Single-cell analysis (SCA) has been increasingly recognized as the key technology for the elucidation of cellular functions, which are not accessible from bulk measurements on the population level. Thus far, SCA has been achieved by miniaturization of established engineering concepts to match the dimensions of a single cell. However, SCA requires procedures beyond the classical approach of upstream processing, fermentation, and downstream processing because the biological system itself defines the technical demands. This review characterizes currently available microfluidics and microreactors for invasive (i.e., chemical) and noninvasive (i.e., biological) SCA. We describe the recent SCA omics approaches as tools for systems biology and discuss the role of SCA in genomics, transcriptomics, proteomics, metabolomics, and fluxomics. Furthermore, we discuss applications of SCA for biocatalysis and metabolic engineering as well as its potential for bioprocess optimization. Finally, we define present and future challenges for SCA and propose strategies to overcome current limitations.

Microscale cultivation systems are important tools to elucidate cellular dynamics beyond the population average and understand the functional architecture of single cells. However, there is scant knowledge about the bias of different microcultivation technologies on cellular functions. We therefore performed a systematic cross-platform comparison of three different microscale cultivation systems commonly harnessed in single-cell analysis: microfluidic non-contact cell traps driven by negative dielectrophoresis, microfluidic monolayer growth chambers, and semi-solid agarose pads. We assessed the specific single-cell growth rates, division rates and morphological characteristics of single Corynebacterium glutamicum cells and microcolonies as a bacterial model organism with medical and biotechnological relevance under standardized growth conditions. Strikingly, the specific single-cell and microcolony growth rates, μmax, were robust and conserved for several cell generations with all three microcultivation technologies, whereas the division rates of cells grown on agarose pads deviated by up to 50% from those of cells cultivated in negative dielectrophoresis traps and monolayer growth chambers. Furthermore, morphological characteristics like cell lengths and division symmetries of individual cells were affected when the cells were grown on agarose pads. This indicated a significant impact of solid cultivation supports on cellular traits. The results demonstrate the impact of microcultivation technology on microbial physiology for the first time and show the need for a careful selection and design of the microcultivation technology in order to allow unbiased analysis of cellular behavior.

ABSTRACTSingularized cells ofPichia pastoris,Hansenula polymorpha, andCorynebacterium glutamicumdisplayed specific growth rates under chemically and physically constant conditions that were consistently higher than those obtained in populations. This highlights the importance of single-cell analyses by uncoupling physiology and the extracellular environment, which is now possible using the Envirostat 2.0 concept.

We present a lab-on-a-chip device, the Envirostat 2.0, which allows for the first time contactless cultivation of a single bacterial cell by negative dielectrophoresis (nDEP) in a precisely controllable microenvironment. Stable trapping in perfusing growth medium was achieved by a miniaturization of octupole electrode geometries, matching the dimensions of bacteria. Temperature sensitive fluorescent measurements showed that these reductions of microelectrode distances led to reduced Joule heating during cell manipulation. The presented miniaturization is not possible with conventional manufacturing processes. Therefore, we present a novel bonding technology, which permits miniaturization of 3D octupole electrode geometry with biocompatible materials. To exclude the influence of other cells and to enable sampling of perfusion medium from the isolated living bacterium under study, computer aided flow simulations were used to develop a microfluidic nDEP isolation procedure. The developed microchannel and microelectrode design integrates for the first time well characterized nDEP cell sorting mechanisms and time-resolved contactless single bacterial cell cultivation in a 1.7 picoliter bioreactor system. The cell type independent trapping is demonstrated with singularized Bacillus subtilis, Escherichia coli, Corynebacterium glutamicum and other industrially relevant microbes. The static and precisely controlled microenvironment resulted in a consistent and significant faster growth compared to maximal growth rates observed on population level. Preventing the influence of surfaces and cell-cell interactions, the Envirostat 2.0 chip permits total microenvironmental control by the experimenter and therefore provides major opportunities for microfluidic based cell analysis of bacteria and small eukaryotes.

Abstract:We have established a novel and scalable methodology for the productive coupling of redox enzymes to reductive electrochemical cofactor regeneration relying on efficient mass transfer of the cofactor to the electron-delivering cathode. Proof of concept is provided by styrene monooxygenase (StyA) catalyzing the asymmetric (S)-epoxidation of styrene with high enantiomeric excess, space-time yields, and current efficiencies. Highly porous reticulated vitreous carbon electrodes, maximized in volumetric surface area, were employed in a flowthrough mode to rapidly regenerate the consumed FADH 2 cofactor required for StyA activity. A systematic investigation of the parameters determining cofactor mass transfer revealed that low FAD concentrations and high flow rates enabled the continuous synthesis of the product (S)-styrene oxide at high rates, while at the same time the accumulation of the side-products acetophenone and phenylacetaldehyde was minimized. At 10 mm FAD and a flow rate of 150 mL min À1, an average space-time yield of 0.35 g L À1 h À1 could be achieved during 2 h with a final (S)-styrene oxide yield of 75.2%. At two-fold lower aeration rates, the electroenzymatic reaction could be sustained for 12 h, albeit at the expense of lower (59%) overall space-time yields. Under these conditions, as much as 20.5% of the utilized current could be channeled into (S)-styrene oxide formation. In comparison with state-of-the-art electroenzymatic methodologies for the same conversion, (S)-styrene oxide synthesis could be improved up to 150-fold with respect to both reaction time and space-time yield. These productivities constitute the most efficient reaction reported for asymmetric in vitro epoxidations of styrene.