Carbon and nitrogen as resources limiting the growth of mono- and mixed cultures of Pseudomonas aeruginosa dissociants

New experiments for detection of resources limiting the growth of mono- and mixed cultures of Pseudomonas aeruginosa dissociants were carried out. The results were analyzed on the basis of the consumption and growth variational model in accordance with the data on the dissociant metabolism special traits. In 83% of cases, the theoretical calculation was confirmed by the experimental results.     

Population size and areas of stratification in the variational model of ecosystem

The formulations of the variational task for finding the relative population size of species in community at a stationary stage of growth and of the theorem of stratification are given. Algorithms of finding the relative size of populations for communities consisting of two and three species consuming two or three resources were obtained. The borders of areas of stratification were described in which one, two, or three resources are limiting. For two species and two resources, the formulae of the dependence of relative size on the ratio of resources were derived, and the shape of this dependence for real requirements of species was demonstrated.     

Development and population structure of mixed (S + M) <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> cultures in the late stationary growth phase

Population growth, the ratio between dissociants, pH, and levels of reducing sugars in the medium were monitored during prolonged (375 h) batch cultivation of Pseudomonas aeruginosa S and M dissociants on mineral medium with glucose. During the stationary growth phase (100–375 h), two scenarios were possible. The first one included extensive cell autolysis coupled to alkalinization of the medium and an increased ratio of the M dissociant. In the second case, acidification of the medium was coupled to the oscillating secondary growth, mostly of the M dissociant; the dynamics of cell numbers of this dissociant correlated with the dynamics of the culture optical density. In this scenario, periodical appearance of reducing sugars in the medium was detected; it was in the opposite phase with the changes of the M dissociant cell numbers. The differences between scenarios of P. aeruginosa growth in the late stationary phase were probably due to the physiological and biochemical characteristics of the S and M dissociants, including different pathways of glucose utilization (respiration or fermentation), resistance to acidification, as well as synthetic (proteolytic) activity and productivity of autoinducers.     

The Requirements of Pseudomonas aeruginosa Dissociants for Carbon,Nitrogen, and Phosphorus

Quantitative data on the nutritional requirements of microorganisms are necessary to predict the behavior of bacterial populations and to control their cultivation. The requirements of the R, S, and M dissociants of P. aeruginosa for carbon, nitrogen, and phosphorus were derived from the results of 88 cultivation experiments. For each of the dissociants, we derived a coefficient that relates the optical density and the number of cells in the dissociant culture, determined the time when the cultures entered the stationary growth phase, studied cultural changes induced by transfer to the stationary phase, and determined what nutrients limit the growth of particular dissociants. The nutritional requirements of the dissociants are discussed in relation to our earlier data.     

The requirements of Pseudomonas aeruginosa dissociants for carbon, nitrogen, and phosphorus

Quantitative data on the nutritional requirements of microorganisms are necessary to predict the behavior of bacterial populations and to control their cultivation. The requirements of the R, S, and M dissociants of Pseudomonas aeruginosa for carbon, nitrogen, and phosphorus were derived from the results of 88 cultivation experiments. For each of the dissociants, we derived a coefficient that relates the optical density and the number of cells in the dissociant culture, determined the time when the cultures entered the stationary growth phase, studied cultural changes induced by transfer to the stationary phase, and determined what nutrients limit the growth of particular dissociants. The nutritional requirements of the dissociants are discussed in relation to our earlier data.     

