A model for light-limited continuous cultures: growth, shading, and maintenance

Light-limited growth in continuous cultures of phototrophic organisms is modeled. It is assumed that light energy up-take rate depends hyperbolically on light intensity and that the maintenance costs are proportional to biomass. Modeling the light distribution caused by shading within the vessel is necessary to explain the existence of steady state in light-limited chemostats. The model fits well to experimental data from literature on light-limited chemostats and turbidostats. Attention is given to the implications of the model for the estimation of the specific maintenance rate constant in light-limited continuous cultures.     

Stability and stabilization for models of chemostats with multiple limiting substrates

We study chemostat models in which multiple species compete for two or more limiting nutrients. First, we consider the case where the nutrient flow and species removal rates and input nutrient concentrations are all given as positive constants. In that case, we use Brouwer degree theory to give conditions guaranteeing that the models admit globally asymptotically stable componentwise positive equilibrium points, from all componentwise positive initial states. Then we use the results to develop stabilization theory for a class of controlled chemostats with two or more limiting nutrients. For cases where the dilution rate and input nutrient concentrations can be selected as controls, we prove that many different componentwise positive equilibria can be made globally asymptotically stable. This extends the existing control results for chemostats with one limiting nutrient. We demonstrate our methods in simulations.     

Stability and stabilization for models of chemostats with multiple limiting substrates

We study chemostat models in which multiple species compete for two or more limiting nutrients. First, we consider the case where the nutrient flow and species removal rates and input nutrient concentrations are all given as positive constants. In that case, we use Brouwer degree theory to give conditions guaranteeing that the models admit globally asymptotically stable componentwise positive equilibrium points, from all componentwise positive initial states. Then we use the results to develop stabilization theory for a class of controlled chemostats with two or more limiting nutrients. For cases where the dilution rate and input nutrient concentrations can be selected as controls, we prove that many different componentwise positive equilibria can be made globally asymptotically stable. This extends the existing control results for chemostats with one limiting nutrient. We demonstrate our methods in simulations.     

Evaluating the effects of chlortetracycline on the proliferation of antibiotic-resistant bacteria in a simulated river water ecosystem

Antibiotics and antibiotic metabolites have been found in the environment, but the biological activities of these compounds are uncertain, especially given the low levels that are typically detected in the environment. The objective of this study was to estimate the selection potential of chlortetracycline (CTC) on the antibiotic resistance of aerobic bacterial populations in a simulated river water ecosystem. Six replicates of a 10-day experiment using river water in continuous flow chemostat systems were conducted. Each replicate used three chemostats, one serving as a control to which no antibiotic was added and the other two receiving low and high doses of CTC (8 microg/liter and 800 microg/liter, respectively). The addition of CTC to the chemostats did not impact the overall level of cultivable aerobic bacteria (P = 0.51). The high-CTC chemostat had significantly higher tetracycline-resistant bacterial colony counts than both the low-CTC and the control chemostats (P < 0.035). The differences in resistance between the low-CTC and control chemostats were highly nonsignificant (P = 0.779). In general a greater diversity of tet resistance genes was detected in the high-CTC chemostat and with a greater frequency than in the low-CTC and control chemostats. Low levels of CTC in this in vitro experiment did not select for increased levels of tetracycline resistance among cultivable aerobic bacteria. This finding should not be equated with the absence of environmental risk, however. Low concentrations of antibiotics in the environment may select for resistant bacterial populations once they are concentrated in sediments or other locations.     

Regulation of tetrapyrrole synthesis by light in chemostat cultures of Rhodobacter sphaeroides.

Control of bacteriochlorophyll (Bchl), magnesium protoporphyrin monomethyl ester (MgPME), cytochromes, and coproporphyrin by light was studied with chemostat cultures of Rhodobacter sphaeroides growing at a constant dilution rate. By increasing the growth-limiting light energy flux from 10 to 55 W/m2, specific Bchl contents decreased from 19.3 to 7.9 nmol/mg of protein. This was strictly proportional to a decrease in the ratio of B800-850 to B875 light-harvesting complexes. MgPME levels increased from 1.5 to 5.3 nmol/mg of protein, while cytochrome as well as coproporphyrin levels stayed constant at 0.46 and 1.95 nmol/mg of protein, respectively. Since in chemostat cultures steady-state levels of a product represent the rate of synthesis, these results infer only slight control of the rate-limiting step of total tetrapyrrol formation by light. In substrate-limited cultures MgPME was accumulated when growth and Bchl formation approached substrate saturation. This suggests that light controls a second step, i.e., MgPME conversion, whenever too much precursor is available, owing to the low sensitivity of the initial step of control. MgPME was preferentially localized in a subcellular fraction with high contents of B875 complexes. A second fraction exhibiting increased contents of B800-850 complexes lacked significant levels of MgPME. These results are discussed in terms of localization of Bchl synthesis in the membrane system of R. sphaeroides.     

