Comparison of properties of collected cells and cells from the culture vessel during continuous culture of Streptococcus mutans Ingbritt

In continuous-culture studies chemostat effluents are usually collected into a receiving flask in an ice bath to obtain enough cells for an experiment. It is assumed that the properties of these are not significantly different from those of the culture in the chemostat vessel. This assumption has been tested for the dental pathogen Streptococcus mutans Ingbritt. Collected supernatant fluid and cells were compared with supernatant fluid and cells taken directly from the culture vessel, for four major groups of culture properties: viability and biomcss, concentrations of metabolites and nutrients, activities of selected enzymes, and glycolytic rates. The assumption held true except for glycolytic rate during endogenous metabolism. It is suggested that comparison of collected and culture vessel cells is an important control which should be done in all continuous culture studies of microbial physiology and biochemistry, but that the properties of Strep, mutans cells collected on ice up to 16 h do reflect those of cells actively growing in the chemostat.     

Comparison of properties of collected cells and cells from the culture vessel during continuous culture of Streptococcus mutans Ingbritt

In continuous-culture studies chemostat effluents are usually collected into a receiving flask in an ice bath to obtain enough cells for an experiment. It is assumed that the properties of these are not significantly different from those of the culture in the chemostat vessel. This assumption has been tested for the dental pathogen Streptococcus mutans Ingbritt. Collected supernatant fluid and cells were compared with supernatant fluid and cells taken directly from the culture vessel, for four major groups of culture properties: viability and biomass, concentrations of metabolites and nutrients, activities of selected enzymes, and glycolytic rates. The assumption held true except for glycolytic rate during endogenous metabolism. It is suggested that comparison of collected and culture vessel cells is an important control which should be done in all continuous culture studies of microbial physiology and biochemistry, but that the properties of Strep. mutans cells collected on ice up to 16 h do reflect those of cells actively growing in the chemostat.     

Biological characteristics of the variants of Bacillus thuringiensis subsp. galleriae formed during continuous culture

Bacillus thuringiensis subsp. galleriae S. variants formed during continuous cultivation differ from the parent culture in certain properties. In contrast to the parent R form, their growth in the chemostat does not yield virulent mutants which can cause their lysis on solid media. The chemostat S forms are resistant against virulent phage mutants produced when the R variants are grown under the conditions of continuous cultivation and against a virulent phage obtained from the parent culture 69-6 under the action of vancomycin. The R forms are sensitive to these phages. When the S forms are grown under the chemostat conditions, they do not revert to the R forms. The R and S forms do not differ noticeably in the character of their growth, formation of spores and crystals, and biological activity.     

Eco-physiological Characterization of Soil Bacterial Populations in Different States of Growth

The method based on characterization of microbial populations in terms of their growth rate in agar plates has been used for testing the prediction of the theory of r- and K-selection in a microbial community from a tropical soil. Conditions which could lead bacterial populations to grow exponentially or to enter into a stationary phase were obtained by growing soil microbial populations in a chemostat and in a chemostat with recycle, respectively. Significant differences in population distribution patterns were observed by comparing results from the two growth systems. When soil community was grown in a chemostat and subjected specifically to well-defined r- and K-conditions, stable associations of organisms with r- and K-type characteristics developed as a consequence of environmental pressure. In contrast, when cultivated in chemostat with recycle under the same r- and K-conditions imposed on chemostat cultures, distribution patterns of r- and K-selected populations appeared very little affected by changes in substrate availability.     

Anaerobic benzene degradation under denitrifying conditions: Peptococcaceae as dominant benzene degraders and evidence for a syntrophic process

An anaerobic microbial community was enriched in a chemostat that was operated for more than 8 years with benzene and nitrate as electron acceptor. The coexistence of multiple species in the chemostat and the presence of a biofilm, led to the hypothesis that benzene-degrading species coexist in a syntrophic interaction, and that benzene can be degraded in syntrophy by consortia with various electron acceptors in the same culture. The benzene-degrading microorganisms were identified by DNA-stable isotope probing with [U-(13) C]-labelled benzene, and the effect of different electron donors and acceptors on benzene degradation was investigated. The degradation rate constant of benzene with nitrate (0.7 day(-1) ) was higher than reported previously. In the absence of nitrate, the microbial community was able to use sulfate, chlorate or ferric iron as electron acceptor. Bacteria belonging to the Peptococcaceae were identified as dominant benzene consumers, but also those related to Rhodocyclaceae and Burkholderiaceae were found to be associated with the anaerobic benzene degradation process. The benzene degradation activity in the chemostat was associated with microbial growth in biofilms. This, together with the inhibiting effect of hydrogen and the ability to degrade benzene with different electron acceptors, suggests that benzene was degraded via a syntrophic process.     