Development and population structure of mixed (S + M) Pseudomonas aeruginosa cultures in the late stationary growth phase

Population growth, the ratio between dissociants, pH, and levels of reducing sugars in the medium were monitored during prolonged (375 h) batch cultivation of Pseudomonas aeruginosa S and M dissociants on mineral medium with glucose. During the stationary growth phase (100-375 h), two scenarios were possible. The first one included extensive cell autolysis coupled to alkalinization of the medium and an increased ratio of the M dissociant. In the second case, acidification of the medium was coupled to the oscillating secondary growth, mostly of the M dissociant; the dynamics of cell numbers of this dissociant correlated with the dynamics of the culture optical density. In this scenario, periodical appearance of reducing sugars in the medium was detected; it was in the opposite phase with the changes of the M dissociant cell numbers. The differences between scenarios of P. aeruginosa growth in the late stationary phase were probably due to the physiological and biochemical characteristics of the S and M dissociants, including different pathways of glucose utilization (respiration or fermentation), resistance to acidification, synthetic (proteolytic) activity, and productivity of autoinducers.     

Characteristics of carbohydrate metabolism of R-, S- i M-dissociants of Pseudomonas aeruginosa]

R and S dissociants of the hydrocarbon-oxidizing strain Pseudomonas aeruginosa K-2 differed but little in their growth in a minimal defined medium with glucose as the source of carbon and energy. At the same time, the number of cells of M dissociant in the late exponential phase was five orders of magnitude less than that of R and S dissociants. The growth of M dissociant was accompanied by the accumulation of formate in the culture liquid and a concurrent decrease in pH. All three dissociants contained the key enzymes of the Entner-Doudoroff pathway of glucose oxidation; however, the activities of these enzymes, especially 6-phosphogluconate dehydrogenase, were low in M dissociant. Conversely, the activity of formate dehydrogenase in cells of M dissociant was higher than in other dissociants. The activity of 6-phosphogluconate dehydrogenase, a key enzyme of the pentosephosphate pathway of glucose oxidation, was detected only in S dissociant. The peculiarities of the carbohydrate metabolism of M dissociant are probably responsible for its poor growth on glucose and determine the more pronounced anaerobic type of its metabolism.     

Selection of Species for the Laboratory-Reared Algal Community by Their Hydrobiological and Biophysical Features

Phytoplankton communities can serve as bioindicators of the water system state. It is necessary to select the appropriate species of microalgae to develop a model of a natural ecosystem that will allow performing multifactor experiments on the influence of physicochemical factors on the biophysical and hydrobiological characteristics of phytoplankton. This study has allowed selecting six species from those available in a museum to establish a model algal community. We have found that similar conditions are required for their optimal growth (light, temperature, and medium nutrients' supply). A medium with low nitrogen content is proposed to be used as a basal medium. Under these conditions, the cells function in a proper way and the cultures show satisfactory growth, while the duration of reaching the stationary stage of growth (10–15 days) allows having more experiments for a limited time. The cells of the selected species have morphological differences that are sufficient for the automated identification within the polyculture. We have obtained the geometric characteristics of cells for the computer counting of each species in the community on the microphotographs.     

Effect of major nutrient elements on the growth and population homogeneity of the R, S and M dissociants of Pseudomonas aeruginosa and the glucose oxidation and fermentation pathways

The population homogeneity of the stationary-phase monocultures of Pseudomonas aeruginosa dissociants was studied as a function of the initial content of major nutrient elements (C, N, and P) in the cultivation medium. The monocultures of the dissociants remained homogeneous during cultivation if the initial concentrations of the major nutrient elements were either sufficiently high or, conversely, very low, but became heterogeneous during cultivation in unbalanced (with respect to the major nutrient elements) media. At the initial concentration of nitrate in the medium equal to 0.07% or phosphate equal to 0.004-0.014%, the initially homogeneous population of R dissociant cultivated to the stationary growth phase turned out to contain 30-40% of S-type cells, whereas the initially homogeneous population of S dissociant was found to contain 50-80% of M-type cells. The population of M dissociant remained homogeneous throughout the cultivation period. R dissociant grew better at sufficiently high concentrations of glucose, nitrate, and phosphate in the medium, whereas M dissociant grew better when the initial concentrations of these nutrients were low. During the cultivation of R dissociant, the pH of the medium changed insignificantly, and the C/P ratio (the ratio of the carbon and phosphorus consumed during growth) was minimal (among the three dissociants), indicating that the R dissociant accomplishes the oxidative pathway of glucose metabolism. During the cultivation of the M dissociant, the pH of the medium dropped to 3.4-3.9, and the C/P ratio was maximal, indicating that this dissociant accomplishes the fermentative pathway of glucose metabolism. During the cultivation of the S dissociant, the pH of the medium and the C/P ratio exhibited variations, indicating that this dissociant triggers its pathways of glucose metabolism.     