Optimization of biomass, vitamins, and carotenoid yield on light energy in a flat-panel reactor using the A-stat technique

Acceleration-stat (A-stat) cultivations in which the dilution rate is continuously changed at a constant acceleration rate, leading to different average light intensities inside the photobioreactor, can supply more information and reduce experimental time compared with chemostat cultivations. The A-stat was used to optimize the biomass and product yield of continuous cultures of the microalgae D. tertiolecta in a flat-panel reactor. In this study, four different accelerations were studied, a pseudo steady state was maintained at acceleration rates of 0.00016 and 0.00029 h(-2) and results were similar to those of the chemostat. An increase in the acceleration rate led to an increase in the deviation between results obtained in the A-stat and in the chemostats. We concluded that it is advantageous to use the A-stat instead of chemostats to determine culture characteristics and optimize a specific photobioreactor. The effect of average light intensity inside the photobioreactor on the production of vitamins C and E, lutein, and beta-carotene was studied using the A-stat. The highest concentrations of these products were 3.48 +/- 0.46, 0.33 +/- 0.06, 5.65 +/- 0.24, and 2.36 +/- 0.38 mg g(-1), respectively. These results were obtained at different average light intensities, showing the importance of optimizing each product on light intensity.     

Evaluating the Effects of Chlortetracycline on the Proliferation of Antibiotic-Resistant Bacteria in a Simulated River Water Ecosystem

Antibiotics and antibiotic metabolites have been found in the environment, but the biological activities of these compounds are uncertain, especially given the low levels that are typically detected in the environment. The objective of this study was to estimate the selection potential of chlortetracycline (CTC) on the antibiotic resistance of aerobic bacterial populations in a simulated river water ecosystem. Six replicates of a 10-day experiment using river water in continuous flow chemostat systems were conducted. Each replicate used three chemostats, one serving as a control to which no antibiotic was added and the other two receiving low and high doses of CTC (8 μg/liter and 800 μg/liter, respectively). The addition of CTC to the chemostats did not impact the overall level of cultivable aerobic bacteria (P = 0.51). The high-CTC chemostat had significantly higher tetracycline-resistant bacterial colony counts than both the low-CTC and the control chemostats (P < 0.035). The differences in resistance between the low-CTC and control chemostats were highly nonsignificant (P = 0.779). In general a greater diversity of tet resistance genes was detected in the high-CTC chemostat and with a greater frequency than in the low-CTC and control chemostats. Low levels of CTC in this in vitro experiment did not select for increased levels of tetracycline resistance among cultivable aerobic bacteria. This finding should not be equated with the absence of environmental risk, however. Low concentrations of antibiotics in the environment may select for resistant bacterial populations once they are concentrated in sediments or other locations.     

Buoyant density changes in the cyanobacterium Microcystis aeruginosa due to changes in the cellular carbohydrate content

Abstract The cyanobacterium Microcystis aeruginosa was grown in light-limited chemostat cultures with various light—dark rhythms providing a total periodicity length of 24 h. The buoyant density of the cells changed in parallel with the carbohydrate content. Short incubation experiments with different light intensities, and experiments with the inhibitors iodoacetic acid and arsenate, showed that the buoyant density changes were due to variations in the cellular carbohydrate content. It seems likely that the low dark-growth yield on carbohydrate, which had been stored during the light period, served to facilitate buoyancy changes.     

