Incomplete mixing effects in a continuous culture laboratory-scale bioreactor

A laboratory bioreactor (1 L working volume) operating in chemostat mode was utilised for the culture of the polymorphic yeast Kluyveromyces marxianus var. marxianus NRRLy2415 undergoing fermentation of cheese whey permeate. Analysis of kinetic data revealed that the experimentally measured lactose concentration profiles in the exit line from the chemostat varied when the inlet feed concentrations ranged from 5–20 g lactose L–1. Further analysis revealed that this was due to about 3.5% of the feed bypassing the mixing zone. This incomplete mixing occurred despite an agitation rate of 800 rpm, an aeration rate of 1 vvm and the presence of internal baffles in the bioreactor. Without allowing for incomplete mixing, the estimation of the apparent Ks value for the organism could be out by a factor of five, over the range of feed concentrations employed. © Rapid Science Ltd. 1998     

The extended Kalman filter applied to a continuous culture model

The temperature-dependent endogenous metabolism model of single species continuous culture dynamics is utilized in the computer simulation of the Kalman filter state estimation technique. Parameters of the nonlinear equations can be “tracked” while variance in measured states can be damped. The state estimator is illustrated in, the context of conventional control strategies.     

Application of a two-compartment model to the wall growth of Pelobacter acidigallici under continuous culture conditions

Continuous cultivation of Pelobacter acidigallici on gallic acid is accompanied by biofilm formation. Steady states can be obtained at dilution rates nearly twice the maximum growth rate. A two-compartment model is presented which allows us to determine rate constants of attachment and detachment. The model is based on the assumption that the biofilm is fully penetrated by the substrate. At high dilution rates the outcome of our experiments is mainly determined by the attachment rate. The influence of the thickness of the biofilm is discussed in order to demonstrate the limitations of the model.     

Resistance ofSchizosaccharomyces pombe to mechanical strain caused by mixing in continuous culture

Schizosaccharomyces pombe is less resistant to mechanical strain caused by mixing thanCandida utilis orSaccharomyces cerevisiae. The highest utilizable frequency of stirring in the continuous culture is 11.9 Hz.     

Application of a competition model to the growth of Streptococcus mutans and Streptococcus sanguis in binary continuous culture

Streptococcus mutans 6715-15 and Streptococcus sanguis 10558 were grown together in continuous culture with glucose as the limiting carbon source. The relationship of growth rate to substrate concentration was determined for pure cultures of each organism in continuous and batch cultures. A model based on competition for a growth-limiting substrate (glucose) was used to predict the proportions of each organism when grown in binary cultures. The results indicate that interactions other than competition for glucose carbon exist between S. mutans and S. sanguis grown under these conditions.     

Application of an improved continuous parallel shaken bioreactor system for three microbial model systems

A continuous parallel shaken bioreactor system, combining the advantages of shaken bioreactors with the advantages of continuous fermentation, was specifically manufactured from quartz glass and provides a geometric accuracy of <1 mm. Two different model systems (facultative anaerobic bacterium C. glutamicum, and Crabtree-negative yeast P. stipitis), whose growth behaviour and metabolite formation are affected by dilution rate and oxygen availability, were studied. The transition from non-oxygen to limited conditions as function of the dilution rate could precisely be predicted applying the approach described by Maier et al. (Biochem Eng J 17:155–167, 2004). In addition, the Crabtree-positive yeast S. cerevisiae was simultaneously studied in the continuous parallel shaken bioreactor system and in a conventional 1-L bioreactor, for comparison. Essentially the same results were obtained in both types of bioreactors. However, many more reading points were obtained with the parallel shaken bioreactor system in the same time at much lower consumption of culture media.     

Application of a competition model to the growth of Streptococcus mutans and Streptococcus sanguis in binary continuous culture.

Streptococcus mutans 6715-15 and Streptococcus sanguis 10558 were grown together in continuous culture with glucose as the limiting carbon source. The relationship of growth rate to substrate concentration was determined for pure cultures of each organism in continuous and batch cultures. A model based on competition for a growth-limiting substrate (glucose) was used to predict the proportions of each organism when grown in binary cultures. The results indicate that interactions other than competition for glucose carbon exist between S. mutans and S. sanguis grown under these conditions.     

Dynamic model development for a continuous culture of Saccharomyces cerevisiae

The problem of dynamically modeling a chemostat is addressed. Using the results of continuous culture experiments for the growth of a strain of Saccharomyces cerevisiae on a glucose-limited medium, a general approach to developing dynamic models is discussed. The approach to develop and verify the model involves three different types of experiments: steady-state, dynamic step response, and feedback identification.     