The energetics of microbes at slow growth rates: Maintenance energies and dormant organisms

In continuous cultures at slow growth rates (less than about 10% maximum) bacterial growth yields from the carbon and energy source are higher than those expected. To account for this deviation it is proposed that dormant or non-viable cells with zero maintenance energy are generated at slow growth rates. From the growth yield variation, it is shown, the dormant fraction of the culture can be calculated. The few quantitative data available on the viability of bacteria in chemostat steady states at very slow growth rates are in agreement with the hypothesis. It is suggested that enzymic, chemical and morphological characters also may be used to distinguish between the growing and dormant fractions of a culture.The model provides a unifying theory for studies of microbial function at slow growth rates, which is a field of great practical importance.     

Evolution of cross-feeding in microbial populations

Although limited by a single resource, microbial populations that grow for long periods in continuous culture (chemostat) frequently evolve stable polymorphisms. These polymorphisms may be maintained by cross-feeding, where one strain partially degrades the primary energy resource and excretes an intermediate that is used as an energy resource by a second strain. It is unclear what selective advantage cross-feeding strains have over a single competitor that completely degrades the primary resource. Here we show that cross-feeding may evolve in microbial populations as a consequence of the following optimization principles: the rate of ATP production is maximized, the concentration of enzymes of the pathway is minimized, and the concentration of intermediates of the pathway is minimized.     

Metabolic engineering under uncertainty--II: analysis of yeast metabolism

Yeast metabolism has been used extensively in scientific investigations and industrial applications. Understanding the properties of the yeast metabolic network is crucial, yet unaccomplished due to its high complexity, the different culture conditions, and the uncertainty associated with kinetic parameters. We recently developed a computational and mathematical framework using Monte Carlo method in which parameter uncertainty can be addressed through large-scale sampling procedure. This framework was applied on the compartmentalized central carbon pathways of Saccharomyces cerevisiae metabolism considering the growth environment of batch and chemostat reactor and integrating information from metabolic flux analysis. Statistical analysis of the results indicates that yeast cells growing in batch culture condition exhibit dramatically different control schemes from those growing in a chemostat. The difference is mainly due to the feedback introduced by the constraints of the chemostat. The control of the enzymes on the rates of the substrate uptake, product excretion, and cell growth and its practical implication are discussed. Clustering of the reaction steps according to the similarity of their responses to enzyme activity perturbations reveals functional coupling of metabolic reactions.     

The impact of feed composition on biodegradation of benzoate under cyclic (aerobic/anoxic) conditions

The response of a mixed microbial culture to different feed compositions, that is, containing benzoate and pyruvate as sole carbon sources at different levels, was studied in a chemostat with a 48-h hydraulic residence time under cyclic aerobic and anoxic (denitrifying) conditions. The cyclic bacterial culture was well adapted to different feed compositions as evidenced by the lack of accumulation of benzoate or pyruvate in the chemostat. Both the benzoate-degrading capabilities and the in vitro catechol 2,3-dioxygenase (C23DO) activities of the cyclic bacterial cultures were in direct proportion to the flux through the chemostat of the substrate degraded by the pathway containing C23DO, with some exceptions. The quantity of C23DO showed a transient decrease during the initial portion of the aerobic period before returning to the level present during the anoxic period. That decrease was most likely caused by the production of H(2)O(2) by the cells upon being returned to aerobic conditions.     

Rapid assessment of bacterial viability and vitality by rhodamine 123 and flow cytometry

A.S. KAPRELYANTS AND D.B. KELL. 1992. The fluorescent dye rhodamine 123 (Rh 123) is concentrated by microbial cells in an uncoupler-sensitive fashion. Steady-state fluorescence measurements with Micrococcus luteus indicated that provided the added dye concentration is below approximately 1 mmol/1, uptake is fully uncoupler-sensitive and is not accompanied by significant self-quenching of the fluorescence of accumulated dye molecules. 'Viable' and 'non-viable' cells are easily and quantitatively distinguished in a flow cytometer by the extent to which they accumulate the dye. The viability of a very slowly growing chemostat culture of Mic. luteus is apparently only about40–50%, as judged by plate counts, but most of the 'non-viable' cells can be resuscitated by incubation of the culture in nutrient medium before plating. The extent to which individual cells accumulate rhodamine 123 can be rapidly assessed by flow cytometry, and reflects the three distinguishable physiological states exhibited by the culture ('non-viable', 'viable' and 'non-viable-but-resuscitable'). Gram-negative bacteria do not accumulate rhodamine 123 significantly because their outer membrane is not permeable to it; a simple treatment overcomes this. Flow cytometry using rhodamine 123 should prove of general utility for the rapid assessment of microbial viability and vitality.     

