Mixed-species Cultures Research Articles

Investigation of Acidithiobacillus ferrooxidans in pure and mixed-species culture for bioleaching of Theisen sludge from former copper smelting.

The aim of this study was to investigate the potential of bioleaching for the treatment of an environmentally hazardous waste, a blast-furnace flue dust designated Theisen sludge. Bioleaching of Theisen sludge was investigated at acidic conditions with Acidithiobacillus ferrooxidans in pure and mixed-species culture with Acidiphilium. In shaking-flask experiments, bioleaching parameters (pH, redox potential, zinc extraction from ZnS, ferrous- and ferric-iron concentration) were controlled regularly. The analysis of the dissolved metals showed that 70% zinc and 45% copper were extracted. Investigations regarding the arsenic and antimony species were performed. When iron ions were lacking, animonate (Sb(V)) and total arsenic concentration were highest in solution. The bioleaching approach was scaled up in stirred-tank bioreactors resulting in higher leaching efficiency of valuable trace elements. Concentrations of dissolved antimony were approx. 23 times, and of cobalt, germanium, and rhenium three times higher in comparison to shaking-flask experiments, when considering the difference in solid load of Theisen sludge. The extraction of base and trace metals from Theisen sludge, despite of its high content of heavy metals and organic compounds, was feasible with iron-oxidizing acidophilic bacteria. In stirred-tank bioreactors, the mixed-species culture performed better. To the best of our knowledge, this study is the first providing an appropriate biological technology for the treatment of Theisen sludge to win valuable elements.

Prediction of Competitive Microbial Growth.

Prediction of competitive microbial growth is becoming important for microbial food safety. There would be two approaches to predict competitive microbial growth with mathematical models. The first approach is the development of a growth model for competitive microbes. Among several candidates for the competition model considered, the combination of the primary growth model of the new logistic (NL) model and the competition model of the Lotka-Vorttera (LV) model showed the best performance in predicting microbial competitive growth in the mixed culture of two species. This system further successfully predicted the growth of three competitive species in mixed culture. The second approach is the application of the secondary model especially for the parameter of the maximum cell population in the primary growth model. The combination of the NL model and a polynomial model for the maximum population successfully predicted Salmonella growth in raw ground beef. This system further successfully predicted Salmonella growth in beef at various initial concentrations and temperatures. The first approach requires microbial growth data in monoculture for analysis. The second approach to the prediction of competitive growth from the viewpoint of microbial food safety would be more suitable for practical application.

Quantitative Polymerase Chain Reaction for Microbial Growth Kinetics of Mixed Culture System.

Microbial growth kinetics is often used to optimize environmental processes owing to its relation to the breakdown of substrate (contaminants). However, the quantification of bacterial populations in the environment is difficult owing to the challenges of monitoring a specific bacterial population within a diverse microbial community. Conventional methods are unable to detect and quantify the growth of individual strains separately in the mixed culture reactor. This work describes a novel quantitative PCR (qPCR)-based genomic approach to quantify each species in mixed culture and interpret its growth kinetics in the mixed system. Batch experiments were performed for both single and dual cultures of Pseudomonas putida and Escherichia coli K12 to obtain Monod kinetic parameters (μmax and Ks). The growth curves and kinetics obtained by conventional methods (i.e., dry weight measurement and absorbance reading) were compared with that obtained by qPCR assay. We anticipate that the adoption of this qPCR-based genomic assay can contribute significantly to traditional microbial kinetics, modeling practice, and the operation of bioreactors, where handling of complex mixed cultures is required.

Culturing oil sands microbes as mixed species communities enhances ex situ model naphthenic acid degradation.