Evaluation of Escherichia coli and Candida utilis as a model continuous, competitive mixed culture system

The yeast Candida utilis and the bacterium Escherichia coli B/r were evaluated as a candidate experimental, continous, competitive mixed culture system under ammonia-nitrogen limited conditions at 30 degrees C. High dilution rates favored yeast dominance, while low dilution rates favored bacterial dominance. The hydrogen ion concentration was also demonstrated to be an effective manipulative variable for control of the yeast-bacterial mixed culture. Through trial-and-error manipulation of the pH for the mixed culture operating at constant dilution rate, it was possible to locate a metastable equilibrium point and to operate in the vicinity of that point for more than 24h. The reproducible emergence of a variant E. coli was also noted in this study.     

Analysis of the productivity of a continuous algal culture system

We describe a first-principles analysis of a system for the continuous culture of the green alga Scenedesmus obliquus under light-limiting conditions. According to this analysis, the productivity of the algal culture is given by the relation Y = E(m)I(0)AK(1 - e(-alphacl)) - GRcV, where Y = yield (g cells/h), E(m) = 0.20 (the maximum attainable photosynthetic conversion on an energy basis), A = illuminated area (m(2)), K = 0.156[(g cells/h/W), the energy equivalent of the algae], I(0) = light intensity (W/m(2)), alpha = extinction coefficient (L/cm/g),c = cell concentration (g/L), I = light path (cm), R = respiration rate (g carbon/g cells/h), V = culture volume (L), and G = ratio of g cells to g carbon (2.04). This formula is completely determined and has no free adjustable parameters. Using parameter values determined independently, the model accurately predicted the relationship of productivity to cell density in the culture system.     

Effect of interstage mixing in multistage culture systems on continuous biomass production

The significance of the interstage mixing on important process parameters of biomass production was studied. The experiments were performed in a multistage tower fermentor and in fermentors in series. The interstage mixing effect can be evaluated under conditions of geometrical similarity, identity of oxygen transfer rate, and identity of dilution rate per stage in the individual stages of both culture systems. Candida utilis was cultivated on a synthetic medium with ethanol as the sole carbon and energy source in the concentration range 10–100 g/liter. Dilution rate, temperature, and pH in each stage of both culture systems were kept constant. It was demonstrated that in the multistage tower fermentor the definite backflow which ensures the permanent reinoculation by adapted cells significantly decreases the inhibitory effect of higher ethanol concentrations on the cell growth and on the rate of ethanol utilization.     

The influence of Staphylococcus aureus on gut microbial ecology in an in vitro continuous culture human colonic model system

An anaerobic three-stage continuous culture model of the human colon (gut model), which represent different anatomical areas of the large intestine, was used to study the effect of S. aureus infection of the gut on the resident faecal microbiota. Studies on the development of the microbiota in the three vessels were performed and bacteria identified by culture independent fluorescence in situ hybridization (FISH). Furthermore, short chain fatty acids (SCFA), as principal end products of gut bacterial metabolism, were measured along with a quantitative assessment of the predominant microbiota. During steady state conditions, numbers of S. aureus cells stabilised until they were washed out, but populations of indigenous bacteria were transiently altered; thus S. aureus was able to compromise colonisation resistance by the colonic microbiota. Furthermore, the concentration of butyric acid in the vessel representing the proximal colon was significantly decreased by infection. Thus infection by S. aureus appears to be able to alter the overall structure of the human colonic microbiota and the microbial metabolic profiles. This work provides an initial in vitro model to analyse interactions with pathogens.     

A continuous culture biofilm model of cariogenic responses

AIMS: To validate an in vitro model for the analysis of physiological and ecological responses to sugar challenge in bacterial populations, and subsequent changes in enamel mineralization. METHODS AND RESULTS: A seven-organism bacterial consortium was grown in a biofilm mode on enamel and hydroxyapatite (HA) surfaces in a continuous culture system and exposed to repeated sucrose challenges. This produced 'pH-cycling' conditions within the system. Populations on HA surfaces were enumerated. Changes in relative proportions of the different populations, and in the total viable count, were observed, between different treatments. Microradiography of the enamel sections showed increasing demineralization with increasing sucrose concentration. The lesions formed were similar to 'white-spot' lesions found in vivo. Differences in the quality of biofilms formed were also observed using Confocal Laser Scanning Microscopy. CONCLUSION: An in vitro model has been validated for the analysis of both physiological and ecological responses to sucrose challenges in bacterial populations, and subsequent changes in enamel mineralization. SIGNIFICANCE AND IMPACT OF THE STUDY: This model should facilitate the study of changes in bacterial populations in response to application of putative anticaries agents and concomitant changes in enamel mineralization.     