On the regulation of the ecological community structure by a variation of the concentration ratios of resources in environment

The possibility to regulate the structure of a community using a variational model of the ecological community was studied. The dependences of relative numbers of microorganisms on the initial ratios of resources of nutrients in environment were obtained. The potential possibility of controlling the community structure was shown. Model calculations and experimental data obtained on a community of Chlorococcales indicated that, as the ratios of resources vary, a change of the dominating species takes place.     

Microcalorimetric study of Escherichia coli aerobic growth: kinetics and experimental enthalpy associated with growth on succinic acid.

A new batch calorimeter was used to study the aerobic growth of Escherichia coli in a minimal containing a growth-limiting concentration of succinic acid. The shape of the thermograms obtained was simple when the energy source was the only growth-limiting factor and showed a stationary phase when the oxygen transfer was limiting. The experimentally determined value of the heat production during growth was found to be significantly lower than the heat of combustion of the succinic acid corrected for the assimilated-substrate fraction. An interpretation has been given to explain the difference between the experimental and the theoretical values.     

Kinetic studies on autohydrogenotrophic growth of <Emphasis Type="Italic">Ralstonia eutropha</Emphasis> with nitrate as terminal electron acceptor

Autohydrogenotrophic batch growth of Ralstonia eutropha H16 was studied in a stirred-tank reactor with nitrate and nitrite as terminal electron acceptors and the sole limiting substrates. Assuming product inhibition by nitrite, saturation kinetics with the two limiting substrates and a simple switching function, which allows growth on nitrite only at low nitrate concentrations, resulted in a kinetic growth model with nine model parameters. The data of two batch experiments were used to identify the kinetic model. The kinetic model was validated with two additional batch experiments. The model predictions are in very good agreement with the experimental data. The maximum nitrite concentration was estimated to be 30.7 mM (total inhibition of growth). After complete reduction of nitrate, the growth rate decreases almost to zero before it increases again because of the following nitrite respiration. The maximum autohydrogenotrophic growth rate of Ralstonia eutropha with nitrate as a final electron acceptor (0.509 d⁻¹) was found to be reduced by 90–95% compared to the so far reported autohydrogenotrophic growth rates with oxygen.     

Changes in Individual Allometry Can Lead to Species Coexistence without Niche Separation

The principle of competitive exclusion is a fundamental tenet of ecology. Commonly used competition models predict that at most only one species per limiting resource can coexist in the same environment at steady state; hence, the upper limit to species diversity depends only on the number of limiting resources and not on the rates of resource supply. We demonstrate that such model behavior is the result of both the growth and biomass turnover functions being proportional to the population biomass. We argue that at least the growth function should be a nonlinear, concave downward function of biomass. This form for the growth function should arise simply because of changes in the allometry of individuals in the population. With this change in model structure, we show that any number of species can coexist at an asymptotically stable steady state, even where there is only one limiting resource. Furthermore, if growth increases nonlinearly with biomass, the steady-state resource concentration and hence the potential for biodiversity increases as the resource supply rate increases. Received 31 August 2001; accepted 10 April 2002.     