Efficiency of the cyclic batch antibiotic fermentation

For a simple mathematical model of microbial product formation, the productivities of stationary and nonstationary cultivation methods (chemostat and cyclic batch) are compared. Conditions of superiority of the cyclic batch cultivation are characterized. The model includes substrate inhibition of the product formation and the age-dependent loss of productivity of the culture.     

Parathion utilization by bacterial symbionts in a chemostat.

A continuous-culture device was used to select and enrich for microorganisms, from sewage and agricultural runoff, that were capable of using the organophosphorus insecticide parathion as a sole growth substrate. Parathion was dissimilated by the highly acclimated symbiotic activities of Pseudomonas stutzeri, which non-oxidatively and cometabolically hydrolyzed the parathion to ionic diethyl thiophosphate and p-nitrophenol, and P. aeruginosa, which utilized the p-nitrophenol as a sole carbon and energy source. Ionic diethyl thiophosphate was found to be inert to any transformations. Methyl parathion was dissimilated in an analogous way. The device functioned as a chemostat with parathion as the growth-limiting nutrient, and extraordinarily high dissimilation rates were attained for parathion (8 g/liter per day) and for p-nitrophenol (7 g/liter per day). This is the first report of parathion utilization by a defined microbial culture and by symbiotic microbial attack and of dissimilation of an organophosphorus pesticide in a chemostat.     

Microbial Ecophysiology of Whey Biomethanation: Comparison of Carbon Transformation Parameters, Species Composition, and Starter Culture Performance in Continuous Culture

Changes in lactose concentration and feed rate altered bacterial growth and population levels in a whey-processing chemostat. The bacterial population and methane production levels increased in relation to increased lactose concentrations comparable to those in raw whey (6%) and converted over 96% of the substrate to methane, carbon dioxide, and cells. Sequential increases in the chemostat dilution rate demonstrated excellent biomethanation performance at retention times as low as 25 h. Retention times shorter than 25 h caused prevalent bacterial populations and methane production to decrease, and intermediary carbon metabolites accumulated in the following order: acetate, butyrate, propionate, lactate, ethanol, and lactose. Bacterial species dominated in the chemostat as a function of their enhanced substrate uptake and growth kinetic properties. The substrate uptake kinetic properties displayed by the mixed chemostat population were equivalent to those of individual species measured in pure culture, whereas the growth kinetic properties of species in mixed culture were better than those measured in pure culture. A designed starter culture consisting of Leuconostoc mesenteroides, Desulfovibrio vulgaris, Methanosarcina barkeri, and Methanobacterium formicicum displayed biomethanation performance, which was similar to that of a diverse adapted mixed-culture inoculum, in a continuous contact digestor system to which 10 g of dry whey per liter was added. Preserved starter cultures were developed and used as inocula for the start-up of a continuous anaerobic digestion process that was effective for biomethanation of raw whey at a retention time of 100 h.     

Parathion utilization by bacterial symbionts in a chemostat.

A continuous-culture device was used to select and enrich for microorganisms, from sewage and agricultural runoff, that were capable of using the organophosphorus insecticide parathion as a sole growth substrate. Parathion was dissimilated by the highly acclimated symbiotic activities of Pseudomonas stutzeri, which non-oxidatively and cometabolically hydrolyzed the parathion to ionic diethyl thiophosphate and p-nitrophenol, and P. aeruginosa, which utilized the p-nitrophenol as a sole carbon and energy source. Ionic diethyl thiophosphate was found to be inert to any transformations. Methyl parathion was dissimilated in an analogous way. The device functioned as a chemostat with parathion as the growth-limiting nutrient, and extraordinarily high dissimilation rates were attained for parathion (8 g/liter per day) and for p-nitrophenol (7 g/liter per day). This is the first report of parathion utilization by a defined microbial culture and by symbiotic microbial attack and of dissimilation of an organophosphorus pesticide in a chemostat.     

Comparative stability of ethanol production by Escherichia coliKO11 in batch and chemostat culture

Differing claims regarding the stability of the recombinant ethanologen E. coli KO11 are addressed here in batch and chemostat culture. In repeat batch culture, the organism was stable on glucose, mannose, xylose and galactose for at least three serial transfers, even in the absence of a selective antibiotic. Chemostat cultures on glucose were remarkably stable, but on mannose, xylose and a xylose/glucose mixture, they progressively lost their hyperethanologenicity. On xylose, the loss was irreversible, indicating genetic instability. The loss of hyperethanologenicity was accompanied by the production of high concentrations of acetic acid and by increasing biomass yields, suggesting that the higher ATP yield associated with acetate production may foster the growth of acetate-producing revertant strains. Plate counts on high chloramphenicol-containing medium, whether directly, or following preliminary growth on non-selective medium, were not a reliable indicator of high ethanologenicity during chemostat culture. In batch culture, the organism appeared to retain its promise for ethanol production from lignocellulosics and concerns that antibiotics may need to be included in all media appear unfounded. Received 13 January 1999/ Accepted in revised form 23 April 1999     

