A versatile bioreactor/fermenter system for 'omics' research on diverse aspects of microbial physiology

The proposed research covers multiple areas of microbial physiology. Single celled microbes such as bacteria and yeasts have to adapt rapidly to changes in their immediate environments. This adaptation capacity depends on their ability to constantly sense a multiplicity of environmental cues (such as temperature, nutritional status, oxygen availability, fluctuations in acidity and alkalinity, and the presence of small molecules that are made by other microbes). Bacteria can use small diffusible molecules as chemical signals reporting the density of bacterial populations (self and competitors). This information can be used to change the adaptive responses of the microbes such that they make new products that might be appropriate to that environment. For example, at high cell density bacteria may become more virulent by making toxins or enzymes that attack their host plants, animals or man. Similary, some microbes can make antibiotics that can kill their competitors and allow them to colonise or defend niches more effectively. Microbes have to respond to diverse environmental stresses - and they often have to do this rapidly as a 'life or death' response. In the genome sequences of microbes, as much as 30-40% of the genes have no predictable biological function so it is important to try to elucidate what these 'unknown' genes actually do for the microbe. Yeast cells are excellent experimental tools for such studies because their genome (genetic material) sequence is known and they are easily grown in the laboratory. Similarly, bacteria are easy to cultivate in laboratory conditions in growth media the composition of which can be easily controlled and defined. Thus, using bacteria and yeast as experimental tools, it is possible to try to understand many different aspects of microbial physiology and metabolism - in particular, how microbes adapt to environmental stresses such as nutritional fluctuations, intercellular chemical signalling and antibiotic production. To study these events in a controlled way it is essential to have apparatus that allows the reproducible growth of microbes in a totally controllable fashion, allowing the experimentalist the opportunity to define the nutrition of the microbe, its growth rate and all other environmenal parameters such as temperature and pH. The only way to achieve this reproducibly is to grow the microbes in a fermenter (bioreactor) that allows total growth control. Only then can experiments be done on the genetics, biochemistry, physiology and metabolism of the microbes to generate robust and reproducible results from which it is possible to deduce how the biology of microbes dictates their responses to their environment. This proposal, therefore, is aimed at securing funds for a simple and flexible fermenter system that can be accessed by five (at least) different research groups in the Department of Biochemistry who are working on a range of problems in the biology of microbes.

We need a robust, simple and versatile fermenter system for the study by multiple research groups of diverse physiological processes in bacteria and yeast, including studies on quorum sensing, secondary metabolism, protein secretion, virulence factor regulation, functional genomics and metabolomics, and metabolic responses to environmental stress. Until recently, commercially-available fermenters had a minimal working volume of around 1 liter. Such fermenters have a minimum turnaround time of 3 days ( to fill and autoclave the vessel, to perform the run, and to kill-off and clean the device for the next run) and so it is difficult to perform more than one or two runs per week. This severely limits the throughput possible with this type of device, and for the purposes of our studies, is wasteful of resources. However, a small-footprint fermenter that can simultaneously grow 4 cultures of ca. 100 mL volume each is now available. We tested this device and its performance characteristics are equal to those of larger, 'conventional' fermenters. The HEL Xplorer device offers system-embedded computer-control over growth parameters including DO, pH, temperature and impeller speed, and has user-friendly software. Each vessel can be fitted with an autoclavable optical density probe to permit on-line measurement of the cell density, so that growth can be monitored in real time. To further minimize perturbation to the system during sample acquisition, we will fit the fermenter vessels with ceramic FISP sampling probes which enable on-line harvesting of sterile culture supernatants (from a few hundred microliters to several milliliters at a time, as required ) without the need for invasive procedures that can alter the transcriptome/proteome/metabolome profiles. This robust set-up minimizes turnaround times and will be sufficiently versatile to allow multiple, simultaneous comparative analyses of growth responses to varying environmental parameters.