are often muted. However, extracellular electron transfer (EET) has historically been difficult to study in high-throughput in a mixed population since extracellular reduction is challenging to trace back to the parent cell and there are no suitable fluorescent readouts for EET. Our method circumvents these challenges by utilizing an aqueous microdroplet emulsion wherein a single cell is statistically isolated in a pico- to nano-liter-sized droplet. Then, via fluorescence obtained from copper reduction, the mixed population can be fluorescently sorted and gated by performance. Utilizing our technique, we characterize two previously unrecognized weak electrogens and .
电活性生物通过细胞外电子转移（EET）促进金属循环、污染物去除及其他氧化还原驱动的环境过程。遗憾的是，建立电活性生物的基因型-表型关系具有挑战性，因为EET必然是从其起源细胞中脱离的。微滴乳液可将单个细胞包裹在水性液滴中，已被用于研究多种细胞外表型，但尚未应用于EET的研究。在此，我们描述了一种微滴乳液系统的开发，用于从复杂群体中筛选和富集具有EET能力的生物。我们使用模式产电菌验证了我们的系统，并描述了用于限氧条件的台式微流控系统的工具。我们展示了从野生型和EET缺陷型混合群体中富集表现出电活性表型的菌株。作为概念验证应用，我们从德克萨斯州奥斯汀市城镇湖的铁沉淀中采集样本，并对其进行微滴富集。我们测量了分选群体中电活性生物的增加，这与以Fe(III)作为唯一电子受体在批量培养中生长的群体明显不同。最后，对两种先前未显示具有EET能力的细菌物种进行了进一步培养和电活性表征。我们的结果证明了微滴乳液在分离和鉴定具有EET能力的细菌方面的实用性。
重要性
这项工作概述了一种从混合群体中鉴定电活性细菌的新的高通量方法。电活性细菌在铁运输、土壤修复和污染物降解中起关键作用。许多现有的鉴定电活性细菌的方法与微生物生长和适应性相关，因此，弱产电菌或生长不良的产电菌的贡献往往被掩盖。然而，由于细胞外还原难以追溯到亲代细胞且没有适用于EET的荧光读数，细胞外电子转移（EET）在混合群体中的高通量研究一直很困难。我们的方法通过利用水性微滴乳液规避了这些挑战，其中单个细胞在皮升至纳升大小的液滴中被统计性地分离。然后，通过铜还原获得的荧光，可以根据性能对混合群体进行荧光分选和门控。利用我们的技术，我们表征了两种先前未被识别的弱电活性菌。
