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NON TECHNICAL SUMMARY:
Biomass is generally recognized as the only source of liquid transportation fuels that may replace the world's finite supply of oil. In addition to reducing our dependence on imported oil, with accompanying increase in energy security and reduction in our trade deficit, cellulosic ethanol production would have positive environmental benefits in the form of reductions in greenhouse gas emissions and air pollution. Moreover, rural economies would benefit with higher incomes and employment due to the increased value of agricultural crops and crop residues and through the introduction of new energy crops. Cellulose-decomposing microbes have received considerable attention because of their potential use in processes that involve the direct conversion of cellulosic biomass to fuels such as ethanol. However, most naturally occurring cellulolytic strains have a limited substrate range, produce low amounts of ethanol, and form fermentation byproducts that reduce ethanol
production efficiency. The research we are proposing focuses on Clostridium phytofermentans, an ethanol-producing cellulose-decomposing bacterium with exceptional nutritional versatility. C. phytofermentans is able to ferment more components of biomass than most other known microbes. Also, cellulose-fermenting cultures of this bacterium produce H2, as well as prodigious amounts of ethanol. Research described in this proposal will advance understanding of the biology of C. phytofermentans to better appreciate its potential in the development of biorefineries for the conversion of biomass to ethanol and other products.
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| APPROACH:
In order to examine the effect of Zymomonas mobilis on ethanol production by Clostridium phytofermentans, stable cocultures will be established. C. phytofermentans will be routinely cultured in medium GS-2, a yeast extract-containing medium, with ball-milled cellulose as growth substrate, and Z. mobilis will be cultured in medium GS-2 supplemented with 2% D-glucose. Cultures will be incubated in an atmosphere of O2-free N2 at 30 C. Co-cultures will be established in GS-2 cellulose medium using several inoculation regimes, varying the amount of inoculum and the relative time of inoculation. Fermentation products will be assayed by gas-liquid chromatography and HPLC. Ethanol concentrations also will be determined enzymatically. Fermentation of five-carbon sugars and five-carbon sugar polymers by C. phytofermentans will be examined by determining the kinetics of growth on these substrates, and quantifying fermentation product formation. Cells will be cultured in medium
GS-2 supplemented with 0.3% (w/v) carbon source, including D-xylose, L-arabinose, and xylan from birchwood, beechwood, and oat spelts. Growth will be monitored turbidimetrically, and fermentation products will be determined as describe above. The potential of the lactococcal pWV01-based conditionally replicating vector system for chromosomal integration in C. phytofermentans will be examined following the techniques of Leenhouts et al. (Leenhouts, K., G. Venema, & J. Kok. 1998. A lactococcal pWV01-based integration toolbox for bacteria. Methods Cell Sci. 20:35-50). C. phytofermentans genomic DNA will be prepared by means of the CTAB method of the DOE Joint Genome Institute. Procedures for the electroporation of C. phytofermentans, which were developed in our laboratory for the introduction of phage DNA, will be modified for the introduction of plasmids. C. phytofermentans mutant strains defective in surface motility on plates of agar media will be selected. Also, mutant strains with
altered fermentation properties will be selected for further study.
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CRIS NUMBER: 0205636
SUBFILE: CRIS
PROJECT NUMBER: MAS00923
SPONSOR AGENCY: NIFA
PROJECT TYPE: HATCH
PROJECT STATUS: TERMINATED
MULTI-STATE PROJECT NUMBER: (N/A)
START DATE: Oct 1, 2005
TERMINATION DATE: Sep 30, 2010
GRANT PROGRAM: (N/A)
GRANT PROGRAM AREA: (N/A)
CLASSIFICATION HEADINGS
KA403 - Waste Disposal, Recycling, and Reuse
S1699 - Pasture and forage crops, general/other
F1103 - Other microbiology
G6.2 - Enhance Soil Quality for Productive Working Lands
RESEARCH EFFORT CATEGORIES
| BASIC |
100% |
| APPLIED |
(N/A)% |
| DEVELOPMENTAL |
(N/A)% |
KEYWORDS: biomass ethanol; clostridium phytofermentans; cellulolytic anaerobe; zymomonas mobilis; renewable energy; cellulose decomposition; xylose fermentation; xylan utilization; energy crops; crop residues; biomass h2 production; genetic tools; pwv01 vector; conditionally replicating
PROGRESS: Oct 1, 2008 TO Sep 30, 2009
OUTPUTS: OUTPUTS. Ethanol production by cocultures of Clostridium phytofermentans and Zymomonas mobilis: As reported previously, cocultures were established and the effects of Z. mobilis on cellulose fermentation and ethanol production were monitored; based on research results, hypotheses for coculture instability and low levels of increased ethanol production were developed. Fermentation of five-carbon sugars and five-carbon sugar polymers by C. phytofermentans: Growth kinetics of C. phytofermentans on xylose, other five-carbon sugars and methyl pentoses were determined and fermentation product formation was quantified. Tools for genetic analyses of C. phytofermentans: Additional potential vectors were identified and isolated and parameters were determined for electroporation of C. phytofermentans; mating and natural competence in C. phytofermentans were also investigated as tools for genetic analyses. DISSEMINATION. Our research efforts have important implications related to the activities of the enzyme system of Clostridium papyrosolvens, genetic manipulations and the potential use of this microbe for the direct conversion of plant biomass to fuels (e.g., ethanol, propanol) and other bioproducts, a process known as "consolidated bioprocessing" (CBP). In order to facilitate dissemination of this information and the advancement of