The activity of ribosome modulation factor during growth of <Emphasis Type="Italic">Escherichia coli</Emphasis> under acidic conditions

Expression of the gene encoding ribosome modulation factor (RMF), as measured using an rmf-lacZ gene fusion, increased with decreasing pH in exponential phase cultures of Escherichia coli. Expression was inversely proportional to the growth rate and independent of the acidifying agent used and it was concluded that expression of rmf was growth rate controlled in exponential phase under acid conditions. Increased rmf expression during exponential phase was not accompanied by the formation of ribosome dimers as occurs during stationary phase. Nor did it appear to have a significant effect on cell survival under acid stress since the vulnerability of an RMF-deficient mutant strain was similar to that of the parent strain. Ribosome degradation was increased in the mutant strain compared to the parent strain at pH 3.75. Also, the peptide elongation rate was reduced in the mutant strain but not the parent during growth under acid conditions. It is speculated that the function of RMF during stress-induced reduction in growth rate is two-fold: firstly to prevent reduced elongation efficiency by inactivating surplus ribosomes and thus limiting competition for available protein synthesis factors, and secondly to protect inactivated ribosomes from degradation.     

The Indole Pulse: A New Perspective on Indole Signalling in Escherichia coli

Indole has diverse signalling roles, including modulation of biofilm formation, virulence and stress responses. Changes are induced by indole concentrations of 0.5–1.0 mM, similar to those found in the supernatant of Escherichia coli stationary phase culture. Here we describe an alternative mode of indole signalling that promotes the survival of E. coli cells during long-term stationary phase. A mutant that has lost the ability to produce indole demonstrates reduced survival under these conditions. Significantly, the addition of 1 mM indole to the culture supernatant is insufficient to restore long-term survival to the mutant. We provide evidence that the pertinent signal in this case is not 1 mM indole in the culture supernatant but a transient pulse of intra-cellular indole at the transition from exponential growth to stationary phase. During this pulse the cell-associated indole reaches a maximum of approximately 60 mM. We argue that this is sufficient to inhibit growth and division by an ionophore-based mechanism and causes the cells to enter stationary phase before resources are exhausted. The unused resources are used to repair and maintain cells during the extended period of starvation.     

The Effect of Major Nutrient Elements on the Growth and Population Homogeneity of the R,S, and M Dissociants of Pseudomonas aeruginosa and the Glucose Oxidation and Fermentation Pathways

The population homogeneity of the stationary-phase monocultures of Pseudomonas aeruginosa dissociants was studied as a function of the initial content of major nutrient elements (C, N, and P) in the cultivation medium. The monocultures of the dissociants remained homogeneous during cultivation if the initial concentrations of the major nutrient elements were either sufficiently high or, conversely, very low, but became heterogeneous during cultivation in unbalanced (with respect to the major nutrient elements) media. At an initial concentration of nitrate in the medium equal to 0.07% or phosphate equal to 0.004–0.014%, the initially homogeneous population of R dissociant cultivated to the stationary growth phase turned out to contain 30–40% of S-type cells, whereas the initially homogeneous population of S dissociant was found to contain 50–80% of M-type cells. The population of M dissociant remained homogeneous throughout the cultivation period. R dissociant grew better at sufficiently high concentrations of glucose, nitrate, and phosphate in the medium, whereas M dissociant grew better when the initial concentrations of these nutrients were low. During the cultivation of R dissociant, the pH of the medium changed insignificantly, and the C/P ratio (the ratio of the carbon and phosphorus consumed during growth) was minimal (among the three dissociants), indicating that the R dissociant accomplishes the oxidative pathway of glucose metabolism. During the cultivation of the M dissociant, the pH of the medium dropped to 3.4–3.9, and the C/P ratio was maximal, indicating that this dissociant accomplishes the fermentative pathway of glucose metabolism. During the cultivation of the S dissociant, the pH of the medium and the C/P ratio exhibited variations, indicating that this dissociant triggers its pathways of glucose metabolism.     

