Microbial single-cell mass spectrometry: status, challenges, and prospects.

Single-cell analysis uncovers phenotypic differences between cells in a population and dissects their individual physiological states and differences on all omics levels from genome to phenome. Spectrometric observation allows label-free analysis of the metabolome and proteome of individual cells, but is still mainly limited to the analysis of mammalian single cells. Recent progress in mass spectrometry approaches now enables the analysis of microbial single cells - mainly by miniaturizing cell handling, incubation, and improving chip-coupling concepts for analyte ionization by interfacing microfluidic chips and mass spectrometers. This review aims at distilling the enabling principles behind microbial single-cell mass spectrometry and puts them into perspective for the future of the field.

Assessing the growth kinetics and stoichiometry of Escherichia coli at the single-cell level.

Microfluidic cultivation and single-cell analysis are inherent parts of modern microbial biotechnology and microbiology. However, implementing biochemical engineering principles based on the kinetics and stoichiometry of growth in microscopic spaces remained unattained. We here present a novel integrated framework that utilizes distinct microfluidic cultivation technologies and single-cell analytics to make the fundamental math of process-oriented biochemical engineering applicable at the single-cell level. A combination of non-invasive optical cell mass determination with sub-pg sensitivity, microfluidic perfusion cultivations for establishing physiological steady-states, and picoliter batch reactors, enabled the quantification of all physiological parameters relevant to approximate a material balance in microfluidic reaction environments. We determined state variables (biomass concentration based on single-cell dry weight and mass density), biomass synthesis rates, and substrate affinities of cells grown in microfluidic environments. Based on this data, we mathematically derived the specific kinetics of substrate uptake and growth stoichiometry in glucose-grown Escherichia coli with single-cell resolution. This framework may initiate microscale material balancing beyond the averaged values obtained from populations as a basis for integrating heterogeneous kinetic and stoichiometric single-cell data into generalized bioprocess models and descriptions.

Controlling microbial co-culture based on substrate pulsing can lead to stability through differential fitness advantages.

Microbial consortia are an exciting alternative for increasing the performances of bioprocesses for the production of complex metabolic products. However, the functional properties of microbial communities remain challenging to control, considering the complex interaction mechanisms occurring between co-cultured microbial species. Indeed, microbial communities are highly dynamic and can adapt to changing environmental conditions through complex mechanisms, such as phenotypic diversification. We focused on stabilizing a co-culture of Saccharomyces cerevisiae and Escherichia coli in continuous cultures. Our preliminary data pointed out that transient diauxic shifts could lead to stable co-culture by providing periodic fitness advantages to the yeast. Based on a computational toolbox called MONCKS (for MONod-type Co-culture Kinetic Simulation), we were able to predict the dynamics of diauxic shift for both species based on a cybernetic approach. This toolbox was further used to predict the frequency of diauxic shift to be applied to reach co-culture stability. These simulations were successfully reproduced experimentally in continuous bioreactors with glucose pulsing. Finally, based on a bet-hedging reporter, we observed that the yeast population exhibited an increased phenotypic diversification process in co-culture compared with mono-culture, suggesting that this mechanism could be the basis of the metabolic fitness of the yeast.

pH Distribution along Growing Fungal Hyphae at Microscale.

Creating unique microenvironments, hyphal surfaces and their surroundings allow for spatially distinct microbial interactions and functions at the microscale. Using a microfluidic system and pH-sensitive whole-cell bioreporters (Synechocystis sp. PCC6803) attached to hyphae, we spatially resolved the pH along surfaces of growing hyphae of the basidiomycete Coprinopsis cinerea. Time-lapse microscopy analysis of ratiometric fluorescence signals of >2400 individual bioreporters revealed an overall pH drop from 6.3 ± 0.4 (n = 2441) to 5.0 ± 0.3 (n = 2497) within 7 h after pH bioreporter loading to hyphal surfaces. The pH along hyphal surfaces varied significantly (p < 0.05), with pH at hyphal tips being on average ~0.8 pH units lower than at more mature hyphal parts near the entrance of the microfluidic observation chamber. Our data represent the first dynamic in vitro analysis of surface pH along growing hyphae at the micrometre scale. Such knowledge may improve our understanding of spatial, pH-dependent hyphal processes, such as the degradation of organic matter or mineral weathering.

