Progress 01/01/08 to 03/31/12
Outputs
OUTPUTS: The specific objectives of this project for Year Two were 1) Comparison of the naturally occurring endophytic yeasts with S. cerevisiae in terms of ethanol and xylitol production rate and productivity during fermentation of five and six carbon sugars. We compared our endophytic strain to the control strain, Candida guillierrdondii and published a manuscript. 2) Examination of requirements for growth and fermentation of the endophytic yeast strains. 3) Testing the tolerance to inhibitors by the endophytic yeast isolates during fermentation of lignocellulosic hydrolysate obtained during steam explosion of lignocellulosic biomass. The analysis was completed and a paper on this research was published. 4) Genomic analysis of the endophytic yeast strains was completed and the manuscript has been published. Vajzovic researched a novel approach of improving fermentation yields by optimizing the steam pretreatment conditions to obtain a low concentration of common fermentation inhibitors, using Candida guilliermondii as the fermenting strain. A systematic study of the effects of low concentrations of fermentation inhibitors on the fermentation of xylose to xylitol and hexoses to ethanol in the steam pretreated hydrolysates from sugarcane bagasse, hybrid poplar, switchgrass, yard waste and giant reed was conducted. Presentation by Azra Vajzovic at the Seventh Conference on Renewable Resources and Biorefineries; Bruges, Belgium 06/08-06/10, 2011. NOVEL APPROACH OF IMPROVING XYLOSE TO XYLITOL CONVERSION BY CANDIDA GUILLIERMONDII AND RHODOTORULA MUCILAGINOSA Azra Vajzovic, Renata Bura, Sharon L. Doty. Ph.D. student Azra Vajzovic and Research Scientist Zareen Khan further developed their skills in fermentation analysis. Xu Ping received her master's degree on the genetic analysis of xylose metabolism by endophytic yeast. Undergraduate researcher, Hannah Morrison, was supervised by Dr. Khan in a project aimed at re-activating PTD3 using plant extracts. We have a patent pending. PARTICIPANTS: Sharon L. Doty, Associate Professor, University of Washington School of Environmental and Forest Sciences. Renata Bura, Assistant Professor, University of Washington School of Environmental and Forest Sciences. Zareen Khan, Research Scientist, University of Washington School of Environmental and Forest Sciences. Azra Vajzovic, Ph.D. student, University of Washington School of Environmental and Forest Sciences. Xu Ping, Master's student, University of Washington School of Environmental and Forest Sciences. Hannah Morrison, undergraduate researcher, University of Washington School of Environmental and Forest Sciences. TARGET AUDIENCES: Target audiences include the Consortium for Plant Biotechnology Research, biofuels researchers, educators, students, and the general public. PROJECT MODIFICATIONS: As described above, after conducting several optimization experiments, our endophytic yeast strain, PTD3, worked initially, fermenting to high levels. However, it then stopped fermenting glucose despite repeated attempts with several stocks of the endophyte strain. We used phytochemicals to re-activate the strain with some success.
Impacts
PTD3 performed well for 2 years but not well in the last year, growing on the substrates rather than fermenting to ethanol or xylitol. Loss of desired activities has been a common problem with fungal isolates from wild samples. As the changes in behavior seemed to be epigenetic, re-activation of the strains using chemicals were tried in these cases and were sometimes successful. However, this did not help to re-activate PTD3 at an azacytidine concentration reported in the literature. We had some success re-activating the strain using plant extracts; however, the results were not fully repeatable. PTD3 was streaked on V8 (vegetable extract with agar) medium. Resulting colonies of PTD3 were then grown in medium supplemented with xylose, KH2PO4, yeast extract, NH4NO3, CaCl2, and MgSO4. This culture was used as an inoculum for the fermentation experiments done in MS media containing xylose and NaNO3. PTD3 consumed xylose and produced xylitol. On day 3, more than 70% xylose was utilized with significant production of xylitol. We also tried to re-activate PTD3 by growing it on poplar extract plates. The resulting colonies of PTD3 were then enriched in rich medium containing yeast extract, peptone, glucose/xylose. After 2 days of enrichment, the inoculum was transferred into fermentation media containing MS salts with either glucose or xylose. Non-reactivated PTD3 was also used as comparison. In glucose fermentations, the reactivated PTD3 consumed 100% of the sugar and produced 65% ethanol, whereas the PTD3 that was not pre-grown on poplar extract made 25% ethanol. In xylose fermentations, the reactivated PTD3 consumed half the sugar in 48 hours and made 18% xylitol compared to 10% by the non-activated strain. This shows that the endophytic yeast required signals from plants in order to trigger normal metabolism. Since the end-use of the strain is to do lignocellulosic fermentation, these signals would most likely be present in the substrate. To test this, experiments have to be repeated. We screened other tree species for xylose-utilizing strains. We isolated a new endophytic yeast strain from alder with superior growth and fermentation properties. Therefore, the idea of using endophytes for biochemical production is a good one that should be further investigated. In her studies, Vazjovic showed improved xylitol yields and as well as ethanol yields compared to the controls for hydrolysates from steam pretreated yard waste and sugarcane bagasse grown in Hawaii. Ethanol yield from hexoses was 100% and xylitol from xylose yields was ~ 90% of theoretical yield for both hydrolysates. Xylitol yields in the hydrolysates were about 20% higher compared to controls. The similar trend was observed when a hydrolysate obtained from sugarcane bagasse grown in Brazil was fermented by C. guilliermondii. Ethanol yield from hexoses was 100% and xylitol from xylose yield was ~87% of theoretical yield. The theoretical yield of produced xylitol was again higher yield compared to controls. This approach will allow us in the future to find the optimum concentration of inhibitors to increase ethanol and xylitol yields during fermentation of hexoses and pentoses.
Publications
- Xu, P., Bura, R., and Doty, S. L. 2011. Genetic analysis of D-xylose metabolism by endophytic yeast strains of Populus. Genetics and Molecular Biology 34(3):471-478.
- Bura, R., Vajzovic, A., and Doty, S. L. 2012. Novel endophytic yeast Rhodotorula mucilaginosa strain PTD3: I. Production of xylitol and ethanol. Journal of Industrial Microbiology & Biotechnology 39(7):1003-1011.
- Vajzovic, A., Bura, R., and Doty, S. L. 2012. Novel endophytic yeast Rhodotorula mucilaginosa strain PTD3: II. Production of xylitol and ethanol in the presence of inhibitors. Journal of Industrial Microbiology & Biotechnology (in press)
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Progress 10/01/07 to 10/01/08
Outputs
OUTPUTS: The results will be discussed at the Society for Industrial Microbiology conference in San Francisco in May 2009. The data have been very exciting, so we are in the process of patenting the use of our novel yeast strains for biofuel and biochemical production. PARTICIPANTS: Graduate students, Azra Vajzovic and Xu Ping, as well as post-doctoral fellow, Zareen Khan, gained valuable experience in microbiology, molecular biology, genome analysis, chemistry, and gas chromatography. TARGET AUDIENCES: This grant allowed us to produce enough data to convince the University of Washington to file a patent on the use of these yeast strains. The target will ultimately be companies interested in bioenergy and biochemicals. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
This grant allowed us to characterize several yeast isolates isolated from within plants. By following the results, we were able to explore an unexpected avenue for the use of the strains, leading to increased commercialization opportunities.
Publications
- No publications reported this period
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