RESUMEN
The concept of modular synthetic co-cultures holds considerable potential for biomanufacturing, primarily to reduce the metabolic burden of individual strains by sharing tasks among consortium members. However, current consortia often show unilateral relationships solely, without stabilizing feedback control mechanisms, and are grown in a shared cultivation setting. Such 'one pot' approaches hardly install optimum growth and production conditions for the individual partners. Hence, novel mutualistic, self-coordinating consortia are needed that are cultured under optimal growth and production conditions for each member. The heterologous production of the antibiotic violacein (VIO) in the mutually interacting E. coli-E. coli consortium serves as an example of this new principle. Interdependencies for growth control were implemented via auxotrophies for L-tryptophan and anthranilate (ANT) that were satisfied by the respective partner. Furthermore, VIO production was installed in the ANT auxotrophic strain. VIO production, however, requires low temperatures of 20-30 °C which conflicts with the optimum growth temperature of E. coli at 37 °C. Consequently, a two-compartment, two-temperature level setup was used, retaining the mutual interaction of the cells via the filter membrane-based exchange of medium. This configuration also provided the flexibility to perform individualized batch and fed-batch strategies for each co-culture member. We achieved maximum biomass-specific productivities of around 6 mg (g h)-1 at 25 °C which holds great promise for future applications.
Asunto(s)
Reactores Biológicos , Técnicas de Cocultivo , Escherichia coli , Indoles , Escherichia coli/metabolismo , Escherichia coli/crecimiento & desarrollo , Indoles/metabolismoRESUMEN
BACKGROUND: The omnipresence of population heterogeneity in industrial bioprocesses originates from prevailing dynamic bioprocess conditions, which promote differences in the expression of cellular characteristics. Despite the awareness, the concrete consequences of this phenomenon remain poorly understood. RESULTS: Therefore, for the first time, a L-phenylalanine overproducing Escherichia coli quadruple reporter strain was established for monitoring of general stress response, growth behavior, oxygen limitation and product formation of single cells based on mTagBFP2, mEmerald, CyOFP1, and mCardinal2 expression measured by flow cytometry. This strain was applied for the fed-batch production of L-phenylalanine from glycerol and ammonia in a stirred-tank bioreactor at homogeneous conditions compared to the same process in a novel two-compartment bioreactor. This two-compartment bioreactor consists of a stirred-tank bioreactor with an initial volume of 0.9 L (homogeneous zone) with a coiled flow inverter with a fixed working volume of 0.45 L as a bypass (limitation zone) operated at a mean hydraulic residence time of 102 s. The product formation was similar in both bioreactor setups with maximum L-phenylalanine concentrations of 21.1 ± 0.6 g L-1 demonstrating the consistency of this study's microbial L-phenylalanine production. However, cell growth was vulnerable to repetitive exposure to the dynamically changing conditions in the two-compartment bioreactor with maximum biomass yields reduced by 21%. The functionality of reporter molecules was approved in the stirred-tank bioreactor cultivation, in which expressed fluorescence levels of all four markers were in accordance with respective process state variables. Additional evaluation of the distributions on single-cell level revealed the presence of population heterogeneity in both bioprocesses. Especially for the marker of the general stress response and the product formation, the corresponding histograms were characterized by bimodal shapes and broad distributions. These phenomena were pronounced particularly at the beginning and the end of the fed-batch process. CONCLUSIONS: The here shown findings confirm multiple reporter strains to be a noninvasive tool for monitoring cellular characteristics and identifying potential subpopulations in bioprocesses. In combination with experiments in scale-down setups, these can be utilized for a better physiological understanding of bioprocesses and support future scale-up procedures.
