Display omitted
•Microfluidics have become a powerful tool for studying single-cell behavior.•Single-cell analysis revealed insights into phenotypic heterogeneity.•Microfluidic single-cell analysis ...is shifting from monitoring towards (quantitative) understanding of cellular mechanisms.•Integrating single-cell tools into biotechnology workflows discloses the impact of single-cell physiology on bioprocesses.
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.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Developing cost-efficient biotechnological processes is a major challenge in replacing fossil-based industrial production processes. The remarkable progress in genetic engineering ensures efficient ...and fast tailoring of microbial metabolism for a wide range of bioconversions. However, improving intrinsic properties such as tolerance, handling, growth, and substrate consumption rates is still challenging. At the same time, synthetic biology tools are becoming easier applicable and transferable to nonmodel organisms. These trends have resulted in the exploitation of new and unconventional microbial systems with sophisticated properties, which render them promising hosts for the bio-based industry. Here, we highlight the metabolic and cellular capabilities of representative prokaryotic newcomers and discuss the potential and drawbacks of these hosts for industrial application.
Changing economic and environmental constraints call for the exploitation of novel microbial systems in industrial biotechnology.Multi-factorial traits like tolerance, growth efficiency, or handling cannot always be engineered into existing model organisms.Microbes with superior intrinsic properties compared to conventional employed cell factories are a more promising route to master future challenges in the bioeconomy.We present key criteria that microbial newcomers have to meet in order to achieve industrial relevance.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Microbial mixed cultures are gaining increasing attention as biotechnological production systems, since they offer a large but untapped potential for future bioprocesses. Effects of secondary ...metabolite induction and advantages of labor division for the degradation of complex substrates offer new possibilities for process intensification. However, mixed cultures are highly complex, and, consequently, many biotic and abiotic parameters are required to be identified, characterized, and ideally controlled to establish a stable bioprocess. In this review, we discuss the advantages and disadvantages of existing measurement techniques for identifying, characterizing, monitoring, and controlling mixed cultures and highlight promising examples. Moreover, existing challenges and emerging technologies are discussed, which lay the foundation for novel analytical workflows to monitor mixed-culture bioprocesses.
Next-generation sequencing and microfluidic analysis can identify and characterize the complex native microbial community in a sample, with little, if any, selection of artifacts.Optical measurement methods are currently the most promising fully scalable techniques for online monitoring and control of mixed-culture processes.The potential of infrared techniques, Raman spectroscopy, and mass spectrometry currently appear to be underestimated and underused for all aspects of the identification, characterization, monitoring, and control of mixed and co-culture processes.
Full text
Available for:
GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Microfluidic single‐cell cultivation (MSCC) is an emerging field within fundamental as well as applied biology. During the last years, most MSCCs were performed at constant environmental conditions. ...Recently, MSCC at oscillating and dynamic environmental conditions has started to gain significant interest in the research community for the investigation of cellular behavior. Herein, an overview of this topic is given and microfluidic concepts that enable oscillating and dynamic control of environmental conditions with a focus on medium conditions are discussed, and their application in single‐cell research for the cultivation of both mammalian and microbial cell systems is demonstrated. Furthermore, perspectives for performing MSCC at complex dynamic environmental profiles of single parameters and multiparameters (e.g., pH and O2) in amplitude and time are discussed. The technical progress in this field provides completely new experimental approaches and lays the foundation for systematic analysis of cellular metabolism at fluctuating environments.
Microfluidic single‐cell cultivation (MSCC) at oscillating and dynamic environmental conditions is of great interest in the research community for the investigation of biologically relevant issues. Here, an overview of this topic is given, and MSCC setups for the realization of dynamic environments and their application in single‐cell research are discussed. Finally, further perspectives of performing MSCC under complex dynamic environmental conditions are discussed.
