Product formation in the continuous culture of microbial populations grown on carbohydrates

Experimental studies of the growth of a natural microbial population on a synthetic liquid effluent containing sugar, sodium alginate, and nutrients showed that: (I) the concentration of substrates in the feed to an activated sludge process exerts a significant effect upon its effluent chemical oxygen demand (COD) and (ii) there is an optimum sludge age for achieving minimum effluent COD, given by the relationship: optimum growth rate ∝ (feed COD)^0.5. These were explained by incorporating the concept of product formation into existing activated-sludge theory, which showed that at sludge ages longer than optimum, effluent COD increased due to product formation; at shorter sludge ages, the effluent COD increased owing to an increased concentration of degradable substrates.     

Coexistence of Chlorobium and Chromatium in a sulfide-limited continuous culture

Competition experiments between Chromatium vinosum and Chlorobium limicola in sulfide-limited continuous culture under photolithoautotrophic conditions resulted in the coexistence of both organisms. The ratio between the two bacteria was dilution-rate as well as pH dependent. The observed coexistence can be explained as a hitherto not reported form of dual substrate limitation. The two substrates involved are the electron donors sulfide (growth-limiting substrate in the reservoir vessel) and extracellular elemental sulfur (formed by Chlorobium as a result of sulfide oxidation). It is argued that, although Chlorobium may have the better affinity for both substrates involved, Chromatium can compete successfully on the basis of its intracellular storage of sulfur. Ecological implication of the observed coexistence with respect to natural blooms are discussed.     

Toward a self-similar theory of microbial populations

A fresh quest is made of segregated cell models of microbial populations with a view to determine whether the multivarite distribution of physiological states, during transient growth, can attain self-similar forms (i.e., become time invariant) when each physiological state variable is scaled with respect to its population average. Such self-similar growth situations are believed to be more general than those of balanced growth. The conditions under which self-similarity is possible are investigated. Thus conditions are stipulated on the synthesis rates of different physiological entities, cell division rate, and the partitioning of the parent cell's components among the daughter cells (assuming binary division) in order for self-similar growth to be attained. Subject to the attainment of self-similar growth, it is shown that cytometric data can be analyzed systematically to determine how the rates of syntheses of various biochemical entities and cell division rates vary with the physiological entities that are measured. Inverse problems, represented by algebraic systems, are identified which will potentially allow flow cytometric data to be inverted to yield quantitative information on the absolute rates of cellular growth and reproductory processes as a function of the cell states chosen for measurement. It is suggested that the methods become more effective when cytometry can be used to make direct observations on dividing cells so that the number of unknowns in the inverse problem can be reduced, thus facilitating its more complete solution. Preliminary analysis of cytometric data obtained in the literature show promise of self-similarity and thus the possibility of application of the methods discussed here. (c) 1994 John Wiley & Sons, Inc.     

Identification of factors regulating Escherichia coli 2,3-butanediol production by continuous culture and metabolic flux analysis

2,3-Butanediol (2,3-BDO) is an organic compound with a wide range of industrial applications. Although Escherichia coli is often used for the production of organic compounds, the wild-type E. coli does not contain two essential genes in the 2,3-BDO biosynthesis pathway, and cannot ferment 2,3-BDO. Therefore, a 2,3-BDO biosynthesis mutant strain of Escherichia coli was constructed and cultured. To determine the optimum culture factors for 2,3-BDO production, experiments were conducted under different culture environments ranging from strongly acidic to neutral pH. The extracellular metabolite profiles were obtained using high-performance liquid chromatography (HPLC), and the intracellular metabolite profiles were analyzed by ultra-performance liquid chromatography and quadruple time-of-flight mass spectrometry (UPLC/ Q-TOF-MS). Metabolic flux analysis (MFA) was used to integrate these profiles. The metabolite profiles showed that 2,3-BDO production favors an acidic environment (pH 5), whereas cell mass favors a neutral environment. Furthermore, when the pH of the culture fell below 5, both the cell growth and 2,3-BDO production were inhibited.     