Impact of Fungal Hyphae on Growth and Dispersal of Obligate Anaerobic Bacteria in Aerated Habitats.

Anoxic microsites arising in fungal biofilms may foster the presence of obligate anaerobes. Here, we analyzed whether and to which degree hyphae of Coprinopsis cinerea thriving in oxic habitats enable the germination, growth, and dispersal of the obligate anaerobic soil bacterium Clostridium acetobutylicum. Time-resolved optical oxygen mapping, microscopy, and metabolite analysis revealed the formation and persistence of anoxic circum hyphal niches, allowing for spore germination, growth, and fermentative activity of the obligate anaerobe in an otherwise inhabitable environment. Hypoxic liquid films containing 80% ± 10% of atmospheric oxygen saturation around single air-exposed hyphae thereby allowed for efficient clostridial dispersal amid spatially separated (>0.5 cm) anoxic sites. Hyphae hence may serve as good networks for the activity and spatial organization of obligate anaerobic bacteria in oxygenated heterogeneous environments such as soil. IMPORTANCE Although a few studies have reported on the presence of anoxic microniches in fungal biofilms, knowledge of the effects of fungal oxygen consumption on bacterial-fungal interactions is limited. Here, we demonstrate the existence and persistence of oxygen-free zones in air-exposed mycelia enabling spore germination, growth, fermentative activity, and dispersal of the obligate anaerobe. Our study points out a previously overlooked role of aerobic fungi in creating and bridging anoxic microniches in ambient oxic habitats. Air-exposed hyphae hence may act as a scaffold for activity and dispersal of strictly anaerobic microbes. Given the short-term tolerance of strict anaerobes to oxygen and reduced oxygen content in the mycosphere, hyphae can promote spatial organization of both obligate anaerobic and aerobic bacteria. Such finding may be important for a better understanding of previously observed co-occurrences of aerobes and anaerobes in well-aerated habitats such as upland soils.

Quantification of Biocatalytic Transformations by Single Microbial Cells Enabled by Tailored Integration of Droplet Microfluidics and Mass Spectrometry.

Improving the performance of chemical transformations catalysed by microbial biocatalysts requires a deep understanding of cellular processes. While the cellular heterogeneity of cellular characteristics, such as the concentration of high abundant cellular content, is well studied, little is known about the reactivity of individual cells and its impact on the chemical identity, quantity, and purity of excreted products. Biocatalytic transformations were monitored chemically specific and quantifiable at the single-cell level by integrating droplet microfluidics, cell imaging, and mass spectrometry. Product formation rates for individual Saccharomyces cerevisiae cells were obtained by i) incubating nanolitre-sized droplets for product accumulation in microfluidic devices, ii) an imaging setup to determine the number of cells in the droplets, and iii) electrospray ionisation mass spectrometry for reading the chemical contents of individual droplets. These findings now enable the study of whole-cell biocatalysis at single-cell resolution.

Fluorescence lifetime activated droplet sorting (FLADS) for label-free sorting of Synechocystis sp. PCC6803.

This study presents the label-free sorting of cyanobacterial cells in droplets with single-cell sensitivity based on their fluorescence lifetime. We separated living and dead cyanobacteria (Synechocystis sp. PCC6803) using fluorescence lifetime signals of the photopigment autofluorescence to indicate their photosynthetic activity. We developed a setup and a chip design to achieve live/dead sorting accuracies of more than 97% at a droplet frequency of 100 Hz with a PDMS-based chip system and standard optics using fluorescence lifetime as the sorting criterion. The obtained sorting accuracies could be experimentally confirmed by cell plating and observing the droplet sorting process via a high-speed camera. The herein presented results demonstrate the capabilities of the developed system for studying the effects of stressors on cyanobacterial physiology and the subsequent deterministic sorting of different stress-response phenotypes. This technology eliminates the need for tedious staining of cyanobacterial cells, which makes it particularly attractive for its application in the field of phototrophic microbial bio(techno)logic and in the context of cell secretion studies.

Microfluidic Single-Cell Analytics.