Oil sands surface mining for bitumen results in the formation of oil sands process water (OSPW), containing acutely toxic naphthenic acids (NAs). Potential exists for OSPW toxicity to be mitigated by aerobic degradation of the NAs by microorganisms indigenous to the oil sands tailings ponds, the success of which is dependent on the methods used to exploit the metabolisms of the environmental microbial community. Having hypothesized that the xenobiotic tolerant biofilm mode-of-life may represent a feasible way to harness environmental microbes for ex situ treatment of OSPW NAs, we aerobically grew OSPW microbes as single and mixed species biofilm and planktonic cultures under various conditions for the purpose of assaying their ability to tolerate and degrade NAs. The NAs evaluated were a diverse mixture of eight commercially available model compounds. Confocal microscopy confirmed the ability of mixed and single species OSPW cultures to grow as biofilms in the presence of the NAs evaluated. qPCR enumeration demonstrated that the addition of supplemental nutrients at concentrations of 1 g L-1 resulted in a more numerous population than 0.001 g L-1 supplementation by approximately 1 order of magnitude. GC-FID analysis revealed that mixed species cultures (regardless of the mode of growth) are the most effective at degrading the NAs tested. All constituent NAs evaluated were degraded below detectable limits with the exception of 1-adamantane carboxylic acid (ACA); subsequent experimentation with ACA as the sole NA also failed to exhibit degradation of this compound. Single species cultures degraded select few NA compounds. The degradation trends highlighted many structure-persistence relationships among the eight NAs tested, demonstrating the effect of side chain configuration and alkyl branching on compound recalcitrance. Of all the isolates, the Rhodococcus spp. degraded the greatest number of NA compounds, although still less than the mixed species cultures. Overall, these observations lend support to the notion that harnessing a community of microorganisms as opposed to targeted isolates can enhance NA degradation ex situ. Moreover, the variable success caused by NA structure related persistence emphasized the difficulties associated with employing bioremediation to treat complex, undefined mixtures of toxicants such as OSPW NAs.

PCR-Based Multiple Species Cell Counting for In Vitro Mixed Culture.

Changes of bacterial profiles in microbial communities are strongly associated with human health. There is an increasing need for multiple species research in vitro. To avoid high cost or measurement of a limited number of species, PCR-based multiple species cell counting (PCR-MSCC) has been conceived. Species-specific sequence is defined as a unique sequence of one species in a multiple species mixed culture. This sequence is identified by comparing a random 1000 bp genomic sequence of one species with the whole genome sequences of the other species in the same artificial mixed culture. If absent in the other genomes, it is the species-specific sequence. Species-specific primers were designed based on the species-specific sequences. In the present study, ten different oral bacterial species were mixed and grown in Brain Heart Infusion Yeast Extract with 1% sucrose for 24 hours. Biofilm was harvested and processed for DNA extraction and q-PCR amplification with the species-specific primers. By comparing the q-PCR data of each species in the unknown culture with reference cultures, in which the cell number of each species was determined by colony forming units on agar plate, the cell number of that strain in the unknown mixed culture was calculated. This technique is reliable to count microorganism numbers that are less than 100,000 fold different from other species within the same culture. Theoretically, it can be used in detecting a species in a mixed culture of over 200 species. Currently PCR-MSCC is one of the most economic methods for quantifying single species cell numbers, especially for the low abundant species, in a multiple artificial mixed culture in vitro.

Listeria monocytogenes-carrying consortia in food industry. Composition, subtyping and numerical characterisation of mono-species biofilm dynamics on stainless steel

