Use Of Chemostat Research Articles

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

The Use of Chemostats in Microbial Systems Biology

Cells regulate their rate of growth in response to signals from the external world. As the cell grows, diverse cellular processes must be coordinated including macromolecular synthesis, metabolism and ultimately, commitment to the cell division cycle. The chemostat, a method of experimentally controlling cell growth rate, provides a powerful means of systematically studying how growth rate impacts cellular processes - including gene expression and metabolism - and the regulatory networks that control the rate of cell growth. When maintained for hundreds of generations chemostats can be used to study adaptive evolution of microbes in environmental conditions that limit cell growth. We describe the principle of chemostat cultures, demonstrate their operation and provide examples of their various applications. Following a period of disuse after their introduction in the middle of the twentieth century, the convergence of genome-scale methodologies with a renewed interest in the regulation of cell growth and the molecular basis of adaptive evolution is stimulating a renaissance in the use of chemostats in biological research.

Regulated expression of polysaccharide utilization and capsular biosynthesis loci in biofilm and planktonic Bacteroides thetaiotaomicron during growth in chemostats

Bacteroides thetaiotaomicron is a prominent member of the human distal gut microbiota that specializes in breaking down diet and host-derived polysaccharides. While polysaccharide utilization has been well studied in B. thetaiotaomicron, other aspects of its behavior are less well characterized, including the factors that allow it to maintain itself in the gut. Biofilm formation may be a mechanism for bacterial retention in the gut. Therefore, we used custom GeneChips to compare the transcriptomes of biofilm and planktonic B. thetaiotaomicron during growth in mono-colonized chemostats. We identified 1,154 genes with a fold-change greater than 2, with confidence greater than or equal to 95%. Among the prominent changes observed in biofilm populations were: (i) greater expression of genes in polysaccharide utilization loci that are involved in foraging of O-glycans normally found in the gut mucosa; and (ii) regulated expression of capsular polysaccharide biosynthesis loci. Hierarchical clustering of the data with different datasets, which were obtained during growth under a range of conditions in minimal media and in intestinal tracts of gnotobiotic mice, revealed that within this group of differentially expressed genes, biofilm communities were more similar to the in vivo samples than to planktonic cells and exhibited features of substrate limitation. The current study also validates the use of chemostats as an in vitro "gnotobiotic" model to study gene expression of attached populations of this bacterium. This is important to gut microbiota research, because bacterial attachment and the consequences of disruptions in attachment are difficult to study in vivo.

Microchemostat array with small-volume fraction replenishment for steady-state microbial culture

A chemostat is a bioreactor in which microorganisms can be cultured at steady-state by controlling the rate of culture medium inflow and waste outflow, thus maintaining media composition over time. Even though many microbial studies could greatly benefit from studying microbes in steady-state conditions, high instrument cost, complexity, and large reagent consumption hamper the routine use of chemostats. Microfluidic-based chemostats (i.e. microchemostats) can operate with significantly smaller reagent consumption while providing accurate chemostatic conditions at orders of magnitude lower cost compared to conventional chemostats. Also, microchemostats have the potential to significantly increase the throughput by integrating arrays of microchemostats. We present a microchemostat array with a unique two-depth culture chamber design that enables small-volume fraction replenishment of culture medium as low as 1% per replenishment cycle in a 250 nl volume. A system having an array of 8 microchemostats on a 40 × 60 mm(2) footprint could be automatically operated in parallel by a single controller unit as a demonstration for potential high throughput microbial studies. The model organism, Saccharomyces cerevisiae, successfully reached a stable steady-state of different cell densities as a demonstration of the chemostatic functionality by programming the dilution rates. Chemostatic functionality of the system was further confirmed by quantifying the budding index as a function of dilution rate, a strong indicator of growth-dependent cell division. In addition, the small-volume fraction replenishment feature minimized the cell density fluctuation during the culture. The developed system provides a robust, low-cost, and higher throughput solution to furthering studies in microbial physiology.

Correlation of gene expression and protein production rate - a system wide study.

BackgroundGrowth rate is a major determinant of intracellular function. However its effects can only be properly dissected with technically demanding chemostat cultivations in which it can be controlled. Recent work on Saccharomyces cerevisiae chemostat cultivations provided the first analysis on genome wide effects of growth rate. In this work we study the filamentous fungus Trichoderma reesei (Hypocrea jecorina) that is an industrial protein production host known for its exceptional protein secretion capability. Interestingly, it exhibits a low growth rate protein production phenotype.ResultsWe have used transcriptomics and proteomics to study the effect of growth rate and cell density on protein production in chemostat cultivations of T. reesei. Use of chemostat allowed control of growth rate and exact estimation of the extracellular specific protein production rate (SPPR). We find that major biosynthetic activities are all negatively correlated with SPPR. We also find that expression of many genes of secreted proteins and secondary metabolism, as well as various lineage specific, mostly unknown genes are positively correlated with SPPR. Finally, we enumerate possible regulators and regulatory mechanisms, arising from the data, for this response.ConclusionsBased on these results it appears that in low growth rate protein production energy is very efficiently used primarly for protein production. Also, we propose that flux through early glycolysis or the TCA cycle is a more fundamental determining factor than growth rate for low growth rate protein production and we propose a novel eukaryotic response to this i.e. the lineage specific response (LSR).

