Progress 09/01/04 to 08/31/07
Outputs
OUTPUTS: Domestic bio-ethanol offers promise for diversifying the national fuel portfolio, decreasing oil consumption, and improving environmental quality. Nearly all bio-ethanol in the United States is currently derived from fermentation of maize starch by yeast, but lifecycle analyses suggest that this production scheme offers little, if any, environmental advantage compared with petroleum-based fuels. Bioconversion of lignocellulosic biomass by thermophilic anaerobic bacteria circumvents some of the problems associated with yeast fermentation of corn. But one of the technological barriers to efficient biomass conversion by bacteria is the relatively low ethanol tolerance that nearly all prokaryotes have when compared to yeast. Other microbial processes of commercial importance are also negatively impacted by fermentation end products, and this sensitivity constrains the technological application of many microorganisms. There is still relatively little detailed information on the
mechanisms responsible for physiological alterations that occur in response to ethanol and other solvents. Until recently, it was tedious and time-consuming to examine protein expression changes in cells responding to an environmental stress such as solvent exposure. However, state-of-the-art proteomic approaches are now available to efficiently identify protein expression changes. Clostridium thermocellum is a thermophilic anaerobic bacterium that directly converts lignocellulosic feedstocks into ethanol and has been proposed as a bio-catalyst in bio-conversion processes. The genome of this bacterium has been sequenced and this bio-informatic resource can be directly used in proteomic analysis. Our hypothesis was that exposure of C. thermocellum to ethanol causes specific alterations in protein expression patterns, and the overall project goal was to identify and characterize these changes using proteomic approaches. We previously adapted a strain of C. thermocellum to tolerate up to
8% (w/v) ethanol and used this novel organism in proteomic investigations. Since our preliminary work indicated that bacterial membrane protein profiles were altered by exposure to ethanol, particular attention was given to this sub-cellular fraction. A combination of gel-based and two-dimensional liquid chromatography protein separation schemes coupled to tandem mass spectrometry (Multi-dimensional Protein Identification Technology; MudPIT) were employed to define alterations in protein expression for bacterial strains that were either sensitive or tolerant of ethanol. The results of these experiments provide information needed to overcome process limitations and to optimize microbial conversion of lignocellulosic biomass to ethanol, solvents and other useful products.
PARTICIPANTS: Drs. Herbert Strobel and Bert Lynn were the project directors and were responsible for overseeing the general design and execution of experiments, interpretation of results, writing manuscripts, and supervising other personnel. There were two post-doctoral associates who were consecutively employed during the granting period. Drs. Xinping Lou and You-Jun Fu each devoted approximately 18 months effort to the project. They were principally responsible for mass spectromety analysis in the initial experimental phase as well as development of the MudPIT approach during the latter portion of the project. Dr. Taufika Williams was the graduate student who developed the novel gel-based technique used to seperate membrane proteins. Several resaearch analysts in Dr. Strobel's laboratory were responsible for the growth of microbial cultures, preoparation of sub-cellular fractions, and gel electrohoresis of samples.
TARGET AUDIENCES: The main target audience is the academic community that is engaged in research dealing with (i) bioconversion of lignocellulosic biomass; (ii) solvent tolerance in microorganisms; (iii) development of proteomic analysis methodologies. Another target audience is commercial research and development scientists engaged in developing and optimizing bioconversion processes that employ microorganisms.
