A novel concept combining experimental and mathematical analysis for the identification of unknown interspecies effects in a mixed culture

Bacteria in natural habitats only occur in consortia together with various other species. Characterization of bacterial species, however, is normally done by laboratory testing of pure isolates. Any interactions that might appear during growth in mixed-culture are obviously missed by this approach. Existing experimental studies mainly focus on two-species mixed cultures with species specifically chosen for their known growth characteristics, and their anticipated interactions. Various theoretical mathematical studies dealing with mixed cultures and possible interspecies effects exist, but often models cannot be validated due to a lack of experimental data. Here, we present a concept for the identification of interspecies effects in mixed cultures with arbitrary and unknown single-species properties. Model structure and parameters were inferred from single-species experiments for the reproduction of mixed-culture experiments by simulation. A mixed culture consisting of the three-species Pseudomonas aeruginosa, Burkholderia cepacia, and Staphylococcus aureus served as a model system. For species-specific enumeration a quantitative terminal restriction length polymorphism (qT-RFLP) assay was used. Based on models fitted to single-species cultivations, the outcome of mixed-culture experiments was predicted. Deviations of simulation results and experimental findings were then used to design additional single-cell experiments, to modify the corresponding growth kinetics, and to update model parameters. Eventually, the resulting mixed-culture dynamics was predicted and compared again to experimental results. During this iterative cycle, it became evident that the observed coexistence of P. aeruginosa and B. cepacia in mixed-culture chemostat experiments cannot be explained on the basis of glucose as the only substrate. After extension of growth kinetics, that is, for use of amino acids as secondary substrates, mixed-culture simulations represented the experimental findings very well. According to the model structure, as motivated by single-species experiments, the growth of P. aeruginosa and B. cepacia on glucose and amino acids could be assumed to be independent of each other. In contrast, both substrates are taken up simultaneously by S. aureus.     

Interspecific interactions in a methane-utilizing mixed culture

A two-member methane-utilizing mixed culture of bacteria, formed by combining two pure cultures isolated from a naturally occurring methane-utilizing mixed culture, was studied in continuous culture. From the nutritional requirements and substrate ranges of the pure cultures, a mechanism for the interspecific interactions occurring in the mixed culture was proposed. Product formation kinetics were determined in continuous culture for each product involved in the proposed mechanism. From this proposed mechanism a mathematical model was derived based on simple material balance equations around a single-stage chemostat. The steady-state predictions of this model were compared to experimental results obtained from continuous-culture experiments with the two-member methane-utilizing mixed culture. Interspecific interactions occurring in two-member methanol-utilizing and three-member methane-utilizing mixed cultures have also been discussed.     

Coexistence in the chemostat as a result of metabolic by-products

Classical chemostat models assume that competition is purely exploitative and mediated via a common, limiting and single resource. However, in laboratory experiments with pathogens related to the genetic disease Cystic Fibrosis, species specific properties of production, inhibition and consumption of a metabolic by-product, acetate, were found. These assumptions were implemented into a mathematical chemostat model which consists of four nonlinear ordinary differential equations describing two species competing for one limiting nutrient in an open system. We derive classical chemostat results and find that our basic model supports the competitive exclusion principle, the bistability of the system as well as stable coexistence. The analytical results are illustrated by numerical simulations performed with experimentally measured parameter values. As a variant of our basic model, mimicking testing of antibiotics for therapeutic treatments in mixed cultures instead of pure ones, we consider the introduction of a lethal inhibitor, which cannot be eliminated by one of the species and is selective for the stronger competitor. We discuss our theoretical results in relation to our experimental model system and find that simulations coincide with the qualitative behavior of the experimental result in the case where the metabolic by-product serves as a second carbon source for one of the species, but not the producer.     

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.     

Growth of Salmonella enterica in model mixed cultures during a two-step enrichment

Growth of Salmonella enterica was studied in model mixed cultures with Citrobacter freundii or Escherichia coli in buffered peptone water (BPW) and in Rappaport-Vassiliadis medium with soya (RVS) with modified concentrations of MgCl2 and malachite green, and at modified incubation temperatures. Selected S. enterica strains were inoculated in BPW (10(0) cfu/ml) together with selected strains of Citrobacter freundii (up to 10(8) cfu/ml) or selected strains of Escherichia coli (up to 10(8) cfu/ml), incubated overnight and then subcultured (1: 100) in RVS variants. Growth of individual bacterial species was followed by the quantitative real-time polymerase chain reaction (PCR). Optimal culture conditions during the second selective step were: MgCl2.6 H2O concentration of 29 g/l, malachite green concentration of 36 mg/1l, and the incubation temperature of 41.5 degrees C. Citr. freundii was found to be a potent competitor and E. coli was a weaker competitor. At optimal culture conditions, competition was reduced and the density of S. enterica cultures reached the level of 10(4) cfu/ml after not later than 2 h of selective enrichment. The results obtained provide a basis for the development of a short two-step enrichment to be used in rapid real-time PCR-based methods for the detection of S. enterica in food and other matrices.     