Coexistence of two microbial populations competing for a renewable resource in a non-predator-prey system

If two microbial populations compete for a single resource in a homogeneous environment with time invariant inputs they cannot coexist indefinitely if the resource competed for is not renewed by biological activity within the system. Mathematical studies have shown that in a predator-prey system, where the resource (prey) is self-renewing, the two competitors (predators) can coexist in a limit cycle. This suggests that if the resource competed for is renewed by biological activity within the system coexistence can occur in any microbial system provided that it exhibits the same features as, but without being, a predator-prey one. A food chain involving commensalism, competition and amensalism is presented here. Two subcases are considered. It is only when maintenance effects are taken into account that coexistence, in limit cycles, can occur for this system. Limit cycle solutions for the system are demonstrated with the help of computer simulations. Some necessary conditions for coexistence are presented, as are some speculations regarding the possible physical explanations of the results.     

Mixed culture biooxidation of phenol. II. Steady state experiments in continuous culture

Experiments are reported in which a mixed population of organisms was continuous cultured on phenol in a 1- to 2-liter well-mixed vessel. Steady state phenol concentrations were measured for a range of inlet concentrations from 100 to 800 mg/liter at various dilution rates. These results were compared with those predicted from a model which incorporates the effect of wall growth. It was found that the effect of growth on the walls of the vessel was considerable and increased by a factor of up to 3 × the dilution rate at which 90% conversion of phenol could be obtained.     

Kinetic model identification in mixed populations using continuous culture data

The kinetic behavior of heterogeneous microbial populations of sewage origin was studied in a single-stage, isothermal, continuous flow, completely mixed aeration tank. A series of experiments were carried out at various dilutions rates using glucose as the limiting substrate. The cell dry weight and substrate concentration in terms of chemical oxygen demand (COD) were continuously monitored. The results indicate that reproducible steady-state conditions can generally be obtained; however, multiple steady states were observed at dilution rates near washout. At low dilution rates (below about 0.1 hr^?1) the contribution of microorganism decay became appreciable. Using the multiresponse data of cell dry weight and COD, the parameter values in various existing growth models were estimated. The analyses of variance and residuals revealed that models proposed by Moser, Monod, and Contois, each with a decay term added, were significantly better than the other models which were tested.     

Mixed populations of marine microalgae in continuous culture: Factors affecting species dominance and biomass productivity

Marine microalgae were grown in multispecies continuous cultures. Under carbon dioxide limitation, blue-green algae dominated. Under nitrate and light limitation, species dominance depended on the initial conditions. When the inoculum consisted primarily of blue-green algae with smaller amounts of other species, blue-green algae and pennate diatoms dominated. When the inoculum consisted of equal amounts of all species, green flagellates and pennate diatoms dominated. Green flagellates and blue-green algae were incompatible and never shared dominance. When nutrient limitations were overcome, the productivity of seawater was increased 100-fold before light limitation occurred. The productivity could be further increased by reducing photorespiration in the culture. The dilution rates studied (0.1, 0.2, and 0.4 day(-1)) had no effect on species dominance, nor did the higher dilution rates select for smaller cells. The maximum productivity occurred at a dilution rate of 0.2 day(-1). Temperature had the greatest effect on species dominance, with green flagellates, pennate diatoms, and blue-green algae dominating at 20 degrees C and only blue-green algae dominating at 35 degrees C. The productivity at 35 degrees C was lower than that at 20 degrees C because of the lower solubility of carbon dioxide at higher temperatures. At 10% salinity, green flagellates and pennate diatoms dominated. The productivity at this salinity was 50% that obtained at the salinity of seawater (3.5%). At 25% salinity, only the green flagellate, Dunaliella salina, survived at a productivity of 1% that obtained at the salinity of seawater.     