In order to find out how real Listeria monocytogenes-carrying biofilms are in industrial settings, a total of 270 environmental samples belonging to work surfaces from fish (n=123), meat (n=75) and dairy industries (n=72) were analysed in order to detect L. monocytogenes. 12 samples were positive for L. monocytogenes and a total of 18 different species were identified as accompanying microbiota in fish and meat industry. No L. monocytogenes was found in samples from dairy industry. Molecular characterisation combining results of AscI and ApaI macrorestriction PFGE assays yielded 7 different subtypes of L. monocytogenes sharing in 71.43% of cases the same serogroup (1/2a–3a). Results from dynamic numerical characterisation between L. monocytogenes monospecies biofilms on stainless steel (SS) using MATLAB-based tool BIOFILMDIVER demonstrated that except in isolate A1, in which a significant increase in the percentage of covered area (CA), average diffusion distance (ADD) and maximum diffusion distance (MDD) was observed after 120h of culture, no significant differences were observed in the dynamics of the rest of the L. monocytogenes isolates. Quantitative dual-species biofilm association experiments performed on SS indicated that L. monocytogenes cell counts presented lower values in mixed-species cultures with certain species at 24 and 48h compared with mono-species culture. However, they remained unaltered after 72h except when co-cultured with Serratia fonticola which presented differences in all sampling times and was also the dominant species within the dual-species biofilm. When considering frequency of appearance of accompanying species, an ecological distribution was demonstrated as Escherichia coli appeared to be the most abundant in fish industry and Carnobacterium spp. in meat industry.

Single- and mixed-species biofilm formation by Escherichia coli O157:H7 and Salmonella, and their sensitivity to levulinic acid plus sodium dodecyl sulfate

The development of single- and mixed-species biofilms formed by Escherichia coli O157:H7 and Salmonella was observed, and the antimicrobial effectiveness of levulinic acid (LVA) plus sodium dodecyl sulfate (SDS) on the cells in single- and dual-species biofilms was determined. Biofilm-forming ability of single- and mixed-species cultures was observed by crystal violet staining and their resistance to levulinic acid plus SDS was determined by enumeration. Fluorescent protein-labeled E. coli O157:H7 and Salmonella were constructed and the bacterial composition of the biofilms after treatment with levulinic acid plus SDS was visualized by confocal laser scanning microscopy (CLSM). E. coli O157:H7 and Salmonella were antagonistic to each other, being more sensitive to levulinic acid plus SDS in mixed-species biofilms. Images captured by CLSM revealed that E. coli O157:H7 and Salmonella were distributed evenly in the single- and dual-species biofilms, and confirmed that the combination of levulinic acid and SDS was effective in inactivating bacterial cells in biofilms. Results revealed that levulinic acid with SDS may be used as a potential biofilm control intervention.

Open Mass Cultures of Marine Microalgae for Biodiesel Production: Laboratory Approach to Study Species Competition in Mixed Cultures

Since the interspecific competition represents a critical issue for biodiesel production in open mass cultures of microalgae, laboratory studies aimed to select competitive species receive significant interest. In this work, a laboratory approach based on the method of multisizing cell counting was developed to monitor the competition course of single algal species grown in mixed cultures. Two marine microalgae, Dunaliella tertiolecta and Phaeodactylum tricornutum, were used to set-up an induced-contamination assay, with the aim to test their competitive capabilities. The evaluation of the oil production by dried biomass extraction was coupled to the growth assay to investigate the effect of competition on oil yield. The adopted experimental approach revealed effective as a method to selectively measure the algal growth of single species in mixed cultures, enabling to evaluate the competitive properties of Dunaliella outgrowing the contaminant species Phaeodactylum. At the end of the experiment the dominant species contributed more than 90% to the total biomass, while no loss of oil production was observed, the oil yield in the mixed being even higher than in the unialgal culture (3.15 vs. 2.28 mg). The outcomes of the induced competition suggest the use of Dunaliella as a competitive oil-producer species and especially support the potential of the experimental approach to be used for preliminary screening to drive species selection for open mass cultures.

Warming shows differential effects on late-season growth and competitive capacity of Elodea canadensis and Potamogeton crispus in shallow lakes