Evolution as a critical component of plankton dynamics

Microevolution is typically ignored as a factor directly affecting ongoing population dynamics. We show here that density-dependent natural selection has a direct and measurable effect on a planktonic predator-prey interaction. We kept populations of Brachionus calyciflorus, a monogonont rotifer that exhibits cyclical parthenogenesis, in continuous flow-through cultures (chemostats) for more than 900 days. Initially, females frequently produced male offspring, especially at high population densities. We observed rapid evolution, however, towards low propensity to reproduce sexually, and by 750 days, reproduction had become entirely asexual. There was strong selection favouring asexual reproduction because, under the turbulent chemostat regime, males were unable to mate with females, produced no offspring, and so had zero fitness. In replicated chemostat experiments we found that this evolutionary process directly influenced the population dynamics. We observed very specific but reproducible plankton dynamics which are explained well by a mathematical model that explicitly includes evolution. This model accounts for both asexual and sexual reproduction and treats the propensity to reproduce sexually as a quantitative trait under selection. We suggest that a similar amalgam of ecological and evolutionary mechanisms may drive the dynamics of rapidly reproducing organisms in the wild.     

Growth of mixed cultures of Actinomyces viscosus and Streptococcus mutans under dual limitation of glucose and oxygen

Abstract Streptococcus mutans and Actinomyces viscosus are among the dominant species in human dental plaque. In their natural environment, carbohydrate- and oxygen-limited conditions are likely to occur frequently. Therfore, mixed cultures of the 2 species were studied under dual limitation of glucose and oxygen. Over a wide range of oxygen-supply rates, coexistence of A. viscosus and S. mutans was observed, within this range A. viscosus increased almost linearly with oxygen supply. A mathematical model based on Monod-type type kinetics and accounting for uncompetitive inhibition of growth by oxygen was developed to simulate these mixed cultures. The model predicted coexistence over a fairly large range of aeration rates. This finding, in combination with the results of the chemostat experiments, led to the conclusion that coexistence of the two species     

Modeling lipid accumulation in oleaginous fungi in chemostat cultures: I. Development and validation of a chemostat model for Umbelopsis isabellina

Lipid-accumulating fungi may be able to produce biodiesel precursors from agricultural wastes. As a first step in understanding and evaluating their potential, a mathematical model was developed to describe growth, lipid accumulation and substrate consumption of the oleaginous fungus Umbelopsis isabellina (also known as Mortierella isabellina) in submerged chemostat cultures. Key points of the model are: (1) if the C-source supply rate is limited, maintenance has a higher priority than growth, which has a higher priority than lipid production; (2) the maximum specific lipid production rate of the fungus is independent of the actual specific growth rate. Model parameters were obtained from chemostat cultures of U. isabellina grown on mineral media with glucose and NH₄ ⁺. The model describes the results of chemostat cultures well for D > 0.04 h⁻¹, but it has not been validated for lower dilution rates because of practical problems with the filamentous fungus. Further validation using literature data for oleaginous yeasts is described in part II of this paper. Our model shows that not only the C/N-ratio of the feed, but also the dilution rate highly influences the lipid yield in chemostat cultures.     

Investigation of a simple amperometric electrode system to rapidly quantify and detect bacteria in foods

A two electrode system mounted as a single probe was developed to measure electrochemically the rate of reduction of a redox mediator (thionine) by bacteria. The system gave a rapid (2 min) bacterial-dependent current above 2.5 x 10(5) cfu/ml with pure cultures of bacteria, but when applied to the measurement of the bacterial contamination in samples of meat and milk it was unable to detect or quantify the contamination reliably. Incubation of samples for a few hours before examination enabled the system to detect bacteria in excess of 10(6) cfu/ml.     