Oscillations in continuous culture populations of Streptococcus pneumoniae: population dynamics and the evolution of clonal suicide

Agents that kill or induce suicide in the organisms that produce them or other individuals of the same genotype are intriguing puzzles for ecologists and evolutionary biologists. When those organisms are pathogenic bacteria, these suicidal toxins have the added appeal as candidates for the development of narrow spectrum antibiotics to kill the pathogens that produce them. We show that when clinical as well as laboratory strains of Streptococcus pneumoniae are maintained in continuous culture (chemostats), their densities oscillate by as much as five orders of magnitude with an apparently constant period. This dynamic, which is unanticipated for single clones of bacteria in chemostats, can be attributed to population-wide die-offs and recoveries. Using a combination of mathematical models and experiments with S. pneumoniae, we present evidence that these die-offs can be attributed to the autocatalytic production of a toxin that lyses or induces autolysis in members of the clone that produces it. This toxin, which our evidence indicates is a protein, appears to be novel; S. pneumoniae genetic constructs knocked out for lytA and other genes coding for known candidates for this agent oscillate in chemostat culture. Since this toxin lyses different strains of S. pneumoniae as well as other closely related species of Streptococcus, we propose that its ecological role is as an allelopathic agent. Using a mathematical model, we explore the conditions under which toxins that kill members of the same clone that produces them can prevent established populations from invasion by different strains of the same or other species. We postulate that the production of the toxin observed here as well as other bacteria-produced toxins that kill members of the same genotype, ‘clonal suicide’, evolved and are maintained to prevent colonization of established populations by different strains of the same and closely related species.     

Factors affecting microbial sulfate reduction by Desulfovibrio desulfuricans in continuous culture: limiting nutrients and sulfide concentration

The effects of sulfate and nitrogen concentrations of the rate and stoichiometry of microbial sulfate reduction were investigated for Desulfovibrio desulfuricans grown on lactate and sulfate in a chemostat at pH 7.0. Maximum specific growth rates (mu(max)), half-saturation coefficients (K(sul)), and cell yield (Y(c/Lac)) of 0.344 +/- 0.007 and 0.352 +/- 0.003 h (-1), 1.8 +/- 0.3 and 1.0 +/- 0.2 mg/L, and 0.020 +/- 0.003 and 0.017 +/- 0.003 g cell/g lactate, respectively, were obtained under sulfate-limiting conditions at 35 degrees C and 43 degrees C. Maintenance energy requirements for D. desulfuricans were significant under sulfate-limiting conditions. The extent of extracellular polymeric substance (EPS) produced was related to the carbon: nitrogen ratio in the medium. EPS production rate increased with decreased nitrogen loading rate. Nitrogen starvation also resulted in decreased cell size of D. desulfuricans. The limiting C : N ratio (w/w) for D. desulfuricans was in the range of 45 : 1 to 120 : 1. Effects of sulfide on microbial sulfate reduction, cell size, and biomass production were also ivestigated at pH 7.0. Fifty percent inhibition of lactate utilization occurred at a total sulfide concentration of approximately 500 mg/L. The cell size of D. desulfuricans decreased with increasing total sulfide concentration. Sulfide inhibition of D. desulfuricans was observed to be a reversible process. (c) 1992 John Wiley & Sons, Inc.     

Studies of cereal nematode populations and cereal yields under continuous or intensive culture

In a field experiment on infested sandland from 1955 to 1967, numbers of Heterodera avenae increased only in 1957 and then decreased under both continuous and rotational spring oats (1955-62) and subsequently under spring barley (1963-67). Fluctuations in numbers of Pratylenchus neglectus and Trichodorus primitivus were recorded from 1963 to 1967. Cereal grain yields were generally unsatisfactory, but were consistently higher for cereals grown in rotation rather than continuously. Extra nitrogen fertilizer gave slightly improved yields with higher post-cropping numbers of H. avenae.     