Prediction of Rate‐Limiting Reactions for Growth‐Associated Production Using a Constraint‐Based Approach

Identification of a rate‐limiting step in pathways is a key challenge in metabolic engineering. Although the prediction of rate‐limiting steps using a kinetic model is a powerful approach, there are several technical hurdles for developing a kinetic model. In this study, an in silico screening algorithm of key enzyme for metabolic engineering is developed to identify the possible rate‐limiting reactions for the growth‐coupled target production using a stoichiometric model without any experimental data and kinetic parameters. In this method, for each reaction, an upper‐bound flux constraint is imposed and the target production is predicted by linear programming. When the constraint decreases the target production at the optimal growth state, the reaction is thought to be a possible rate‐limiting step. For validation, this method is applied to the production of succinate or 1,4‐butanediol (1,4‐BDO) in Escherichia coli, in which the experimental engineering for eliminating rate‐limiting steps has been previously reported. In succinate production from glycerol, nine reactions including phosphoenolpyruvate carboxylase are predicted as the rate‐limiting steps. In 1,4‐BDO production from glucose, eight reactions including pyruvate dehydrogenase are predicted as the rate‐limiting steps. These predictions include experimentally identified rate‐limiting steps, which would contribute to metabolic engineering as a practical tool for screening candidates of rate‐limiting reactions.     

Bacterial Competition in Activated Sludge: Theoretical Analysis of Varying Solids Retention Times on Diversity

A mechanistic model for activated sludge sewage treatment was developed to predict exploitative competition of six aerobic heterotrophic bacterial species competing for three essential resources. The central hypothesis of the model is that in a multispecies/limiting resource system the number of coexisting bacterial species, N, exceeds the number of limiting resources, K, available for them. The explanation for this is that for certain species combinations, the dynamics of the competition process generate oscillations in the abundances of species, and these oscillations allow the coexistence of greater number of species than the number of limiting resources (N > K). This result is a direct contradiction of an existing activated sludge steady state competition theory, the principle of competitive exclusion, which states that the competition process proceeds to equilibrium, allowing only N K species to coexist. The model was used to investigate the effect of varying solids retention times on the diversity of species using the conventional, completely mixed activated sludge configuration. The results of model simulations showed that for a certain range of solids retention times (2.28–5.66 days) the competition of six species for three essential resources produces oscillations within the structure of the bacterial community allowing for the sustained growth of more than three species on three resources.This revised version was published online in November 2004 with corrections to Volume 48.     

Contrasting patterns of trait‐based community assembly in lianas and trees from temperate Australia

Trait variation in plant communities is thought to be constrained by two opposing community assembly processes operating at discrete spatial scales: habitat filtering and limiting similarity between coexisting species. Filtering processes cause convergence in ecological strategy as species are excluded from unsuitable sites, whilst limiting similarity leads to the divergence of trait values between co‐occurring species in order to alleviate competition for finite resources. Levels of alpha (within‐site) and beta (among‐site) trait variation can be indicative of the strength of these community assembly processes. We used trait‐gradient analysis to explicitly compare evidence of community assembly patterns in lianas (woody vines) and trees. These two growth forms exhibit striking differences in carbon capture and regeneration strategies, yet trait‐based mechanisms that maintain their coexistence remain understudied. Using data for four functional traits – leaf mass per area, leaf nitrogen content (N_mass), leaf area and seed mass – we partitioned interspecific trait variation in lianas and trees into alpha and beta components. Our three key findings were: 1) lianas and trees exhibit divergent patterns of trait‐based habitat filtering, due to differences in the relationship between leaf size and the other three traits examined (LMA, N_mass and seed mass), 2) on average, liana species possess smaller seeds, lower LMA and higher N_mass than do trees, but there was no clear difference in leaf area between the two growth forms, and 3) soil fertility was correlated with trait variation (leaf area, seed mass) at the site‐level in trees, but not in lianas. These results provide evidence that dominant growth forms can be filtered into the same habitat on the basis of different combinations of traits. Our findings have important implications for community assembly and co‐existence theory and for more pragmatic matters such as using trait‐based principles to inform community restoration.