The physiology and pathogenicity of Mycobacterium tuberculosis grown under controlled conditions in a defined medium

A chemically-defined culture medium was developed which supported batch growth of Mycobacterium tuberculosis, strain H37Rv, at a minimum doubling time of 14.7 h. This medium also facilitated chemostat culture of M. tuberculosis at a constant doubling time of 24 h. Chemostat growth was optimized at a dissolved oxygen tension of 20% (v/v) and 0.2% (v/v) Tween-80. Chemostat cultures were dispersed suspensions of single bacilli (1.5-3 microm long), or small aggregates, at a mean density of log10 8.3 cfu ml-1. A limited number of amino acids was utilized (alanine, asparagine, aspartate and serine were depleted by >50%; glycine, arginine, isoleucine, leucine and phenylalanine, by approximately 40%). Chemostat-grown cells were pathogenic in aerosol-infected guinea pigs, producing disseminated infection similar to that caused by plate-grown cells. Cells from chemostat culture were significantly more invasive for J774A.1 mouse macrophages than agar- or batch-grown cells. This study demonstrates the suitability of chemostat culture for the growth of pathogenic mycobacteria in a defined physiological state with potential applications for the controlled production of mycobacterial components for therapeutic and vaccine applications.     

Using terminal restriction fragment length polymorphism (T-RFLP) to identify mycorrhizal fungi: a methods review

Terminal restriction fragment length polymorphism (T-RFLP) is an increasingly widely used technique in mycorrhizal ecology. In this paper, we review the technique as it is used to identify species of mycorrhizal fungi and distinguish two different versions of the technique: peak-profile T-RFLP (the original version) and database T-RFLP. We define database T-RFLP as the use of T-RFLP to identify individual species within samples by comparison of unknown data with a database of known T-RFLP patterns. This application of T-RFLP avoids some of the pitfalls of peak-profile T-RFLP and allows T-RFLP to be applied to polyphyletic functional groups such as ectomycorrhizal fungi. The identification of species using database T-RFLP is subject to several sources of potential error, including (1) random erroneous matches of peaks to species, (2) shared T-RFLP profiles across species, and (3) multiple T-RFLP profiles within a species. A mathematical approximation of the risk of the first type of error as a function of experimental parameters is discussed. Although potentially less accurate than some other methods such as clone libraries, the high throughput of database T-RFLP permits much greater replication and may, therefore, be preferable for many ecological questions, particularly when combined with other techniques such as cloning.     

Development of a diagnostic test for Yersinia pestis by the polymerase chain reaction

A 501 bp caf1 gene fragment and a 443 bp of pla gene fragment carried by 100 kb (pFra) and 10 kb (pPst) species-specific extrachromosomal replicons, respectively, were used as targets to study the conditions under which DNA amplification by polymerase chain reaction (PCR) may be applied to detect and identify Yersinia pestis DNA in cell lysates of pure cultures and biological samples. The sensitivity limit of PCR with the crude cell lysates of Y. pestis EV was estimated as 10–50 cfu in reaction mixture. When target Y. pestis EV cells were mixed with fresh blood of white mice, which contained 0.4% potassium citrate, the PCR detection level varied from 400 to 100 cfu ml^-1 of blood depending on the method used for preparing the sample. In our tests PCR was effective for the detection of yersinia in the blood of white laboratory mice experimentally infected with virulent Y. pestis KM638 strain. This method can be considered convenient for routine detection and identification of Y. pestis.     

Determination of in vivo oxygen uptake and carbon dioxide evolution rates from off-gas measurements under highly dynamic conditions

In vivo kinetics of Saccharomyces cerevisiae are studied, in a time window of 150 s, by analyzing the response of O(2) and CO(2) in the fermentor off-gas after perturbation of chemostat cultures by metabolite pulses. Here, a new mathematical method is presented for the estimation of the in vivo oxygen uptake rate (OUR) and carbon dioxide evolution rate (CER) directly from the off-gas data in such perturbation experiments. The mathematical construction allows effective elimination of delay and distortion in the off-gas measurement signal under highly dynamic conditions. A black box model for the fermentor off-gas system is first obtained by system identification, followed by the construction of an optimal linear filter, based on the identified off-gas model. The method is applied to glucose and ethanol pulses performed on chemostat cultures of S. cerevisiae. The estimated OUR is shown to be consistent with the independent dissolved oxygen measurement. The estimated in vivo OUR and CER provide valuable insights into the complex dynamic behavior of yeast and are essential for the establishment and validation of in vivo kinetic models of primary metabolism.     