Impact of dilution on microbial community structure and functional potential: comparison of numerical simulations and batch culture experiments

A series of microcosm experiments was performed using serial dilutions of a sewage microbial community to inoculate a set of batch cultures in sterile sewage. After inoculation, the dilution-defined communities were allowed to regrow for several days and a number of community attributes were measured in the regrown assemblages. Based upon a set of numerical simulations, community structure was expected to differ along the dilution gradient; the greatest differences in structure were anticipated between the undiluted-low-dilution communities and the communities regrown from the very dilute (more than 10(-4)) inocula. Furthermore, some differences were expected among the lower-dilution treatments (e.g., between undiluted and 10(-1)) depending upon the evenness of the original community. In general, each of the procedures used to examine the experimental community structures separated the communities into at least two, often three, distinct groups. The groupings were consistent with the simulated dilution of a mixture of organisms with a very uneven distribution. Significant differences in community structure were detected with genetic (amplified fragment length polymorphism and terminal restriction fragment length polymorphism), physiological (community level physiological profiling), and culture-based (colony morphology on R2A agar) measurements. Along with differences in community structure, differences in community size (acridine orange direct counting), composition (ratio of sewage medium counts to R2A counts, monitoring of each colony morphology across the treatments), and metabolic redundancy (i.e., generalist versus specialist) were also observed, suggesting that the differences in structure and diversity of communities maintained in the same environment can be manifested as differences in community organization and function.     

Simple generic model for dynamic experiments with Saccharomyces cerevisiae in continuous culture: decoupling between anabolism and catabolism

The dynamic behavior of a continuous culture of Saccharomyces cerevisiae subjected to a sudden increase in the dilution rate has been successfully modelled for anaerobic growth on glucose, and for aerobic growth on acetate, on ethanol, and on glucose. The catabolism responded by an immediate jump whereas biosynthesis did not. Thus catabolism was in excess to anabolism. The model considers the decoupling between biosynthesis and catabolism, both types of reactions being modelled by first-order kinetic expressions evolving towards maximal values. Yield parameters and maximal reaction rates were identified in steady state continuous cultures or during batch experiments. Only the time constant of biosynthesis regeneration, tauX, and the time constant of catabolic capacity regeneration, taucat, had to be identified during transient experiments. In most experiments tauX was around 3 h, and taucat varied between 2 and 2.5 h for the different metabolisms investigated.     

Competition of two suspension-feeding protozoan populations for a growing bacterial population in continuous culture

Mathematical studies for ecosystems involving 2 predators competing for a growing prey population have shown that the 2 competitors can coexist in a state of sustained oscillations for a range of values of the system parameters. For the case of 1 suspension-feeding protozoan population, recent experimental observations suggest that the predator-prey interaction is complicated by the ability of the bacteria to grow on products produced by the lysis of protozoan cells. This situation is studied here for the case where 2 suspension-feeding protozoan populations compete for a growing bacterial population in a chemostat. Computer simulations show that the 2 protozoan populations can coexist over a range of the operating parameters. Some necessary conditions for coexistence are presented as are some speculations regarding the possible physical explanations of results.     

Development of a novel continuous culture device for experimental evolution of bacterial populations

The availability of a robust and reliable continuous culture apparatus that eliminates wall growth problems would lead to many applications in the microbial field, including allowing genetically engineered strains to recover high fitness, improving biodegradation strains, and predicting likely antibiotic resistance mechanisms. We describe the design and implementation of a novel automated continuous culture machine that can be used both in time-dependent mode (similar to a chemostat) and turbidostat modes, in which wall growth is circumvented through the use of a long, variably divisible tube of growth medium. This tube can be restricted with clamps to create a mobile growth chamber region in which static portions of the tube and the associated medium are replaced together at equal rates. To functionally test the device as a tool for re-adaptation of engineered strains, we evolved a strain carrying a highly deleterious deletion of Elongation Factor P, a gene involved in translation. In 200 generations over 2 weeks of dilution cycles, the evolved strain improved in generation time by a factor of three, with no contaminations and easy manipulation.