What is the impact of cellular heterogeneity on process performance? How do individual cells contribute to averaged process productivity? Single-cell analysis is a key technology for answering such key questions of biotechnology, beyond bulky measurements with populations. The analysis of cellular individuality, its origins, and the dependency of process performance on cellular heterogeneity has tremendous potential for optimizing biotechnological processes in terms of metabolic, reaction, and process engineering. Microfluidics offer unmatched environmental control of the cellular environment and allow massively parallelized cultivation of single cells. However, the analytical accessibility to a cell's physiology is of crucial importance for obtaining the desired information on the single-cell production phenotype. Highly sensitive analytics are required to detect and quantify the minute amounts of target analytes and small physiological changes in a single cell. For their application to biotechnological questions, single-cell analytics must evolve toward the measurement of kinetics and specific rates of the smallest catalytic unit, the single cell. In this chapter, we focus on an introduction to the latest single-cell analytics and their application for obtaining physiological parameters in a biotechnological context from single cells. We present and discuss recent advancements in single-cell analytics that enable the analysis of cell-specific growth, uptake, and production kinetics, as well as the gene expression and regulatory mechanisms at a single-cell level.

The Metabolic Flux Probe (MFP)-Secreted Protein as a Non-Disruptive Information Carrier for 13C-Based Metabolic Flux Analysis.

Novel cultivation technologies demand the adaptation of existing analytical concepts. Metabolic flux analysis (MFA) requires stable-isotope labeling of biomass-bound protein as the primary information source. Obtaining the required protein in cultivation set-ups where biomass is inaccessible due to low cell densities and cell immobilization is difficult to date. We developed a non-disruptive analytical concept for 13C-based metabolic flux analysis based on secreted protein as an information carrier for isotope mapping in the protein-bound amino acids. This "metabolic flux probe" (MFP) concept was investigated in different cultivation set-ups with a recombinant, protein-secreting yeast strain. The obtained results grant insight into intracellular protein turnover dynamics. Experiments under metabolic but isotopically nonstationary conditions in continuous glucose-limited chemostats at high dilution rates demonstrated faster incorporation of isotope information from labeled glucose into the recombinant reporter protein than in biomass-bound protein. Our results suggest that the reporter protein was polymerized from intracellular amino acid pools with higher turnover rates than biomass-bound protein. The latter aspect might be vital for 13C-flux analyses under isotopically nonstationary conditions for analyzing fast metabolic dynamics.

Microfluidic device for concentration and SERS-based detection of bacteria in drinking water.

There is a constant need for the development of easy-to-operate systems for the rapid and unambiguous identification of bacterial pathogens in drinking water without the requirement for time-consuming culture processes. In this study, we present a disposable and low-cost lab-on-a-chip device utilizing a nanoporous membrane, which connects two stacked perpendicular microfluidic channels. Whereas one of the channels supplies the sample, the second one attracts it by potential-driven forces. Surface-enhanced Raman spectrometry (SERS) is employed as a reliable detection method for bacteria identification. To gain the effect of surface enhancement, silver nanoparticles were added to the sample. The pores of the membrane act as a filter trapping the bodies of microorganisms as well as clusters of nanoparticles creating suitable conditions for sensitive SERS detection. Therein, we focused on the construction and characterization of the device performance. To demonstrate the functionality of the microfluidic chip, we analyzed common pathogens (Escherichia coli DH5α and Pseudomonas taiwanensis VLB120) from spiked tap water using the optimized experimental parameters. The obtained results confirmed our system to be promising for the construction of a disposable optical platform for reliable and rapid pathogen detection which couples their electrokinetic concentration on the integrated nanoporous membrane with SERS detection.

Illuminate the hidden: in vivo mapping of microscale pH in the mycosphere using a novel whole-cell biosensor.

The pH of an environment is both a driver and the result of diversity and functioning of microbial habitats such as the area affected by fungal hyphae (mycosphere). Here we used a novel pH-sensitive bioreporter, Synechocystis sp. PCC6803_peripHlu, and ratiometric fluorescence microscopy, to spatially and temporally resolve the mycosphere pH at the micrometre scale. Hyphae of the basidiomycete Coprionopsis cinerea were allowed to overgrow immobilised and homogeneously embedded pH bioreporters in an agarose microcosm. Signals of >700 individual cells in an area of 0.4 × 0.8 mm were observed over time and used to create highly resolved (3 × 3 µm) pH maps using geostatistical approaches. C. cinerea changed the pH of the agarose from 6.9 to ca. 5.0 after 48 h with hyphal tips modifying pH in their vicinity up to 1.8 mm. pH mapping revealed distinct microscale spatial variability and temporally stable gradients between pH 4.4 and 5.8 over distances of ≈20 µm. This is the first in vivo mapping of a mycosphere pH landscape at the microscale. It underpins the previously hypothesised establishment of pH gradients serving to create spatially distinct mycosphere reaction zones.