Submerged macrophytes are likely to be affected by climate changes through changes in water temperatures and length of growing season. We conducted a lab experiment to examine the influence of a late-season temperature increase on growth, biomass allocation, and acclimation of 2 submerged macrophyte species, Elodea canadensis and Potamogeton crispus. We also ran competitive interaction experiments between the 2 species with mono- and mixed-species cultures in pots placed in outdoor heated mesocosms (5 years at ambient temperature and a higher temperature following the IPCC A2 scenario downscaled to local conditions but enhanced by 50%). In the lab, macrophytes collected in the 2 types of mesocosms were grown at ambient temperatures (12 °C in September and 8 °C in October) and 4 °C higher. Warming had an overall stronger effect on E. candensis than P. crispus, particularly within the low temperature range studied. Hence, the relative growth rate (RGR) of E. canadensis acclimated to ambient mesocosm conditions increased 6-fold from low (8 °C) to high (16 °C) temperature, whereas the RGR of P. crispus increased <2-fold. In the competitive interaction experiment, warming increased the biomass and RGR of E. canadensis in the monoculture. In addition, warming increased shoot elongation of the plant in both the monoculture and mixed culture. P. crispus was generally unaffected by warming when grown in both monoculture and mixed culture, but competition decreased the elongation of shoots pre-adapted to ambient conditions and grown in the warmer mesocosms. The decomposition rate of E. canadensis accelerated with warming but was unaffected in P. crispus. We conclude that E. canadensis is a stronger competitor than P. crispus under warmer late-season conditions; however, it may have a higher demand for oxygen due to the increased decomposition rates at higher temperatures, particularly in the peripheral growing season, with potential profound effects on lake ecosystems. Although acclimatisation was evident, we suggested that temperature changes will affect the growth pattern of the 2 plant species and thereby perhaps induce a switch in macrophyte species dominance.

Біоплівкова форма стафілококів у моно- та бівидовій культурі в поєднанні з лактобацилами

Біоплівкова форма стафілококів у моно- та бівидовій культурі в поєднанні з лактобацилами

Metabolism of n-C10:0 and n-C11:0 fatty acids by Trichoderma koningii, Penicillium janthinellum and their mixed culture: I. Biomass and CO2 production, and allocation of intracellular lipids

The capacity of two soil fungi, Trichoderma koningii and Penicillium janthinellum, to oxidize n-C10:0 and n-C11:0 fatty acids to CO2 and store intracellular lipids during growth is unknown. This article reports for the first time the metabolism of decanoic acid (DA, C10:0), undecanoic acid (UDA, n-C11:0), a mixture of the acids (UDA+DA) and a mixture of UDA+ potato dextrose broth (PDB) by T. koningii and P. janthinellum and their mixed culture. A control PDB complex substrate was used as a substrate control treatment. The fungal cultures were assayed for their capacity to: (1) oxidize n-C10:0 and n-C11:0 fatty acids to CO2 and (2) store lipids intracellularly during growth. On all four fatty acid substrates, the mixed T. koningii and P. janthinellum culture produced more biomass and CO2 than the individual fungal cultures. Per 150 mL culture, the mixed species culture grown on UDA+PDB and on PDB alone produced the most biomass (7,567 mg and 11,425 mg, respectively). When grown in DA, the mixed species culture produced the least amount of biomass (6,400 mg), a quantity that was lower than those obtained in UDA (7,550 mg) or UDA+DA (7,270 mg). Amounts of CO2 produced ranged from 210 mg under DA to 618 mg under PDB, and these amounts were highly correlated with biomass (r2 = 0.99). Fluorescence microscopy of stained lipids in the mixed fungal cell cultures growing during the exponential phase demonstrated larger fungal cells and higher accumulation of lipids in membranes and storage bodies than those observed during the lag and stationary phases. T. koningii and P. janthinellum grown on n-C10:0 and n-C11:0 fatty acids produced lower amounts of biomass and CO2, but stored higher amounts of intracellular lipids, than when grown on PDB alone.