Full text
Available for:
BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The control of root-feeding wireworms has become more challenging as synthetic soil insecticides have been progressively phased out due to environmental risk concerns. Innovative microbial control ...alternatives such as the so-called attract-and-kill strategy depend on the rapid and successful development of dried encapsulated microorganisms, which is initiated by rehydration. Casein is a functional additive that is already used in food or pharmaceutical industry due to its water binding capacity. Cross-linked forms such as formalin-casein (FC), exhibit altered network structures. To determine whether FC influences the rehydration of alginate beads in order to increase the efficacy of an attract-and-kill formulation for wireworm pest control, we incorporated either casein or FC in different alginate/starch formulations. We investigated the porous properties of alginate/starch beads and subsequently evaluated the activities of the encapsulated entomopathogenic fungus
Metarhizium brunneum
and the CO
2
producing yeast
Saccharomyces cerevisiae
. Adding caseins altered the porous structure of beads. FC decreased the bead density from (1.0197 ± 0.0008) g/mL to (1.0144 ± 0.0008) g/mL and the pore diameter by 31%. In contrast to casein, FC enhanced the water absorbency of alginate/starch beads by 40%. Furthermore, incorporating FC quadrupled the spore density on beads containing
M. brunneum
and
S. cerevisiae
, and simultaneous venting increased the spore density even by a factor of 18. Moreover, FC increased the total CO
2
produced by
M. brunneum
and
S. cerevisiae
by 29%. Thus, our findings suggest that rehydration is enhanced by larger capillaries, resulting in an increased water absorption capacity. Our data further suggest that gas exchange is improved by FC. Therefore, our results indicate that FC enhances the fungal activity of both fungi
M. brunneum
and
S. cerevisiae
, presumably leading to an enhanced attract-and-kill efficacy for pest control.
Graphic abstract
Full text
Available for:
CEKLJ, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Microfluidics and novel lab-on-a-chip applications have the potential to boost biotechnological research in ways that are not possible using traditional methods. Although microfluidic tools were ...increasingly used for different applications within biotechnology in recent years, a systematic and routine use in academic and industrial labs is still not established. For many years, absent innovative, ground-breaking and “out-of-the-box” applications have been made responsible for the missing drive to integrate microfluidic technologies into fundamental and applied biotechnological research. In this review, we highlight microfluidics’ offers and compare them to the most important demands of the biotechnologists. Furthermore, a detailed analysis in the state-of-the-art use of microfluidics within biotechnology was conducted exemplarily for four emerging biotechnological fields that can substantially benefit from the application of microfluidic systems, namely the phenotypic screening of cells, the analysis of microbial population heterogeneity, organ-on-a-chip approaches and the characterisation of synthetic co-cultures. The analysis resulted in a discussion of potential “gaps” that can be responsible for the rare integration of microfluidics into biotechnological studies. Our analysis revealed six major gaps, concerning the lack of interdisciplinary communication, mutual knowledge and motivation, methodological compatibility, technological readiness and missing commercialisation, which need to be bridged in the future. We conclude that connecting microfluidics and biotechnology is not an impossible challenge and made seven suggestions to bridge the gaps between those disciplines. This lays the foundation for routine integration of microfluidic systems into biotechnology research procedures.
Interspecies interactions inside microbial communities bear a tremendous diversity of complex chemical processes that are by far not understood. Even for simplified, often synthetic systems, the ...interactions between two microbes are barely revealed in detail. Here, we present a microfluidic co-cultivation platform for the analysis of growth and interactions inside microbial consortia with single-cell resolution. Our device allows the spatial separation of two different microbial organisms inside adjacent microchambers facilitating sufficient exchange of metabolites via connecting nanochannels. Inside the cultivation chambers cell growth can be observed with high spatio-temporal resolution by live-cell imaging. In contrast to conventional approaches, in which single-cell activity is typically fully masked by the average bulk behavior, the small dimensions of the microfluidic cultivation chambers enable accurate environmental control and observation of cellular interactions with full spatio-temporal resolution. Our method enables one to study phenomena in microbial interactions, such as gene transfer or metabolic cross-feeding. We chose two different microbial model systems to demonstrate the wide applicability of the technology. First, we investigated commensalistic interactions between an industrially relevant l-lysine-producing Corynebacterium glutamicum strain and an l-lysine auxotrophic variant of the same species. Spatially separated co-cultivation of both strains resulted in growth of the auxotrophic strain due to secreted l-lysine supplied by the producer strain. As a second example we investigated bacterial conjugation between Escherichia coli S17-1 and Pseudomonas putida KT2440 cells. We could show that direct cell contact is essential for the successful gene transfer via conjugation and was hindered when cells were spatially separated. The presented device lays the foundation for further studies on contactless and contact-based interactions of natural and synthetic microbial communities.