Impacts
The protein expression patterns of ethanol sensitive and tolerant C. thermocellum strains were compared using sophisticated proteomic approaches. We first designed a novel gel-based separation method that circumvented problems associated with traditional membrane protein analysis. Approximately 60% of membrane proteins identified by mass spectrometry were differentially expressed. Most (73%) of these proteins were down-regulated in ethanol-tolerant cells, and a significant proportion were involved with carbohydrate transport and metabolism. Overall, the results suggest that many membrane-associated proteins in the tolerant strain were either being synthesized in lower quantities or failed to properly incorporate into the membrane. Although our method was useful, protein resolution and detection are limited in gel-based separations, particularly for those molecules that are high molecular weight, very basic, or hydrophobic. Therefore, we developed a two-dimensional
liquid chromatography separation scheme coupled to tandem mass spectrometry (Multi-dimensional Protein Identification Technology; MudPIT). This non-gel approach was refined to provide quantification by using a metabolic labeling strategy. Ethanol-sensitive cells were grown in the presence of isotopically labeled ammonium sulfate (15-N) while the tolerant strain was grown in medium containing non-labeled ammonium sulfate (14-N). The 15-N labeled cells served as internal standards, and this minimized experimental errors and provided better quantitative comparison of differences in protein levels. MudPIT analysis identified 168 and 172 proteins from membrane and cytosolic fractions, respectively. Many proteins were up-regulated in tolerant cells and a striking fraction (more than 20%) were ribosomal proteins. Significant up-regulation in tolerant cells was also noted for enzymes found in key energy transduction pathways, oxidation-reduction reactions, and those responsible for protein
folding. Tolerant cells were quite different from sensitive cells when viewed via light microscopy. Subsequent analysis with electron microscopy indicated major changes in the ultra-structure of the cell envelope in the tolerant strain including an inability to properly divide. Interestingly, several cell division proteins were apparently down-regulated in the tolerant cells. Based on these results, an integrative model was developed to describe the global changes that that occur in response to ethanol exposure. In summary, we have developed a reliable analysis platform that can be used to profile the C. thermocellum proteome, and this is the first quantitative proteomic analysis of the organism using the MudPIT approach. This project provides information needed to overcome present process limitations and to optimize microbial conversion of lignocellulosic biomass. It is estimated that there are nearly 11 million tons of agricultural, forestry and urban-waste fibrous biomass that
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 could replace a significant quantity of the gasoline utilized in the state.
Publications
- Williams, T., Lynn, B., Combs, J., and Strobel, H. J. (2005). Proteomic Profile Changes in Membranes of Ethanol-Tolerant Clostridium thermocellum. General Meeting American Society for Microbiology, Atlanta, GA, May 1-6, 2005.
- Williams, T., Lynn, B., Combs, J., and Strobel, H. J. (2005). The membrane proteome of Clostridium thermocellum. by MALDI-TOF-MS. American Society of Mass Spectrometry Meeting. San Antonio, TX.
- Williams, T. I., Combs, J. C., Thakur,A. P., Strobel,H. J., and Lynn, B. C (2006). A novel bicine running buffer system for doubled sodium dodecyl sulfate polyacrylamide gel electrophoresis of membrane proteins. Electrophoresis 27:2984-2995.
- Liu, X. P., Lynn, B. C., Combs, J. C., and and Strobel, H. J. (2006). Proteomic Analysis of Clostridium thermocellum Using Two-Dimensional Liquid Chromatography Separation and Tandem Mass Spectrometry. General Meeting American Society for Microbiology, Orlando, FL, May 21-26, 2006.
- Liu, X. P., Strobel, H. J., Combs, J. C., and Lynn, B. C. (2006). Quantitative Proteomic Analysis of Clostridium thermocellum Using 15N-Metabolic Labeling . American Society of Mass Spectrometry Meeting. Seattle, WA.
- Fu, Y- J., Strobel, H. J., and Lynn, B. C. (2007). Proteomic Analysis of Clostridium thermocellum sub-cellular fractions using 15N-metabolic labeling strategy. American Society of Mass Spectrometry Meeting. Indianapolis, In.
- Williams, T. I., Combs, J. C., Lynn, B. C., and Strobel, H. J. (2007). Proteomic profile changes in membranes of ethanol-tolerant Clostridium thermocellum. Applied Microbiology and Biotechnology 74:422-432.