Conversion Efficiencies of a Few Living Microbial Cells Detected at a High Throughput by Droplet-Based ESI-MS.

The label-free and sensitive detection of synthesis products from single microbial cells remains the bottleneck for determining the specific turnover numbers of individual whole-cell biocatalysts. We demonstrate the detection of lysine synthesized by only a few living cells in microfluidic droplets via mass spectrometry. Biocatalyst turnover numbers were analyzed using rationally designed reaction environments compatible with mass spectrometry, which were decoupled from cell growth and showed high specific turnover rates (∼1 fmol/(cell h)), high conversion yields (25%), and long-term catalyst stability (>14h). The heterogeneity of the cellular reactivity of only 15 ± 5 single biocatalysts per droplet could be demonstrated for the first time by parallelizing the droplet incubation. These results enable the resolution of biocatalysis beyond averages of populations. This is a key step toward quantifying specific reactivities of single cells as minimal functional catalytic units.

Microfluidic single-cell analysis in biotechnology: from monitoring towards understanding.

Our understanding of the microbial cell is based on averaged values from bulks. Microfluidic single-cell analysis holds the promise of understanding cellular processes from a single cell perspective. But what is needed to measure single-cell physiology and to disclose the consequences of individuality for biotechnology? Current single-cell research is not yet able to provide all the necessary insights, but innovative approaches now emerge that propel the field towards a better understanding of cellular processes via quantitative physiology. Here, we critically review novel single-cell technologies that enable us to control cellular input parameters such as environmental conditions and to measure intracellular processes, as well as novel approaches that enable for the first time to quantify non-averaged cell-specific rates and yields. Finally, we demonstrate how integrating microfluidic single-cell analysis into established population-based experimental workflows might unlock its full potential for biotechnology research in the future.

Quantifying a Biocatalytic Product from a Few Living Microbial Cells Using Microfluidic Cultivation Coupled to FT-ICR-MS.

The in vivo quantification of metabolic products from microbial single cells is one of the last grand challenges in (bio)analytical chemistry. To date, no label-free analytical concept exists that is powerful enough to detect or even quantify the minute amounts of secreted low molecular weight compounds produced by living and isolated single bacteria or yeast cells. Coupling microfluidic cultivation systems with ultrahigh resolution electrospray-ionization mass spectrometry with its exquisite sensitivity and specificity offers the prospect of single-cell product analysis and quantification, but has not been successfully implemented yet. We report an analytical framework that interfaces noninvasive microfluidic trapping and cultivation of a few bacterial single cells with the analysis of their catalytic products by Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). Cell trapping was performed with the microfluidic Envirostat platform for cultivating bacterial cells under continuous perfusion via negative dielectrophoresis (nDEP). A total of 1.5 µL of product-containing cell supernatant was sampled into microcapillaries using a dead volume-reduced world-to-chip interface. The samples were analyzed with a nanoESI ion source coupled to a FT-ICR-MS (limit of detection for lysine: 0.5 pg). As a biocatalytic model system, we analyzed few Corynebacterium glutamicum DM 1919 pSenLys cells that synthesized l-lysine from d-glucose. Secreted lysine was quantified from a few cells (down to 19). Single-cell specific lysine productivities were 2 and 10 fmol/cell/h. This demonstrates that coupling microfluidics and mass spectrometry (SIC-MS) now enables the quantification of catalytic products and extracellular metabolites from only a few living microbial cells.

l-Arabinose triggers its own uptake via induction of the arabinose-specific Gal2p transporter in an industrial Saccharomyces cerevisiae strain.