Selection Affects Genes Involved in Replication during Long-Term Evolution in Experimental Populations of the Bacteriophage φX174

Observing organisms that evolve in response to strong selection over very short time scales allows the determination of the molecular mechanisms underlying adaptation. Although dissecting these molecular mechanisms is expensive and time-consuming, general patterns can be detected from repeated experiments, illuminating the biological processes involved in evolutionary adaptation. The bacteriophage φX174 was grown for 50 days in replicate chemostats under two culture conditions: Escherichia coli C as host growing at 37°C and Salmonella typhimurium as host growing at 43.5°C. After 50 days, greater than 20 substitutions per chemostat had risen to detectable levels. Of the 97 substitutions, four occurred in all four chemostats, five arose in both culture conditions, eight arose in only the high temperature S. typhimurium chemostats, and seven arose only in the E. coli chemostats. The remaining substitutions were detected only in a single chemostat, however, almost half of these have been seen in other similar experiments. Our findings support previous studies that host recognition and capsid stability are two biological processes that are modified during adaptation to novel hosts and high temperature. Based upon the substitutions shared across both environments, it is apparent that genome replication and packaging are also affected during adaptation to the chemostat environment, rather than to temperature or host per se. This environment is characterized by a large number of phage and very few hosts, leading to competition among phage within the host. We conclude from these results that adaptation to a high density environment selects for changes in genome replication at both protein and DNA sequence levels.     

Protease productivity in chemostat fermentations with retention of biomass on suspended particles

Retention of bacterial biomass (Bacillus firmus) in a chemostat by a new carrier material, Luxopor, led to increased productivity of protease. Luxopor is a porous mineral product of irregular shape. When these particles are put into a fermenter, aeration and stirring make them float. Fermenters with Luxopor loadings of 200 and 500 g l^?1 were run as chemostats parallel to a control chemostat without it. The Luxopor particles contained >50% of the biomass in the chemostats (50 mg dry cell weight g^?1), which had a higher biomass and protease activity in the culture fluid than the control chemostat. The overall protease productivity was up to four times higher than that of the control.     

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 selection of microbial communities by constant or fluctuating temperatures

Abstract The diversity of bacterial communities isolated from Antarctic lake sediment in chemostats under constant low temperature (8°C) or diurnally fluctuating temperature (1°C to 16°C) was examined. The median optimum temperature for growth of the freshwater bacteria isolated from the fluctuation chemostat was significantly lower ( P < 1%) than that for those from the constant temperature chemostat. The diversity of the enriched bacterial community isolated in the chemostat culture subjected to short-term temperature fluctuations was greater than that enriched under constant temperature. At least 4 different groups of bacteria, that occupied separate 'temperature niches', were isolated from the fluctuating chemostat compared to only one group isolated from the stable chemostat. Furthermore, a pseudomonad from the fluctuating chemostat was shown to out-compete another pseudomonad from the stable chemostat when both were subjected to the fluctuating temperature regime. However, the pseudomonad of constant (8°C) temperature origin out-competed that isolated under fluctuating conditions when subjected to a stable temperature regime.     

Chaos in a periodically forced chemostat with algal mortality

We study the possibility of chaotic dynamics in the externally driven Droop model. This model describes a phytoplankton population in a chemostat under periodic nutrient supply. Previously, it has been proven under very general assumptions, that such systems are not able to exhibit chaotic dynamics. We show that the simple introduction of algal mortality may lead to chaotic oscillations of algal density in the forced chemostat. Our numerical simulations show that the existence of chaos is intimately related to plankton overshooting in the unforced model. We provide a simple measure, based on stability analysis, for estimating the amount of overshooting. These findings are not restricted to the Droop model but also hold for other chemostat models with mortality. Our results suggest periodically driven chemostats as a simple model system for the experimental verification of chaos in ecology.     

Competition between Nitrospira spp. and Nitrobacter spp. in nitrite-oxidizing bioreactors

In this work the question was addressed if in nitrite-oxidizing activated sludge systems the environmental competition between Nitrobacter spp. and Nitrospira spp., which only recently has been discovered to play a role in these systems, is affected by the nitrite concentrations. Two parallel chemostats were inoculated with nitrifying-activated sludge containing Nitrospira and operated under identical conditions. After addition of Nitrobacter to both chemostats, the nitrite concentration in the influent of one of the chemostats was increased such that nitrite peaks in the bulk liquid of this reactor were detected. The other chemostat served as control reactor, which always had a constant nitrite influent concentration. The relative cellular area (RCA) of Nitrospira and Nitrobacter was determined by quantitative fluorescence in situ hybridization (FISH). The nitrite perturbation stimulated the growth of Nitrobacter while in the undisturbed control chemostat Nitrospira dominated. Overall, the results of this experimental study support the hypothesis that Nitrobacter is a superior competitor when resources are abundant, while Nitrospira thrive under conditions of resource scarcity. Interestingly, the dominance of Nitrobacter over Nitrospira, caused by the elevated nitrite concentrations, could not be reverted by lowering the available nitrite concentration to the original level. One possible explanation for this result is that when Nitrobacter is present at a certain cell density it is able to inhibit the growth of Nitrospira. An alternative explanation would be that the length of the experimental period was not long enough to observe an increase of the Nitrospira population.     