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Progress 01/01/06 to 12/31/06
Outputs
Increased bio-fuel production in the United States is being prompted by recognition that continued use of fossil fuels has negative consequences for the environment, economy, and national security. Bio-ethanol offers promise for diversifying the national fuel portfolio, decreasing oil consumption, reducing environmental impacts, and increasing domestic markets for agricultural commodities. Nearly all bio-ethanol in the United States is currently produced using yeast fermentation of maize starch, but there are constraints to the cost-effective and long-term use of this approach. In addition, some lifecycle analyses suggest that producing bio-ethanol from corn offers little, if any, environmental advantage to petroleum-based fuels. An alternative strategy is to use thermophilic anaerobic bacteria for the conversion of cellulosic biomass to ethanol. Using cellulosic feedstocks has the potential to be economically and environmentally viable in the long-term. Clostridium
thermocellum is a thermophilic anaerobic bacterium that directly converts fibrous feedstocks into ethanol. However, a technological barrier to efficient biomass conversion by this organism is its relatively low ethanol tolerance. We have selected an ethanol-resistant strain of C. thermocellum and are using state-of-the-art protein analysis techniques to characterize the molecular basis of ethanol resistance. Our initial studies utilized traditional gel-based protein separations, and we demonstrated that protein expression in the tolerant strain was dramatically different than in the sensitive wild-type. However, gel-based separations are limited by their ability to detect and resolve proteins that are high molecular weight, highly basic, or hydrophobic. With this in mind, we developed a two-dimensional liquid chromatography separation scheme coupled to tandem mass spectrometry (Multi-dimensional Protein Identification Technology; MudPIT). Our most recent work has refined this non-gel
approach to provide better quantification by using a metabolic labeling strategy. Wild-type (ethanol-sensitive) cells were grown in the presence of isotopically labeled ammonium sulfate (15-N) while the ethanol-tolerant strain was grown in medium containing non-labeled ammonium sulfate (14-N). The 15-N labeled cells served as internal standards; this minimized experimental errors and provided better quantitative comparison of differences in protein levels. MudPIT analysis revealed that the expression of more than 60 proteins was significantly affected by adaptation to ethanol. These proteins take part in a wide variety of functional processes including regulation of transcription, translation, signal transduction, metabolism, transportation, post-translational modification. This is the first quantitative proteomic analysis of C. thermocellum based on the MudPIT approach. In summary, we have a reliable MudPIT analysis platform that can be used to profile the C. thermocellum proteome.
These experiments will provide information needed to overcome present process limitations and to optimize microbial conversion of cellulosic biomass to alcohol, solvents and other useful products.
Impacts
Continued dependence on fossil-based fuels has negative impacts on the environment, economy, and national security. The conversion of cellulosic 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 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 field of proteomics to examine the metabolism of anaerobic bacteria under industrially relevant conditions. The results of this work will be useful in designing economically relevant bio-based processes that
utilize cellulosic biomass. The University of Kentucky Mass Spectrometry Facility is a partner in this project.
Publications
- Williams, T. I., Combs,J. C., Thakur,A. P., Strobel,H. J., and B. C. Lynn. 2006. A novel bicine running buffer system for doubled sodium dodecyl sulfate polyacrylamide gel electrophoresis of membrane proteins. Electrophoresis 27:2984-2995.
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Progress 01/01/05 to 12/31/05
Outputs
The production of bio-based chemicals, materials, and fuels from agricultural and forestry feedstocks has the potential to reduce the nation's dependence on fossil fuels. Clostridium thermocellum is a thermophilic anaerobic bacterium that has received considerable attention because of its ability to directly convert fibrous feedstocks into ethanol. However, commercial exploitation of this organism is currently limited by several factors including its relatively low tolerance to ethanol in the fermentation environment. Work with previously isolated ethanol-adapted strains has continued and state-of-the-art proteomic approaches have been employed to investigate the nature of ethanol sensitivity and tolerance. Since initial studies suggested that there were significant alterations in the membrane proteome of ethanol-adapted organisms, further studies were conducted using a two-stage SDS-polyacrylamide gel protocol developed in our laboratory. This methodology was
designed to circumvent problems associated with membrane protein analysis using traditional gel-based proteomics approaches. Membranes from wild type and ethanol-adapted C. thermocellum cells displayed similar spot diversity in the high and low molecular weight ranges, but approximately 60% of proteins identified by mass spectrometry were differentially expressed between the two strains. A majority (73%) of these proteins were down-regulated in the ethanol-adapted bacterium, and a significant proportion of these down-regulated proteins were involved with carbohydrate transport and metabolism. Of the proteins that were up-regulated in the ethanol-adapted organism, a number were associated with chemotaxis and signal transduction. Overall, the results suggested that many membrane-associated proteins in the ethanol-adapted strain were either being synthesized in lower quantities or the bacterium failed to properly incorporate them into the cell membrane. Work is on-going to further
elucidate changes in the membrane proteome. In an effort to completely obviate the need for gel-based approaches, a two-dimensional liquid chromatography separation scheme coupled to tandem mass spectrometry is currently being developed to profile the C. thermocellum proteome. Preliminary studies have identified 187 unique proteins from a 25 microgram sample using a multi-dimensional protein identification technology (MudPIT). A sizable fraction (25%) of the identified proteins had high molecular weight (>100 kDa), basic pI value (>9), or were hydrophobic and membrane-associated; such proteins are often very difficult to separate and identify using traditional techniques. The MudPIT approach also permitted relative protein abundance information and provided the first three-dimensional information map (i.e., molecular weight, pI, and relative abundance) of protein distribution in C. thermocellum. Further work will refine the MudPIT approach and provide a more expansive and detailed
proteomic profile of the organism. These experiments will provide information needed to overcome present process limitations and to optimize microbial conversion of biomass to alcohol, solvents and other useful products.
