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Abstract

We study a mixed culture consisting of three bacterial species Pseudomonas aeruginosa, Burkholderia cepacia, and Staphylococcus aureus as model system relevant for medical applications. Our approach is to experimentally characterize species-specific growth as community and to mathematically analyze these growth dynamics. We use modeling and simulation to describe and better understand our model community focusing on possible interspecies interactions. In contrast to the high number of pure modeling results in literature for up to n species, experimental validation to our knowledge has not yet been performed for a system comprising more than 2-species. Plate dilution for determination of cell number is reported in literature for 2-species mixed culture studies. As this method was not applicable for our model system considering consumption of time and material, we chose a quantitative T-RFLP analysis method on a multicapillary sequencer (ABI 3100Avant, Applied Biosystems, Germany). For measuring specific species cell numbers from mixed samples a T-RFLP method established by Trotha et al. [1] was adapted. Comparison to selective-plate colony counts showed that our method is less laborious and highly reproducible. In preliminary studies several chemically defined media were tested and pure culture growth characteristics of different bacterial strains were determined. Comparison of results lead to the selection of the three strains mentioned above for mixed culture experiments. The chosen medium ensures carbon-limited growth conditions for the three strains and guarantees the possibility of quantifying every component that might be used up or produced. To provide defined and homogeneous growth conditions for mixed culture experiments we use a 2 L-stirred tank reactor (Biostat B2, B. Braun, Germany). Process parameters are controlled and monitored online by a digital process control system (PCS 7, Siemens AG, Germany). Online-analysis of exhaust gas is used for calculation of OTR, CTR and RQ during cultivations. We will present results of growth characteristics in chemostat mode of our 3-species mixed culture in comparison to results from pure cultures. Experimental data will be compared to modeling results based on parameter estimation from pure cultures. [1] Trotha R., Reichl U., Thies F. L., Sperling D., Koenig W., and Koenig B. (2002). Adaptation of a Fragment Analysis Technique to an Automated High-throughput Multicapillary Electrophoresis Device for the Precise Qualitative and Quantitative Characterization of Microbial Communities. Electrophoresis 23:1070-1079