Evolution of specialists in an experimental microcosm

The impact of adaptation on the persistence of a balanced polymorphism was explored using the lactose operon of Escherichia coli as a model system. Competition in chemostats for two substitutable resources, methylgalactoside and lactulose, generates stabilizing frequency-dependent selection when two different naturally isolated lac operons (TD2 and TD10) are used. The fate of this balanced polymorphism was tracked over evolutionary time by monitoring the frequency of fhuA-, a linked neutral genetic marker that confers resistance to the bacteriophage T5. In four out of nine chemostats the lac polymorphism persisted for 400-600 generations when the experiments were terminated. In the other five chemostats the fhuA polymorphism, and consequently the lac operon polymorphism, was lost between 86 and 219 generations. Four of 13 chemostat cultures monomorphic for the lac operon retained the neutral fhuA polymorphism for 450-550 generations until they were terminated; the remainder became monomorphic at fhuA between 63 and 303 generations. Specialists on each galactoside were isolated from chemostats that maintained the fhuA polymorphism, whether polymorphic or monomorphic at the lac operon. Strains isolated from three of four chemostats in which the lac polymorphism was preserved had switched their galactoside preference. Most of the chemostats where the fhuA polymorphism was lost also contained specialists. These results demonstrate that the initial polymorphism at lac was of little consequence to the outcome of long-term adaptive evolution. Instead, the fitnesses of evolved strains were dominated by mutations arising elsewhere in the genome, a fact confirmed by showing that operons isolated from their evolved backgrounds were alone unable to explain the presence of both specialists. Our results suggest that, once stabilized, ecological specialization prevented selective sweeps through the entire population, thereby promoting the maintenance of linked neutral polymorphisms.     

Regulation of glucose catabolism in Thiobacillus A2 grown in the chemostat under dual limitation by succinate and glucose

In contrast to its diauxic behaviour in batch culture, Thiobacillus A2 grew in chemostat culture using glucose and succinate as dual limiting substrates. Biomass production under dual limitations was the sum of that on single substrates with each substrate being oxidized and assimilated to similar extents in single and dual substrate-limited cultures. In glucose and glucose + succinate-limited cultures glucose was oxidized largely by the Entner-Doudoroff and pentose phosphate pathways, but other mechanisms also contributed and the ratios of pathways depended on substrate ratios and the previous substrate-history of the culture. Variations in specific activities of enzymes of carbohydrate metabolism following switches from single to mixed substrates were considerable, ranging from fourfold for fructose diphosphate aldolase to more than 200-fold for hexokinase, fructose diphosphatase, glucose 6-phosphate and 6-phosphogluconate dehydrogenases. Changes in specific activities occurred only over prolonged time periods in the chemostat, probably reflecting low concentrations of free substrates in carbon-limited cultures and consequent low levels of catabolite repression.     

Homeostatic adjustment and metabolic remodeling in glucose-limited yeast cultures

We studied the physiological response to glucose limitation in batch and steady-state (chemostat) cultures of Saccharomyces cerevisiae by following global patterns of gene expression. Glucose-limited batch cultures of yeast go through two sequential exponential growth phases, beginning with a largely fermentative phase, followed by an essentially completely aerobic use of residual glucose and evolved ethanol. Judging from the patterns of gene expression, the state of the cells growing at steady state in glucose-limited chemostats corresponds most closely with the state of cells in batch cultures just before they undergo this "diauxic shift." Essentially the same pattern was found between chemostats having a fivefold difference in steady-state growth rate (the lower rate approximating that of the second phase respiratory growth rate in batch cultures). Although in both cases the cells in the chemostat consumed most of the glucose, in neither case did they seem to be metabolizing it primarily through respiration. Although there was some indication of a modest oxidative stress response, the chemostat cultures did not exhibit the massive environmental stress response associated with starvation that also is observed, at least in part, during the diauxic shift in batch cultures. We conclude that despite the theoretical possibility of a switch to fully aerobic metabolism of glucose in the chemostat under conditions of glucose scarcity, homeostatic mechanisms are able to carry out metabolic adjustment as if fermentation of the glucose is the preferred option until the glucose is entirely depleted. These results suggest that some aspect of actual starvation, possibly a component of the stress response, may be required for triggering the metabolic remodeling associated with the diauxic shift.     