Asunto(s)
Reactores Biológicos , Escherichia coli , Escherichia coli/metabolismo , Fermentación , Biomasa , Oxígeno/metabolismoRESUMEN
Hythane has been well known as a mixture of hydrogen and methane gases but their production is mostly in a different way. The present study dealt with the potential biohythane production in a two-compartment (lower, hydrogenesis; upper, methanogenesis) reactor via a single-stage anaerobic fermentation at mesophilic temperature. The effect of hydraulic retention time (HRT) was tested at 10-2â¯d using food waste substrate. HRT 2â¯d resulted in (1) maximum removal efficiencies for COD, carbohydrate, lipid and protein contents with values of 58.5, 58.4, 62.6 and 79.1%, respectively; (2) peak hydrogen and methane production rates of 714 and 254â¯mL/L-d, respectively; and (3) biogas contents of hydrogen 8.6% and methane 48.0% in the produced gas. At this HRT, Clostridium sensu stricto 2 and Methanosaeta were dominant species in H2 and CH4 compartments, respectively. The novelty of this work is creating a novel two-compartment reactor for single-stage anaerobic biohythane fermentation.
Asunto(s)
Reactores Biológicos , Hidrógeno , Anaerobiosis , Biocombustibles , Fermentación , MetanoRESUMEN
Exosomes are membrane-enclosed vesicles released by different cell types into the extracellular space. As mediators of intercellular communication, they are involved in multiple physiological processes, but they are also associated with the pathogenesis of human malignancies including leukemia. Isolation of exosomes enables the characterization of their role in microenvironment modulation as well as their participation in disease pathology. A variety of strategies and techniques exists to purify exosomes from many biological fluids (e.g., blood, urine, and saliva). Here, we describe the efficient production of large quantities of exosomes from leukemic cell lines by using CELLine bioreactors based on two-compartment technology, as well as their isolation and purification by combining differential centrifugation and ultracentrifugation through a density gradient (17% OptiPrep™ cushion). Thus, exosomes are appropriately prepared for characterization by western blotting to detect exosome markers or imaging flow cytometry (ImageStream), and for downstream analyses such as the internalization in microenvironmental cells by confocal imaging or flow cytometry, methods which are also described in this chapter.
Asunto(s)
Reactores Biológicos , Técnicas de Cultivo de Célula/instrumentación , Fraccionamiento Celular/métodos , Exosomas/patología , Leucemia/patología , Técnicas de Cultivo de Célula/métodos , Fraccionamiento Celular/instrumentación , Línea Celular Tumoral , Centrifugación por Gradiente de Densidad/instrumentación , Centrifugación por Gradiente de Densidad/métodos , Citometría de Flujo/instrumentación , Citometría de Flujo/métodos , Colorantes Fluorescentes/química , Humanos , Separación Inmunomagnética/instrumentación , Separación Inmunomagnética/métodos , Leucemia/inmunología , Microscopía Confocal/instrumentación , Microscopía Confocal/métodos , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Microambiente Tumoral/inmunologíaRESUMEN
Large-scale bioreactors are inhomogeneous systems, in which the fluid phase expresses concentration gradients. They depend on the mass transfer and fluid dynamics in the reactor, the feeding strategy, the cell-specific substrate uptake parameters, and the cell density. As high cell densities are only obtained at low specific growth rates, it is necessary to investigate the cellular responses to oscillations in particular under such conditions, an issue which is mostly neglected. Instead, the feed oscillations are often started directly after the batch phase, when the specific growth rate is close to the maximum. We show here that the cultivation mode before oscillations are started has a tremendous effect on the metabolic responses. In difference to cells, which were pre-grown under batch conditions at a high growth rate, Escherichia coli cells that were pre-grown under glucose limitation at a low growth rate accumulate short-chain fatty acids (acetate, lactate, succinate) and glycolysis-related amino acids to a higher extent in a two-compartment scale-down bioreactor. Thus, cells which enter oscillations from a lower specific growth rate seem to react more sensitive to oscillations than cells that are subjected to oscillations directly after a batch phase. These results are interesting in designing reliable scale-down systems, which better reflect large-scale bioprocesses.