BACKGROUND Cell-based experimentation in microfluidic droplets is becoming increasingly popular among biotechnologists and microbiologists, since inherent characteristics of droplets allow high throughput at low cost and space investment. The range of applications for droplet assays is expanding from single cell analysis toward complex cell-cell incubation and interaction studies. As a result of cellular metabolism in these setups, relevant physicochemical alterations frequently occur before functional assays are conducted. However, to use droplets as truly miniaturized bioreactors, parameters like pH and oxygen availability should be controlled similar to large-scale fermentation to ensure reliable research. RESULTS Here, we introduce a comprehensive strategy to monitor and control pH for large droplet populations during long-term incubation. We show the correlation of fluorescence intensity of 6-carboxyfluorescein and pH in single droplets and entire droplet populations. By taking advantage of inter-droplet transport of pH-mediating molecules, the average pH value of several million droplets is simultaneously adjusted in an a priori defined direction. To demonstrate the need of pH control in practice, we compared the fermentation profiles of two E. coli strains, a K12-strain and a B-strain, in unbuffered medium with 5 g/L glucose for standard 1 L bioreactors and 180 pL droplets. In both fermentation formats, the commonly used B-strain E. coli BL21 is able to consume glucose until depletion and prevent a pH drop, while the growth of the K12-strain E. coli MG1655 is soon inhibited by a low pH caused by its own high acetate production. By regulating the pH during fermentation in droplets with our suggested strategy, we were able to prevent the growth arrest of E. coli MG1655 and obtained an equally high biomass yield as with E. coli BL21. CONCLUSION We demonstrated a comparable success of pH monitoring and regulation for fermentations in 1 L scale and 180 pL scale for two E. coli strains. This strategy has the potential to improve cell-based experiments for various microbial systems in microfluidic droplets and opens the possibility for new functional assay designs.

中文翻译：

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微生物培养过程中微流液滴pH的监测和外部控制。

背景技术由于微滴的固有特性允许以低成本和空间投资实现高通量，因此在微流体微滴中进行基于细胞的实验变得越来越受生物技术人员和微生物学家的欢迎。液滴测定的应用范围正在从单细胞分析扩展到复杂的细胞间孵育和相互作用研究。由于这些设置中的细胞代谢，在进行功能测定之前，经常发生相关的理化变化。但是，要将液滴用作真正的微型生物反应器，应像大规模发酵一样控制pH和氧气利用率等参数，以确保可靠的研究。结果在这里，我们介绍了一种在长期孵育过程中监测和控制大液滴种群pH的综合策略。我们显示了单个液滴和整个液滴种群中6-羧基荧光素的荧光强度与pH的相关性。通过利用pH介导分子的小滴间转运，可以在事先定义的方向上同时调节数百万个小滴的平均pH值。为了证明在实践中需要控制pH，我们比较了两种大肠杆菌菌株，即K12菌株和B菌株在5 g / L葡萄糖的非缓冲培养基中对于标准1 L生物反应器和180 pL液滴的发酵曲线。 。在这两种发酵形式中，常用的B株大肠杆菌BL21能够消耗葡萄糖直至耗尽并防止pH下降，而K12株MG1655的生长很快就被其细菌引起的低pH抑制了。自己的醋酸盐产量高。通过使用我们建议的策略调节液滴在发酵过程中的pH，我们能够防止大肠杆菌MG1655的生长停滞，并获得了与大肠杆菌BL21相同的高生物量产量。结论我们证明了对两种大肠杆菌菌株在1 L规模和180 pL规模发酵中pH监测和调节的可比成功。该策略具有改进微流液滴中各种微生物系统基于细胞的实验的潜力，并为新的功能分析设计提供了可能性。