Quantitative Mixed Culture Studies with Three Bacterial Species

Microbial communities are the common mode of existence of bacteria in their natural habitats. As such complex systems gain more and more interest for Biotechnology as well as Medical and Ecological Research, there is an increasing need for detailed knowledge, e.g. of community growth characteristics and their complex interactions for better understanding and improved control. Results obtained by investigation of pure cultures are not easily transferred due to interferences between species that cannot be predicted. To our knowledge, quantitative growth studies of defined mixed cultures of three and more bacterial species have not yet been accomplished. We study a mixed culture consisting of three bacterial species (Pseudomonas aeruginosa, Burkholderia cepacia, Staphylococcus aureus) as model system relevant for medical applications. One of our aims is to characterize growth characteristics and to mathematically describe such a model community focusing on possible interspecies interactions. Determination of cell number by plate colony counting in literature is often reported for 2-species mixed culture studies. To apply this methode to our model system was ineligible considering consumption of time and reliability of obtained data. We chose a quantitative T-RFLP analysis method on a multicapillary sequencer (ABI 3100Avant, Applied Biosystems, Germany) for measuring specific species cellnumbers from mixed samples. Therefore, we adapted an T-RFLP analysis method established by Trotha et al. [1] for mixed species quantification. Comparison to selective-plate colony counts show that our method is less laborious and highly reproducible. Detailed results on quality and statistics of the method will be given. 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 three strains mentioned above for mixed culture experiments. The selected medium ensures carbon-limited growth conditions for the three strains. To provide defined and homogeneous growth conditions for mixed culture experiments we use a 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. Here, we present some first results of growth characteristics of our 3-species model community in comparison to results 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 : 1070-107