Research Accomplishment Reports 2007

Post-Genomic Characterization of Anaerobic Bacterial Metabolism

H.J. Strobel
Department of Animal and Food Sciences

 

Project Description

There is considerable interest in the development of technologies for producing chemicals, materials, and energy using biologically-based processes. Much of this is prompted by the realization that continued use of fossil fuel will have negative impacts on the environment, economy, and national security. So called 'green-technologies' are generally more sustainable than current practices, but there remain significant gaps in fundamental scientific knowledge and application-based information.

This project involves work with an anaerobic bacteria that has considerable promise in the bioprocessing of agricultural and forestry biomass to bio-fuels, namely ethanol. Given Kentucky's considerable biomass resources, this project has relevance to development of locally based bioprocessing industries and the emergence of the state as a producer of bio-fuels. The physiology and metabolism of the bacterium Clostridium thermocellum will be studied using state-of-the-art bio-analytical techniques, and the project results will help develop more effective biological catalysts that can be used for production of bio-materials and bio-fuels. The overall goal is to apply emerging post-genomic technologies in the characterization of anaerobic bacterial metabolism. Clostridium thermocellum will be used since this anaerobic, thermophilic bacterium has potential utility as a bio-catalyst in bio-fuels production. Specifically, proteomic and metabolomic analyses of ethanol sensitive and ethanol tolerant C. thermocellum strains will be conducted.

Low tolerance to ethanol is one barrier that currently limits the commercial application of the organism. Emerging techniques that do not utilize traditional gel-based methods will be used to separate and identify proteins expressed by the different strains. These techniques will permit the more rapid and complete profiling of protein expression in response to ethanol. In addition, mass spectrometry-based analysis of metabolite production will be conducted to develop a more in depth understanding of metabolic responses that occur in the two different strains.

We are currently expanding our understanding of C. thermocellum physiology by quantitatively profiling small molecule metabolites. Such metabolomic analysis provides a physiological interpretation of proteomic and genomic information. We have coupled capillary electrophoresis-based techniques to ion trap mass spectrometry to provide rapid and sensitive identification of metabolites. Conditions were optimized for sensitive detection, and calibration plots were obtained for 12 targeted metabolites to permit quantitative study. A rapid filtration and extraction protocol was then developed that involves exposure of cells to a boiling ethanol-buffer solution. We are using this approach to compare metabolite levels in wild type and ethanol adapted strains.

Impact

Continued dependence on fossil-based fuels has negative impacts on the environment, economy, and national security. The conversion of biomass by microorganisms to bio-based products and bio-energy is a sustainable alternative. Estimates are that nearly 11 million tons of agricultural, forestry and urban-waste fibrous biomass could be used each year for bio-fuel production in Kentucky. Bio-conversion of these feedstocks could yield 600 million gallons of ethanol, and this production could replace a significant quantity of the gasoline utilized in the state. However, there are still significant barriers that prevent the economical implementation of bio-based technologies. Our studies combine the information contained in genomic sequence databases with the emerging fields of proteomics and metabolomics to examine the physiology of anaerobic bacteria under industrially relevant conditions. The results of this work will be useful in designing economically relevant bio-based processes.