Bioethanol production processes with Saccharomyces cerevisiae using lignocellulosic biomass as feedstock are challenged by the simultaneous utilization of pentose and hexose sugars from biomass hydrolysates. The pentose uptake into the cell represents a crucial role for the efficiency of the process. The focus of the here presented study was to understand the uptake and conversion of the pentose l-arabinose in S. cerevisiae and reveal its regulation by d-glucose and d-galactose. Gal2p-the most prominent transporter enabling l-arabinose uptake in S. cerevisiae wild-type strains-has an affinity for the transport of l-arabinose, d-glucose, and d-galactose. d-Galactose was reported for being mandatory for inducing GAL2 expression. GAL2 expression is also known to be regulated by d-glucose-mediated carbon catabolite repression, as well as catabolite inactivation. The results of the present study demonstrate that l-arabinose can be used as sole carbon and energy source by the recombinant industrial strain S. cerevisiae DS61180. RT-qPCR and RNA-Seq experiments confirmed that l-arabinose can trigger its own uptake via the induction of GAL2 expression. Expression levels of GAL2 during growth on l-arabinose reached up to 21% of those obtained with d-galactose as sole carbon and energy source. l-Arabinose-induced GAL2 expression was also subject to catabolite repression by d-glucose. Kinetic investigations of substrate uptake, biomass, and product formation during growth on a mixture of d-glucose/l-arabinose revealed impairment of growth and ethanol production from l-arabinose upon d-glucose depletion. The presence of d-glucose is thus preventing the fermentation of l-arabinose in S. cerevisiae DS61180. Comparative transcriptome studies including the wild-type and a precursor strain delivered hints for an increased demand in ATP production and cofactor regeneration during growth of S. cerevisiae DS61180 on l-arabinose. Our results thus emphasize that cofactor and energy metabolism demand attention if the combined conversion of hexose and pentose sugars is intended, for example in biorefineries using lignocellulosics.

Beyond the bulk: disclosing the life of single microbial cells.

Microbial single cell analysis has led to discoveries that are beyond what can be resolved with population-based studies. It provides a pristine view of the mechanisms that organize cellular physiology, unbiased by population heterogeneity or uncontrollable environmental impacts. A holistic description of cellular functions at the single cell level requires analytical concepts beyond the miniaturization of existing technologies, defined but uncontrolled by the biological system itself. This review provides an overview of the latest advances in single cell technologies and demonstrates their potential. Opportunities and limitations of single cell microbiology are discussed using selected application-related examples.

Taking control over microbial populations: Current approaches for exploiting biological noise in bioprocesses.

Phenotypic plasticity of microbial cells has attracted much attention and several research efforts have been dedicated to the description of methods aiming at characterizing phenotypic heterogeneity and its impact on microbial populations. However, different approaches have also been suggested in order to take benefit from noise in a bioprocess perspective, e.g. by increasing the robustness or productivity of a microbial population. This review is dedicated to outline these controlling methods. A common issue, that has still to be addressed, is the experimental identification and the mathematical expression of noise. Indeed, the effective interfacing of microbial physiology with external parameters that can be used for controlling physiology depends on the acquisition of reliable signals. Latest technologies, like single cell microfluidics and advanced flow cytometric approaches, enable linking physiology, noise, heterogeneity in productive microbes with environmental cues and hence allow correctly mapping and predicting biological behavior via mathematical representations. However, like in the field of electronics, signals are perpetually subjected to noise. If appropriately interpreted, this noise can give an additional insight into the behavior of the individual cells within a microbial population of interest. This review focuses on recent progress made at describing, treating and exploiting biological noise in the context of microbial populations used in various bioprocess applications.

The MOX promoter in Hansenula polymorpha is ultrasensitive to glucose-mediated carbon catabolite repression.

Redesigning biology towards specific purposes requires a functional understanding of genetic circuits. We present a quantitative in-depth study on the regulation of the methanol-specific MOX promoter system (PMOX) at the single-cell level. We investigated PMOX regulation in the methylotrophic yeast Hansenula (Ogataea) polymorpha with respect to glucose-mediated carbon catabolite repression. This promoter system is particularly delicate as the glucose as carbon and energy source in turn represses MOX promoter activity. Decoupling single cells from population activity revealed a hitherto underrated ultrasensitivity of the MOX promoter to glucose repression. Environmental control with single-cell technologies enabled quantitative insights into the balance between activation and repression of PMOX with respect to extracellular glucose concentrations. While population-based studies suggested full MOX promoter derepression at extracellular glucose concentrations of ∼1 g L(-1), we showed that glucose-mediated catabolite repression already occurs at concentrations as low as 5 × 10(-4) g L(-1) These findings demonstrate the importance of uncoupling single cells from populations for understanding the mechanisms of promoter regulation in a quantitative manner.

Technical bias of microcultivation environments on single-cell physiology.

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.