Impacts
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. It is estimated that there are nearly 11 million tons of agricultural, forestry and urban-waste fibrous biomass that 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 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 field of proteomics to examine the metabolism of anaerobic bacteria under industrially relevant conditions. The results of this work will be useful in designing economically relevant bio-based processes.
The University of Kentucky Mass Spectrometry Facility is a partner in this project.
Publications
- No publications reported this period
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Progress 09/01/04 to 12/31/04
Outputs
There is renewed interest in the development of technologies for producing chemicals, materials, and energy using biologically-based processes. Much of this has been prompted by recognition that continued increases in fossil fuel use will have negative impacts on the environment, economy, and national security. The production of bio-ethanol offers considerable promise for decreasing fuel oil consumption and increasing domestic markets for agricultural and forestry commodities. Bioconversion of fibrous organic material by thermophilic anaerobic bacteria circumvents some of the problems associated with currently used yeast fermentations. Perhaps the greatest advantage of anaerobic bacteria is their ability to degrade and metabolize structural plant carbohydrates. This latter advantage is particularly important since fibrous biomass is relatively inexpensive and is expected to become the predominant feedstock for liquid fuels within the next 20 years. However, one of the
technological barriers to efficient biomass conversion by bacteria is the relatively low ethanol tolerance that nearly all bacteria have when compared to yeast. The effects of ethanol on some thermophilic bacteria have been examined and putative modes of inhibition have been proposed, but there is still relatively little detailed information on the mechanisms causing cellular alterations. State-of-the-art proteomic approaches are now available to efficiently characterize protein expression changes. Our basic hypothesis is that exposure of thermophilic anaerobic bacteria to ethanol causes alterations in protein expression patterns. The overall goal of this project is to identify and define these changes using proteomic approaches. Several organisms will be used to model the effects that ethanol has on the proteomes of thermophilic anaerobes. Specifically, Clostridium thermocellum and Thermoanaerobacter ethanolicus produce ethanol, degrade polymeric carbohydrates, and have been proposed
for use in biomass conversion systems. The specific objectives are to: (i) characterize alterations in the proteomic profile of C. thermocellum and T. ethanolicus in response to ethanol challenge; (ii) determine the proteomic profile of ethanol resistant strains; (iii) examine whether proteomic changes elicited by ethanol are similar to those caused by other environmental stresses including altered temperature, pH, and organic solvents; and (iv) evaluate alternative approaches to identify and quantify changes in proteomes of thermophilic bacteria. The recent completion of the C. thermocellum genome sequence will greatly aid in the identification and putative function of isolated proteins. Basic and specific knowledge will be developed on organisms that can be used to develop more sustainable energy technologies. These experiments will provide information needed to overcome present process limitations and to optimize microbial conversion of biomass to alcohol, solvents and other useful
products.
Impacts
Continued dependence on fossil-based fuels has negative impacts on the environment, economy, and national security. The conversion of biomass by mircoorganisms to bio-based products and bio-energy is a sustainable alternative. 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 field of proteomics to examine the metabolism of anaerobic bacteria under industrially relevant conditions. The results of this work will be useful in designing economically relevant bio-based processes. The University of Kentucky Mass Spectrometry Facility is a partner in this project.
Publications
- No publications reported this period
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