Abstract
Cell factories converting bio-based precursors to chemicals present an attractive avenue to a sustainable economy, yet screening of genetically diverse strain libraries to identify the ...best-performing whole-cell biocatalysts is a low-throughput endeavor. For this reason, transcriptional biosensors attract attention as they allow the screening of vast libraries when used in combination with fluorescence-activated cell sorting (FACS). However, broad ligand specificity of transcriptional regulators (TRs) often prohibits the development of such ultra-high-throughput screens. Here, we solve the structure of the TR LysG of
Corynebacterium glutamicum
, which detects all three basic amino acids. Based on this information, we follow a semi-rational engineering approach using a FACS-based screening/counterscreening strategy to generate an
l
-lysine insensitive LysG-based biosensor. This biosensor can be used to isolate
l
-histidine-producing strains by FACS, showing that TR engineering towards a more focused ligand spectrum can expand the scope of application of such metabolite sensors.
A series of coarse-grained models, with different levels of structural resolution, were tested to calculate the steric contributions to protein osmotic second virial coefficients (B 22,S) for ...proteins ranging from small single-domain molecules to large multidomain molecules, using the recently developed Mayer sampling method. B 22,S was compared for different levels of coarse-graining: four-beads-per-amino-acid (4bAA), one-bead-per-amino-acid (1bAA), one-sphere-per-domain (1sD), and one-sphere-per-protein (1sP). Values for the 1bAA and 4bAA models were quantitatively indistinguishable for both spherical and nonspherical proteins, and the agreement with values from all-atom models improved with increasing protein size, making the CG approach attractive for large proteins of biotechnological interest. Interestingly, in the absence of detailed structural information, the hydrodynamic radius (R h) along with a simple 1sP approximation provided reasonably accurate values for B 22,S for both globular and highly asymmetric protein structures, while other 1sP approximations gave poorer agreement; this helps to justify the currently empirical practice of estimating B 22,S from R h for large proteins such as antibodies. The results also indicate that either 1bAA or 4bAA CG models may be good starting points for incorporating short-range attractions. Comparison of gD-crystallin B 22 values including both sterics and short-range attractions shows that 1bAA and 4bAA models give equivalent results when properly scaled to account for differences in the number of surface beads in the two CG descriptions. This provides a basis for future work that will also incorporate long-ranged electrostatic attractions and repulsions.
Full text
Available for:
IJS, KILJ, NUK, PNG, UL, UM
The majority of biotechnologically relevant metabolites do not impart a conspicuous phenotype to the producing cell. Consequently, the analysis of microbial metabolite production is still dominated ...by bulk techniques, which may obscure significant variation at the single-cell level. In this study, we have applied the recently developed Lrp-biosensor for monitoring of amino acid production in single cells of gradually engineered L-valine producing Corynebacterium glutamicum strains based on the pyruvate dehydrogenase complex-deficient (PDHC) strain C. glutamicum ΔaceE. Online monitoring of the sensor output (eYFP fluorescence) during batch cultivation proved the sensor's suitability for visualizing different production levels. In the following, we conducted live cell imaging studies on C. glutamicum sensor strains using microfluidic chip devices. As expected, the sensor output was higher in microcolonies of high-yield producers in comparison to the basic strain C. glutamicum ΔaceE. Microfluidic cultivation in minimal medium revealed a typical Gaussian distribution of single cell fluorescence during the production phase. Remarkably, low amounts of complex nutrients completely changed the observed phenotypic pattern of all strains, resulting in a phenotypic split of the population. Whereas some cells stopped growing and initiated L-valine production, others continued to grow or showed a delayed transition to production. Depending on the cultivation conditions, a considerable fraction of non-fluorescent cells was observed, suggesting a loss of metabolic activity. These studies demonstrate that genetically encoded biosensors are a valuable tool for monitoring single cell productivity and to study the phenotypic pattern of microbial production strains.
Full text
Available for:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
PDF