Occurence of lactose-negative mutants in chemostat cultures of lactic streptococci.

Batch and chemostat cultures of Streptococcus cremoris HP and Streptococcus lactis 829 were examined for lactose-hegative (lac-)mutants on indicator agar. In batch cultures, S. cremoris HP gave less than 1% of the total count as lac- colonies while S. lactis 829 consistently contained about 15% of the total as lac- colonies. In chemostat cultures of S. cremoris HP in 2% skim milk containing casamino acids and yeast extract (0.1% each), the percentage of lac- colonies increased markedly when the temperature of growth was 18 degrees C but not when the temperature of growth was 25 degrees C. The percentage of lac- colonies in chemostat cultures in the skim milk medium at 25 degrees C was about the same as that in batch cultures. On the other hand, when chemostat cultures of S. lactis 829 in the skim milk medium were grown at several temperatures between 18 and 33 degrees C, the percentage of lac- colonies was markedly lower than that found in batch cultures of this organism. Cultivation of S. cremoris HP in chemostats with yeast extract-glucose broth at low temperature (18 degrees C) resulted in a selection of cells giving lac- colonies and atypical (small) lac+ colonies. The results show that cultivation of S. cremoris HP and S. lactis 829 in chemostats sometimes gave rise to altered populations. Conditions causing a change in one organism did not necessarily cause a similar change in the other. The results indicate that the successful propagation of lactic streptococci in chemostats for use as starter cultures in the dairy industry will require the careful establishment of optimum conditions for every strain so as to minimize the possible selection of undesirable populations.     

Competition for Light between Toxic and Nontoxic Strains of the Harmful Cyanobacterium Microcystis

The cyanobacterium Microcystis can produce microcystins, a family of toxins that are of major concern in water management. In several lakes, the average microcystin content per cell gradually declines from high levels at the onset of Microcystis blooms to low levels at the height of the bloom. Such seasonal dynamics might result from a succession of toxic to nontoxic strains. To investigate this hypothesis, we ran competition experiments with two toxic and two nontoxic Microcystis strains using light-limited chemostats. The population dynamics of these closely related strains were monitored by means of characteristic changes in light absorbance spectra and by PCR amplification of the rRNA internal transcribed spacer region in combination with denaturing gradient gel electrophoresis, which allowed identification and semiquantification of the competing strains. In all experiments, the toxic strains lost competition for light from nontoxic strains. As a consequence, the total microcystin concentrations in the competition experiments gradually declined. We did not find evidence for allelopathic interactions, as nontoxic strains became dominant even when toxic strains were given a major initial advantage. These findings show that, in our experiments, nontoxic strains of Microcystis were better competitors for light than toxic strains. The generality of this finding deserves further investigation with other Microcystis strains. The competitive replacement of toxic by nontoxic strains offers a plausible explanation for the gradual decrease in average toxicity per cell during the development of dense Microcystis blooms.     

Chemostat cultivation as a tool for studies on sugar transport in yeasts.

Chemostat cultivation enables investigations into the effects of individual environmental parameters on sugar transport in yeasts. Various means are available to manipulate the specific rate of sugar uptake (qs) in sugar-limited chemostat cultures. A straightforward way to manipulate qs is variation of the dilution rate, which, in substrate-limited chemostat cultures, is equal to the specific growth rate. Alternatively, qs can be varied independently of the growth rate by mixed-substrate cultivation or by variation of the biomass yield on sugar. The latter can be achieved, for example, by addition of nonmetabolizable weak acids to the growth medium or by variation of the oxygen supply. Such controlled manipulation of metabolic fluxes cannot be achieved in batch cultures, in which various parameters that are essential for the kinetics of sugar transport cannot be controlled. In sugar-limited chemostat cultures, yeasts adapt their sugar transport systems to cope with the low residual sugar concentrations, which are often in the micromolar range. Under the conditions, yeasts with high-affinity proton symport carriers have a competitive advantage over yeasts that transport sugars via facilitated-diffusion carriers. Chemostat cultivation offers unique possibilities to study the energetic consequences of sugar transport in growing cells. For example, anaerobic, sugar-limited chemostat cultivation has been used to quantify the energy requirement for maltose-proton symport in Saccharomyces cerevisiae. Controlled variation of growth conditions in chemostat cultures can be used to study the differential expression of genes involved in sugar transport and as such can make an important contribution to the ongoing studies on the molecular biology of sugar transport in yeasts.