Transcriptional responses ofSaccharomyces cerevisiaeto preferred and nonpreferred nitrogen sources in glucose-limited chemostat cultures

Aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae grown with six different nitrogen sources were subjected to transcriptome analysis. The use of chemostats enabled an analysis of nitrogen-source-dependent transcriptional regulation at a fixed specific growth rate. A selection of preferred (ammonium and asparagine) and nonpreferred (leucine, phenylalanine, methionine and proline) nitrogen sources was investigated. For each nitrogen source, distinct sets of genes were induced or repressed relative to the other five nitrogen sources. In total, 131 such 'signature transcripts' were identified in this study. In addition to signature transcripts, genes were identified that showed a transcriptional coresponse to two or more of the six nitrogen sources. For example, 33 genes were transcriptionally upregulated in leucine-grown, phenylalanine-grown and methionine-grown cultures; this was partly attributed to the involvement of common enzymes in the dissimilation of these amino acids. In addition to specific transcriptional responses elicited by individual nitrogen sources, their impact on global regulatory mechanisms such as nitrogen catabolite repression (NCR) were monitored. NCR-sensitive gene expression in the chemostat cultures showed that ammonium and asparagine were 'rich' nitrogen sources. By this criterion, leucine, proline and methionine were 'poor' nitrogen sources, and phenylalanine showed an 'intermediate' NCR response.

A cultural divide on the use of chemostats

A cultural divide on the use of chemostats

Biochemical responses and photosynthetic performance of Gracilaria sp. (Rhodophyta) from Cádiz, Spain, cultured under different inorganic carbon and nitrogen levels

Photosynthetic acclimation and the interactions between carbon (C) and nitrogen (N) metabolism have been studied in the red macroalga Gracilaria sp. from Cádiz, Spain, cultured under different inorganic C and N levels. The use of chemostats and buffered medium allowed continuous restoration of the alkaline reserve and constancy of pH during the experiments. The N:C ratios and phycobiliprotein, chlorophyll a and soluble protein contents decreased when Gracilaria sp. was grown at low N levels. Algae grown in a high inorganic C concentration (5% CO2) displayed a higher soluble carbohydrate concentration and maximum photosynthesis rates but a lower photosynthesic affinity for inorganic C, and lower phycobiliprotein and Rubisco contents, than those cultured at low inorganic C levels (air CO2). The inorganic C enrichment also affected the N uptake and assimilation in Gracilaria sp., causing a decrease in the N uptake rate even under conditions of N sufficiency. These results reflect the significant influence of the inorganic C growth regime on N assimilation in Gracilaria sp.

Use of chemostat for enhanced production of β-glucosidase by newly isolated anaerobic cellulolyticClostridium strain RT9

A new anaerobic, mesophilic, spore-forming, cellulolyticClostridium strain, RT9, was isolated from bovine rumen fluid. This strain has the ability to hydrolyze cellulose (Sigma cell-100), carboxymethyl-cellulose (CMC), and cellobiose into ethanol, acetate, butyrate, lactate, acetone, H2, and Co2 as the major fermentation products. The bacterium also exhibited endoglucanase, exoglucanase, and β-glucosidase activities in batch culture during growth on chemically defined medium with cellulose as the sole carbon source. Chemostat experiments were conducted under pH, dilution rate, and carbohydrate limitations to study the effect of various parameters on enzymes and product formation. In chemostat with 0.5% cellobiose as the limiting nutrient, maximum β-glucosidase activity and ethanol production occurred at pH values of 5.5 and 6.5, respectively. At low pH, acid accumulation was higher. β-glucosidase production increased up to 17-fold at a low dilution rate, and ethanol raised twofold. Relative activity of β-glucosidase on a percent basis was maximum at pH 6.5 and 45 °C. In a carbon-limited, continuous-culture experiment, when CMC was used as an inducer in cellobiose-growing culture, β-glucosidase activity was higher than the cellobiose-limited chemostat. On the other hand, enzyme declined twofold, and ethanol production increased in glucose limitation.

Use of chemostat for selection ofStreptomyces chrysomallus mutants altered in the induction of D-glucose isomerase.

D-glucose isomerase ofStreptomyces chrysomallus PL45 is inducible by D-xylose only. In mutants obtained by means of a selection procedure in a chemostat the isomerase was induced in xylose-free medium containing glucose as carbon source.

Use of chemostat for selection ofStreptomyces hygroscopicus mutants altered in regulation of maltose utilization

Streptomyceshygroscopicus mutants showing altered fermentation kinetics were isolated using a selection procedure in a chemostat. Several mutants were obtained which differed in their capacity to produce the macrolide antibiotic turimycin.

DNA-MEDIATED TRANSFORMATION IN BACILLUS SUBTILIS WITH SPECIAL REFERENCE TO THE METHOD FOR PREPARING COMPETENT CELLS

A routine method of DNA-mediated transformation is described. Cells of tryptophan dependent strain (No. 160 Sr) of Bacillus subtilis, harvested at an early stationary phase of growth in a glucose minimal medium containing 0.1% yeast extract, are found to be the most competent for transformation (1st culture). When transforming DNA is added to the cells suspended in a fresh medium (2nd culture), transformation occurs linearly with the time lapse after a short lag period. The process of transformation seems independent of cell growth, although the acquisition of competence by the cells is under the control of delicate physiological conditions of the cells which are terminating exponential growth. The yeast extract which is required for the cell growth in the early 2nd culture, when added in excess, lowers the efficiency of transformation. The inhibitory effect of the supplemented medium is eliminated during the growth of cells. The similar inhibition is demonstrated by the addition of nucleotides or amino acids.Physiological control of cell growth in the 1st culture by changing incubation conditions improves the competence. The use of chemostat or the incubation at low temperatures were proved to be an efficient way for obtaining competent cells.