Metabolism of nC11 fatty acid fed to Trichoderma koningii and Penicillium janthinellum II: Production of intracellular and extracellular lipids

Little is known about the fungal metabolism of nC10 and nC11 fatty acids and their conversion into lipids. A mixed batch culture of soil fungi, T. koningii and P. janthinellum, was grown on undecanoic acid (UDA), a mixture of UDA and potato dextrose broth (UDA+PDB), and PDB alone to examine their metabolic conversion during growth. We quantified seven intracellular and extracellular lipid classes using Iatroscan thin-layer chromatography with flame ionization detection (TLC-FID). Gas chromatography with flame ionization detection (GC-FID) was used to quantify 42 individual fatty acids. Per 150 mL culture, the mixed fungal culture grown on UDA+PDB produced the highest amount of intracellular (531 mg) and extracellular (14.7 mg) lipids during the exponential phase. The content of total intracellular lipids represented 25% of the total biomass-carbon, or 10% of the total biomass dry weight produced. Fatty acids made up the largest class of intracellular lipids (457 mg/150 mL culture) and they were synthesized at a rate of 2.4 mg/h during the exponential phase, and decomposed at a rate of 1.8 mg/h during the stationary phase, when UDA+PDB was the carbon source. Palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2) and vaccenic acid (C18:1) accounted for >80% of the total intracellular fatty acids. During exponential growth on UDA+PDB, hydrocarbons were the largest pool of all extracellular lipids (6.5 mg), and intracellularly they were synthesized at a rate of 64 μg/h. The mixed fungal species culture of T. koningii and P. janthinellum produced many lipids for potential use as industrial feedstocks or bioproducts in biorefineries.

Impacts of Carpobrotus edulis (L.) N.E.Br. on the germination, establishment and survival of native plants: a clue for assessing its competitive strength.

Does Carpobrotus edulis have an impact on native plants? How do C. edulis’ soil residual effects affect the maintenance of native populations? What is the extent of interspecific competition in its invasion process? In order to answer those questions, we established pure and mixed cultures of native species and C. edulis on soil collected from invaded and native areas of Mediterranean coastal dunes in the Iberian Peninsula. We examined the impact of the invader on the germination, growth and survival of seeds and adult plants of two native plant species (Malcolmia littorea (L.) R.Br, and Scabiosa atropurpurea L.) growing with ramets or seeds of C. edulis. Residual effects of C. edulis on soils affected the germination process and early growth of native plants in different ways, depending on plant species and density. Interspecific competition significantly reduced the germination and early growth of native plants but this result was soil, density, timing and plant species dependent. Also, at any density of adult individuals of C. edulis, established native adult plants were not competitive. Moreover, ramets of C. edulis had a lethal effect on native plants, which died in a short period of time. Even the presence of C. edulis seedlings prevents the recruitment of native species. In conclusion, C. edulis have strong negative impacts on the germination, growth and survival of the native species M. littorea and S. atropurpurea. These impacts were highly depended on the development stages of native and invasive plants. Our findings are crucial for new strategies of biodiversity conservation in coastal habitats.

The use of real-time PCR to study Penicillium chrysogenum growth kinetics on solid food at different water activities

Fungal growth on solid foods can make them unfit for human consumption, but certain specialty foods require fungi to produce their characteristic properties. In either case, a reliable way of measuring biomass is needed to study how various factors (e.g. water activity) affect fungal growth rates on these substrates. Biochemical markers such as chitin, glucosamine or ergosterol have been used to estimate fungal growth, but they cannot distinguish between individual species in mixed culture. In this study, a real-time polymerase chain reaction (rt-PCR) protocol specific for a target fungal species was used to quantify its DNA while growing on solid food. The measured amount of DNA was then related to the biomass present using an experimentally determined DNA-to-biomass ratio. The highly sensitive rt-PCR biomass assay was found to have a wide range, able to quantify the target DNA within a six orders-of-magnitude difference. The method was used to monitor germination and growth of Penicillium chrysogenum spores on a model porous food (cooked wheat flour) at 25°C and different water activities of 0.973, 0.936, and 0.843. No growth was observed at 0.843, but lag, exponential and stationary phases were identified in the growth curves for the higher water activities. The calculated specific growth rates (μ) during the exponential phase were almost identical, at 0.075/h and 0.076/h for aw=0.973 and 0.936, respectively. The specificity of the method was demonstrated by measuring the biomass of P. chrysogenum while growing together with Aspergillus niger on solid media at aw=0.973.