related research, interested investigators on the UMass Amherst campus have coalesced as The Institute for Massachusetts Biofuels Research (TIMBR) to pursue funding for further research, as well as the establishment of the Biofuels Research and Development Laboratory that will supply facilities and research capacity for collaborative bioenergy research and development projects in Massachusetts. The Laboratory will provide a physical and intellectual platform for the participants in TIMBR to directly interface with each other, their collaborators at other institutions, and with industrial partners. As such, the Laboratory will serve as the catalyst for multidisciplinary research and promote the translation of TIMBR research into the private sector. We have also continued to develop the Massachusetts Bioenergy Partners (MBP) organization. This component of TIMBR consists of an Advisory Board comprising bioenergy professionals from industry and academics, and a professional staff focused on promoting and facilitating technology transfer, workforce development, and industry, market and organizational research all aimed at converting agricultural residues, forest debris, biomass crops, and other non-food sources of biomass into biofuels and other bioproducts. TIMBR participants have established a number of industry partner relationships with Massachusetts companies (e.g., Qteros, Anellotech, Renewable Oil Int., Northeast Biofuels, CeraMem, Berkeley Biofuels). Our goal is to further develop these relationships into research and technology transfer collaborations. Furthermore, MBP outreach will aid in identifying and fostering additional industry and agricultural collaborations. PARTICIPANTS: PARTICIPANT INDIVIDUALS: PI/PD: Susan Leschine Summer Student Intern: David Sokolowski TRAINING AND PROFESSIONAL DEVELOPMENT: The summer internship provided training and experience in laboratory research, including experience in microbiology, biochemistry, bioinformatics. TARGET AUDIENCES: TARGET AUDIENCES include 1) microbiologists and other scientists working in the fields of biofuels and bioproducts research; 2) plant biologists investigating potential biomass crops; and 3) the biofuels and bioproducts industry through their interest in new technologies that may be developed through research conducted as part of the project. PROJECT MODIFICATIONS: Not relevant to this project.
IMPACT: 2008-10-01 TO 2009-09-30
Ethanol production by cocultures of Clostridium phytofermentans and Zymomonas mobilis: As reported previously, we established cocultures and monitored the effects of Z. mobilis on cellulose fermentation and ethanol production. Hypotheses for coculture instability and low levels of increased ethanol production were developed, tested, and further modified. Results of our studies indicated that Z. mobilis cells were lost from cocultures after three transfers of cocultures to fresh medium, and ethanol production was not significantly increased. Culture conditions were varied to encourage the growth and activity of Z. mobilis. By means of a reducing sugar assay, we determined that reducing sugars increased near the end of growth on media containing cellulose. Analyses of these sugars revealed that glucose and cellobiose were not detectable using various enzymatic and chromatographic methods. Thin layer chromatography revealed that higher sugar oligomers accumulated in cultures at the end of the growth cycle. These unexpected results have directed our studies to the cellulase enzyme system produced by C. phytofermentans, which we determined is very different from cellulosomal systems of other cellulolytic clostridia. We hypothesize that C. phytofermentans-Z. mobilis cocultures are unstable because activity of the cellulase system of C. phytofermentans does not produce substrates that Z. mobilis is capable of transporting into cells and/or metabolizing. We are approaching these questions with genomics and biochemical approaches to characterize the cellulase system of C. phytofermentans. Fermentation of five-carbon sugars and five-carbon sugar polymers by C. phytofermentans: Growth kinetics of C. phytofermentans on xylose were determined and fermentation product formation was quantified. We have determined that C. phytofermentans is capable of fermenting xylose and producing ethanol as the major fermentation product. We had previously determined that C. phytofermentans is capable of fermenting xylan, and continue to investigate growth and product formation on various other components of the hemicellulose fraction of plant biomass, including methyl pentoses. Tools for genetic analyses of C. phytofermentans: We isolated appropriate plasmids and determined parameters suggesting successful electroporation of C. phytofermentans; we have begun to investigate mating and natural competence in C. phytofermentans as means for genetic transfer. We have continued using Bacillus subtilis W26T as donor in filter mating experiments to transfer various genetic elements to C. phytofermentans. We initiated investigations of three transconjugates that showed altered motility were also affected in their ability to form biofilms and degrade cellulose. We also continued to investigate electroporation as a means to transfer plasmids to C. phytofermentans. Our results indicate that conjugative transposon mutagenesis may be an effective genetic tool for C. phytofermentans. In planned future studies, additional modifications will be examined to facilitate the study of cellulose degradation and metabolism in this environmentally and economically important microbe.
PUBLICATION INFORMATION: 2008-10-01 TO 2009-09-30
Alonso, A. N., Pomposiello, P.J. and S. B. Leschine. 2008. Biofilm formation in the life cycle of the cellulolytic actinomycete Thermobifida fusca. Biofilms. Published online by Cambridge University Press 19 Nov 2008 doi:10.1017/S1479050508002238.
PROJECT CONTACT INFORMATION
| NAME: |
Cromack, P. |
| PHONE: |
413-545-4204 |
| FAX: |
413-577-0242 |
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