Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 24, 2006
Publication Date: May 5, 2006
Citation: Slininger, P.J., Dien, B.S., Gorsich, S.W., Liu, Z. 2006. Nitrogen source and mineral optimization enhance D-xylose conversion to ethanol by the yeast Pichia stipitis NRRL Y-7124. Applied Microbiology and Biotechnology. 72(6):1285-1296. Interpretive Summary: Efficient fermentation processes to produce ethanol from the hexose and pentose sugars available from low-cost plant biomass (i.e., from crop and forest residues, energy crops, or municipal wastes) are sought to support the expansion of the biofuels industry. Pichia stipitis NRRL Y-7124 is one of the natural yeast strains best able to convert the pentose sugar, xylose, to ethanol. To develop a more efficient pentose fermentation process, nutrition-based strategies for improving yeast vitality and fermentation efficiency were explored by studying cultures varied in nutrients such as: amino acids, urea, vitamins, and minerals. Mineral and nitrogen source optimizations were found key to maximizing ethanol production efficiency. Such new information will be useful to other researchers involved in fermentation process and technology development and to the biofuels industry where it can be applied to the design of a functional, low-cost fermentation medium for ethanol production from renewable biomass. This new technology contributes to our national priorities of achieving energy independence, strengthening our rural economy, and preserving our environment.
Technical Abstract: Efficient fermentation processes to produce ethanol from hexose and pentose sugars available in low-cost lignocellulosic biomass are sought to support the expansion of the biofuels industry. Pichia stipitis NRRL Y-7124 is a natural yeast able to convert xylose to ethanol, a capability not possessed by traditional Saccharomyces strains used to produce ethanol from corn starch. To investigate nutrition-based strategies for improving xylose to ethanol conversion by P. stipitis, kinetic studies were conducted on growing and non-growing cultures provided a defined medium which was varied in nitrogen, vitamin, purine/pyrimidine and mineral content. The interaction of minerals with amino acids and/or urea was key to optimizing ethanol production by cells in both growing and stationary phase cultures; and although a vitamin mix (including biotin) was required to sustain significant yeast growth, vitamins had no impact on the ethanol accumulation in either growing or non-growing stationary phase cultures. Surprisingly, stationary phase cultures were unable to ferment xylose (or glucose) to ethanol without the addition of a nitrogen source such as amino acids. When comparing culture media with and without individual amino acids, ethanol accumulation significantly improved with added arginine, alanine, aspartic acid, glutamic acid, glycine, histidine, leucine, and tyrosine, but declined with isoleucine. Only arginine and histidine improved both ethanol and cell yields, while proline significantly improved only cell yield. Ethanol production from 150 g/L xylose was significantly enhanced by optimizing the combination of urea and amino acids to supply 40-80 percent nitrogen from urea and 60-20 percent from casamino acids. When either urea or casamino acids was used as sole nitrogen source, ethanol accumulation dropped to 11 or 24 g/L, respectively, from the maximum of 46 g/L for the optimal nitrogen combination. Iron sulfate, manganese chloride, and magnesium sulfate were key to both biomass and ethanol production, while calcium chloride or zinc sulfate impacted either biomass or ethanol accumulation, respectively. Ethanol accumulations in non-growing cultures with casamino acids increased from 24 g/L to 53 g/L with the addition of an optimized mineral supplement. These results allow design of a defined medium for research or a low-cost commercial medium for xylose fermentation.