Predicting stability of mixed microbial cultures from single species experiments: 2. Physiological model

In this paper, we study the equilibria of a physiological model describing the continuous culture in which two microbial populations compete for two substitutable resources. This work is an extension of the stability analysis of the phenomenological model of mixed microbial growth [M.M. Ballyk, G.S.K. Wolkowicz, Exploitative competition in the chemostat for two perfectly substitutable resources, Math. Biosci. 118 (1993) 127-180; S.S. Pilyugin, G.T. Reeves, A. Narang, Predicting stability of mixed microbial cultures from single species experiments: 2. Phenomenological model]. Here, we investigate the influence of the peripheral enzymes that catabolize the substrate uptake on the stability of the mixed culture. We show that, under steady state conditions, an increase in the concentration of one substrate inhibits the uptake of the other substrate(s). We present the criteria for existence, uniqueness, and stability of various types of equilibria. We formulate these criteria in terms of growth isoclines and consumption curves for each of the competing species. Since both types of curves can be obtained from a single species experiment, our approach provides a direct connection between theory and experiment and allows one to infer the dynamics of mixed cultures from the dynamics of single species cultures. By expressing the stability criteria in terms of intracellular properties, the model establishes a link between ecology and molecular biology.     

Investigation of a simple amperometric electrode system to rapidly quantify and detect bacteria in foods

A two electrode system mounted as a single probe was developed to measure electrochemically the rate of reduction of a redox mediator (thionine) by bacteria. The system gave a rapid (2 min) bacterial-dependent current above 2.5×10⁵ cfu/ml with pure cultures of bacteria, but when applied to the measurement of the bacterial contamination in samples of meat and milk it was unable to detect or quantify the contamination reliably. Incubation of samples for a few hours before examination enabled the system to detect bacteria in excess of 10⁶ cfu/ml.     

A combined PCR and selective enrichment method for rapid detection of Listeria monocytogenes.

Development of a routine detection assay for Listeria monocytogenes in foods that uses the polymerase chain reaction (PCR) and enrichment cultures was investigated. Oligonucleotide primers were chosen to amplify a 3' region of L. monocytogenes hlyA gene spanning a conserved HindIII site. PCR detection sensitivity for L. monocytogenes in dilutions of pure enrichment cultures was between 50 and 500 colony forming units. A short enrichment period before PCR amplification allowed detection of the organisms in a range of complex foods contaminated with 10(4) cfu/g. Detection sensitivity for the assay in the presence of chicken skin and soft cheese was determined at 10-100 cfu/g. Utilization of enrichment cultures and PCR allowed identification of the organism within 24 h or 2 days.     

Development of a PCR method for mycoplasma testing of Chinese hamster ovary cell cultures used in the manufacture of recombinant therapeutic proteins

Chinese hamster ovary (CHO) cell cultures used to produce biopharmaceuticals are tested for mycoplasma contamination as part of the ensurance of a safe and pure product. The current U.S. Food and Drug Administration (FDA) regulatory guideline recommends using two procedures: broth/agar cultures and DNA staining of indicator cell cultures. Although these culture methods are relatively sensitive to most species, theoretically capable of detecting as few as 1-10 cfu/ml of most species, the overall procedure is lengthy (28 d), costly and less sensitive to noncultivable species. The detection of mycoplasma using the polymerase chain reaction (PCR) method has been considered an alternative method because it is relatively fast (1-2 d), inexpensive, and independent of culture conditions, however, limitations in sensitivity (limit of detection >/=1000 cfu/ml) and the risk of false positive and false negative results have prevented PCR from replacing the traditional culture methods in the industrial setting. In this report, we describe a new PCR assay for mycoplasma detection that appears to resolve these issues while being sufficiently simple and inexpensive for routine use. This assay applies readily available techniques in DNA extraction together with a modified single-step PCR using a previously characterized primer pair that is homologous to a broad spectrum of mycoplasma species known to infect mammalian cell cultures. Analysis is made easy by the detection of only a single amplification product within a narrow size range, 438-470 bp. A high sensitivity and specificity for mycoplasma detection in CHO cell production cultures is made possible through the combination of three key techniques: 8-methoxypsoralen and UV light treatment to decontaminate PCR reagents of DNA; hot-start Taq DNA polymerase to reduce nonspecific priming events; and touchdown- (TD-) PCR to increase sensitivity while also reducing nonspecific priming events. In extracts of mycoplasma DNA, the limit of detection for eight different mycoplasma species is 10 genomic copies. In CHO cell production cultures containing gentamicin, the limit of detection for a model organism, gentamicin-resistant M. hyorhinis, is 1 cfu/ml. The sensitivity and specificity of this PCR assay for mycoplasma detection in CHO cell production cultures appear similar to the currently used culture methods and thus should be considered as an alternative method by the biopharmaceutical industry.