Does temperature-mediated reproductive success drive the direction of species displacement in two invasive species of leafminer fly?

Liriomyza sativae and L. trifolii (Diptera: Agromyzidae) are two highly invasive species of leafmining flies, which have become established as pests of horticultural crops throughout the world. In certain regions where both species have been introduced, L. sativae has displaced L. trifolii, whereas the opposite has occurred in other regions. These opposing outcomes suggest that neither species is an inherently superior competitor. The regions where these displacements have been observed (southern China, Japan and western USA) are climatically different. We determined whether temperature differentially affects the reproductive success of these species and therefore if climatic differences could affect the outcome of interspecific interactions where these species are sympatric. The results of life table parameters indicate that both species can develop successfully at all tested temperatures (20, 25, 31, 33°C). L. sativae had consistently higher fecundities at all temperatures, but L. trifolii developed to reproductive age faster. Age-stage specific survival rates were higher for L. sativae at low temperatures, but these were higher for L. trifolii at higher temperatures. We then compared the net reproductive rates (R 0) for both species in pure and mixed cultures maintained at the same four constant temperatures. Both species had significantly lower net reproductive rates in mixed species cultures compared with their respective pure species cultures, indicating that both species are subject to intense interspecific competition. Net reproductive rates were significantly greater for L. sativae than for L. trifolii in mixed species groups at the lower temperatures, whereas the opposite occurred at the higher temperature. Therefore, interactions between the species are temperature dependent and small differences could shift the competitive balance between the species. These temperature mediated effects may contribute to the current ongoing displacement of L. sativae by the more recent invader L. trifolii in warm climatic areas of China.

Interactions between Kluyveromyces marxianus and Saccharomyces cerevisiae in tequila must type medium fermentation

Traditional tequila fermentation is a complex microbial process performed by different indigenous yeast species. Usually, they are classified in two families: Saccharomyces and Non-Saccharomyces species. Using mixed starter cultures of several yeasts genera and species is nowadays considered to be beneficial to enhance the sensorial characteristics of the final products (taste, odor). However, microbial interactions occurring in such fermentations need to be better understood to improve the process. In this work, we focussed on a Saccharomyces cerevisiae/Kluyveromyces marxianus yeast couple. Indirect interactions due to excreted metabolites, thanks to the use of a specific membrane bioreactor, and direct interaction due to cell-to-cell contact have been explored. Comparison of pure and mixed cultures was done in each case. Mixed cultures in direct contact showed that both yeast were affected but Saccharomyces rapidly dominated the cultures whereas Kluyveromyces almost disappeared. In mixed cultures with indirect contact the growth of Kluyveromyces was decreased compared to its pure culture but its concentration could be maintained whereas the growth of Saccharomyces was enhanced. The loss of viability of Kluyveromyces could not be attributed only to ethanol. The sugar consumption and ethanol production in both cases were similar. Thus the interaction phenomena between the two yeasts are different in direct and indirect contact, Kluyveromyces being always much more affected than Saccharomyces.

Differentiation of the Emerging Human Pathogens Trichosporon asahii and Trichosporon asteroides from Other Pathogenic Yeasts and Moulds by Using Species-Specific Monoclonal Antibodies