Estimation of steady-state culture characteristics during acceleration-stats with yeasts

Steady-state culture characteristics are usually determined in chemostat cultivations, which are very time-consuming. In contrast, acceleration-stat (A-stat) cultivations in which the dilution rate is continuously changed with a constant acceleration rate are not so time-consuming, especially at high acceleration rates. Therefore, the A-stat could be advantageous to use instead of the chemostat. However, the highest acceleration rate, meaning the fastest A-stat that can be applied for estimating steady-state culture characteristics, is not known yet. Experimental results obtained with Zygosaccharomyces rouxii, an important yeast in soy sauce processes, showed that the culture characteristics during the A-stat with an acceleration rate of 0.001 h(-2) were roughly comparable to those of the chemostat. For higher acceleration rates the deviation between the culture characteristics in the A-stat and those in the chemostat obtained at the same dilution rate generally started to increase. The source of these deviations was examined by simulation for Saccharomyces cerevisiae. The simulations demonstrated that this deviation was not only dependent on the metabolic adaptation rate of the yeast, but also on the rate of change in environmental substrate concentrations during A-stats. From this work, it was concluded that an A-stat with an acceleration rate of 0.001 h(-2) is attractive to be used instead of chemostat whenever a rough estimation of steady-state culture characteristics is acceptable.     

Removal of Cadmium by Microorganisms in a Two-Stage Chemostat

A two-stage chemostat was used to study removal of cadmium by microorganisms in continuous culture. The medium was contaminated with 0.8 mg of Cd per liter. At 20°C, most of the microbial biomass formed aggregates which settled in the second stage of the chemostat. Effluent was free of bacteria. Up to 80% of the metal contained in the inlet flux was removed by the biomass, with 20% remaining in solution. At 10°C and with a shorter retention time, flocculation was poorer and metal removal by settling biomass did not exceed 35%.     

Competition of two microbial populations for a single resource in a chemostat when one of them exhibits wall attachment

It is known that two microbial populations competing for a single resource in a homogeneous environment with time-invariant inputs cannot coexist in a steady state. The case where two microbial populations compete for a single resource in a chemostat but one of them exhibits attachment to the chemostat walls is studied theoretically. Because of the cells' attachment to the walls, the environment is no longer homogeneous. The present article considers the case where the attached cells form no more than a monolayer. Other situations occur, often frequently, but we do not consider them here. Two models are used to represent the attachment to the walls: the Topiwala-Hamer model and a model which assumes that the attachment of microbial cells to the solid surfaces is a reversible process. The first model does not allow the population that exhibits wall attachment to wash out from the chemostat, in contrast to the second model (which nevertheless reduces to the first one in the limit). It has been found that in most of the possible cases for both models, the two competitors can coexist in a stable steady state for a wide range of the operating parameters space. The results of the stability analysis are discussed and analytical expressions for the conditions and the boundaries of the domains of stable coexistence are given for all the possible situations that may arise.     

Studies of the Utilization of Coconut Water Waste for the Production of the Food Yeast Saccharomyces fragilis

The accepted food yeast Saccharomyces fragilis was grown in batch and chemostat culture on coconut water and on a simulated coconut-water medium containing glucose, fructose, sucrose and sorbitol, to provide kinetic data for a feasibility study of microbial protein production. Analyses of growth on individual and mixed carbon substrates were made to determine sugar assimilation patterns in batch and chemostat cultures on coconut water. Growth on the polyol produced a much reduced specific growth rate, assimilation rate, growth yield and productivity compared to growth on the sugars. In mixed substrate fermentations a sequential utilization of the carbohydrates occurred. Both the monosaccharides repressed invertase synthesis and all three sugars repressed sorbitol assimilation. Complete carbon assimilation was only obtained by prolonged batch fermentation or in chemostat cultures at low dilution rates (<0.10 h^-1). Supplementation of coconut water with biotin and nicotinic acid increased biomass yields in chemostat cultures.     

Teflon chemostat for studies of trace metal metabolism in Streptococcus mutans and other bacteria.

A teflon chemostat constructed for studies of microbial trace metal metabolism is described. The utility of this continuous culture system was demonstrated with Streptococcus mutans, in which iron and manganese stimulated growth in ranges of 0.18 to 0.45 and 18 to 54 microM, respectively. This device should facilitate studies of the effect of trace metals on a variety of physiological functions.