Evolution of bacteriophage in continuous culture: a model system to test antiviral gene therapies for the emergence of phage escape mutants

The emergence of viral escape mutants is usually a highly undesirable phenomenon. This phenomenon is frequently observed in antiviral drug applications for the treatment of viral infections and can undermine long-term therapeutic success. Here, we propose a strategy for evaluating a given antiviral approach in terms of its potential to provoke the appearance of resistant virus mutants. By use of Q beta RNA phage as a model system, the effect of an antiviral gene therapy, i.e., a virus-specific repressor protein expressed by a recombinant Escherichia coli host, was studied over the course of more than 100 generations. In 13 experiments carried out in parallel, 12 phage populations became resistant and 1 became extinct. Sequence analysis revealed that only two distinct phage mutants emerged in the 12 surviving phage populations. For both escape mutants, sequence variations located in the repressor binding site of the viral genomic RNA, which decrease affinity for the repressor protein, conferred resistance to translational repression. The results clearly suggest the feasibility of the proposed strategy for the evaluation of antiviral approaches in terms of their potential to allow resistant mutants to appear. In addition, the strategy proved to be a valuable tool for observing virus-specific molecular targets under the impact of antiviral drugs.     

Effect of acidic pH and salt on acid end-products by Lactobacillus plantarum in aerated, glucose-limited continuous culture

In aerated, glucose-limited continuous culture, Lactobacillus plantarum altered its metabolic pathways of acid production in response to changes in environmental pH. Under acidic conditions, acetate and L-lactate production decreased with decreasing pH, whereas levels of D-lactate increased. In the presence of 6% NaCl, acetate and lactate production continued, but no acetate was detected at pH 4.5.     

Production of Bacillus thuringiensis spores in total cell retention culture and two-stage continuous culture using an internal ceramic filter system

The production of Bacillus thuringiensis spores was investigated in a bioreactor incorporating a ceramic membrane filter to improve spore concentration and volumetric productivity. Two cultivation methods were used in this study: a total cell retention culture (TCRC), and a two-stage continuous culture with partial cell bleeding. In the TCRC, fed by 50 g/L of glucose, a spore concentration of 1.6 x 10(10) CFU/mL was obtained with a spore percentage of greater than 95% and a maximum cell mass of 82.2 g/L. The volumetric productivity was four times higher than that obtained from batch cultivation. In the two-stage continuous culture with partial cell bleeding spore concentration was strongly dependent on the bleed ratio. The spore concentration of 1.8 x 10(9) CFU/mL and the spore percentage of 70% were obtained at the second stage when a bleed ratio of 0.33 and a dilution rate of 0.23 h(-1) were used. (c) 1993 John Wiley & Sons, Inc.     

Application of the functional-form model to the culture of seaweeds

Selecting the most appropriate species or strains is an important first step in the development of most algal cultivation systems and is usually a tedious, time-consuming, and expensive step. The functional-form model, first developed to synthesize the adaptive significance of easily assessed thallus-form attributes relative to the productivity and survival of benthic macroalgae, is applicable to the culture of seaweeds and can expedite species or strain selection. The production ecology aspects of the model are useful particularly for applications where the desired product is not species-specific, e.g., systems in which the emphasis is on algal production, such as algal biomass farms and wastewater treatment. A thallus-form with a high surface area: volume ratio is more suited for rapid production and nutrient uptake. The utility of this model to strain selection is demonstrated with the red alga Gracilaria tikvahiae, a species that has been considered a maricultural candidate for a number of utilizations. A continuum of surface area: volume ratios for eight clones of G. tikvahiae showed that this ratio decreased as morphological complexity increased and was a good predictor of both short-term photosynthesis and long-term growth rate. Clones near opposite ends of the surface area: volume ratio spectrum had significant differences for both photosynthesis and growth. Each clone of G. tikvahiae possesses concomitant combinations of benefits as well as costs, which should be carefully evaluated for the cultivation application of interest. Knowledge of functional-form relationships in seaweeds can significantly expedite their successful cultivation.     

Glucose-stat, a glucose-controlled continuous culture

A predictive and feedback proportional control algorithm, developed for fed-batch fermentations and described in a companion paper (G. L. Kleman, J. J. Chalmers, G. W. Luli, and W. R. Strohl, Appl. Environ. Microbiol. 57:910-917, 1991), was used in this work to control a continuous culture on the basis of the soluble-glucose concentration (called the glucose-stat). This glucose-controlled continuous-culture system was found to reach and maintain steady state for 11 to 24 residence times when four different background glucose concentrations (0.27, 0.50, 0.7, and 1.5 g/liter) were used. The predictive-plus-feedback control system yielded very tight control of the continuous nutristat cultures; glucose concentrations were maintained at the set points with less than 0.003 standard error. Acetate production by Escherichia coli B in glucose-stats was found not to be correlated with the level of steady-state soluble-glucose concentration.