The fungal genus Trichosporon contains emerging opportunistic pathogens of humans, and is the third most commonly isolated non-candidal yeast from humans. Trichosporon asahii and T. asteroides are the most important species causing disseminated disease in immunocompromised patients, while inhalation of T. asahii spores is the most important cause of summer-type hypersensitivity pneumonitis in healthy individuals. Trichosporonosis is misdiagnosed as candidiasis or cryptococcosis due to a lack of awareness and the ambiguity of diagnostic tests for these pathogens. In this study, hybridoma technology was used to produce two murine monoclonal antibodies (MAbs), CA7 and TH1, for detection and differentiation of Trichosporon from other human pathogenic yeasts and moulds. The MAbs react with extracellular antigens from T. asahii and T. asteroides, but do not recognise other related Trichosporon spp., or unrelated pathogenic yeasts and moulds including Candida, Cryptococcus, Aspergillus, Fusarium, and Scedosporium spp., or the etiologic agents of mucormycosis. Immunofluorescence and Western blotting studies show that MAb CA7, an immunoglobulin G1 (IgG1), binds to a major 60 kDa glycoprotein antigen produced on the surface of hyphae, while TH1, an immunoglobulin M (IgM), binds to an antigen produced on the surface of conidia. The MAbs were used in combination with a standard mycological growth medium (Sabouraud Dextrose Agar) to develop an enzyme-linked immunosorbent assay (ELISA) for differentiation of T. asahii from Candida albicans and Cryptococcus neoformans in single and mixed species cultures. The MAbs represent a major advance in the identification of T. asahii and T. asteroides using standard mycological identification methods.

Prediction of microbial growth in mixed culture with a competition model.

Prediction of microbial growth in mixed culture was studied with a competition model that we had developed recently. The model, which is composed of the new logistic model and the Lotka-Volterra model, is shown to successfully describe the microbial growth of two species in mixed culture using Staphylococcus aureus, Escherichia coli, and Salmonella. With the parameter values of the model obtained from the experimental data on monoculture and mixed culture with two species, it then succeeded in predicting the simultaneous growth of the three species in mixed culture inoculated with various cell concentrations. To our knowledge, it is the first time for a prediction model for multiple (three) microbial species to be reported. The model, which is not built on any premise for specific microorganisms, may become a basic competition model for microorganisms in food and food materials.

Development of a competition model for microbial growth in mixed culture.

A novel competition model for describing bacterial growth in mixed culture was developed in this study. Several model candidates were made with our logistic growth model that precisely describes the growth of a monoculture of bacteria. These candidates were then evaluated for the usefulness in describing growth of two competing species in mixed culture using Staphylococcus aureus, Escherichia coli, and Salmonella. Bacterial cells of two species grew at initial doses of 10(3), 10(4), and 10(5) CFU/g at 28ºC. Among the candidates, a model where the Lotka-Volterra model, a general competition model in ecology, was incorporated as a new term in our growth model was the best for describing all types of growth of two competitors in mixed culture. Moreover, the values for the competition coefficient in the model were stable at various combinations of the initial populations of the species. The Baranyi model could also successfully describe the above types of growth in mixed culture when it was coupled with the Gimenez and Dalgaard model. However, the values for the competition coefficients in the competition model varied with the conditions. The present study suggested that our model could be a basic model for describing microbial competition.

Biofilm development and enhanced stress resistance of a model, mixed-species community biofilm

Most studies of biofilm biology have taken a reductionist approach, where single-species biofilms have been extensively investigated. However, biofilms in nature mostly comprise multiple species, where interspecies interactions can shape the development, structure and function of these communities differently from biofilm populations. Hence, a reproducible mixed-species biofilm comprising Pseudomonas aeruginosa, Pseudomonas protegens and Klebsiella pneumoniae was adapted to study how interspecies interactions affect biofilm development, structure and stress responses. Each species was fluorescently tagged to determine its abundance and spatial localization within the biofilm. The mixed-species biofilm exhibited distinct structures that were not observed in comparable single-species biofilms. In addition, development of the mixed-species biofilm was delayed 1-2 days compared with the single-species biofilms. Composition and spatial organization of the mixed-species biofilm also changed along the flow cell channel, where nutrient conditions and growth rate of each species could have a part in community assembly. Intriguingly, the mixed-species biofilm was more resistant to the antimicrobials sodium dodecyl sulfate and tobramycin than the single-species biofilms. Crucially, such community level resilience was found to be a protection offered by the resistant species to the whole community rather than selection for the resistant species. In contrast, community-level resilience was not observed for mixed-species planktonic cultures. These findings suggest that community-level interactions, such as sharing of public goods, are unique to the structured biofilm community, where the members are closely associated with each other.