Production of xylitol and ethanol from lignocellulosics
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Lignocelluloses as agricultural, industrial, forest residuals, food-processing waste or paper wastes have the potential to serve as low-cost and abundant source of raw materials for the production of different biochemicals which can serve as future energy sources. These potentially valuable materials consist of sugars and lignin that can be converted via biological pathway into many valuable bioproducts and biochemicals. Two valuable biochemicals that can be fermented from sugars derived from lignocellulosic biomass are xylitol and ethanol. Compared with glucose, which can be readily fermented by well studied yeast (<italic>Saccharomyces cerevisiae</italic>) and bacterial (<italic>Zymomonas mobilis</italic>) strains, xylose is more difficult to ferment because a lack of industrially suitable microorganism able to rapidly and efficiently metabolize xylose in presence of six carbon sugars. In order to keep biochemicals production cost at minimum, all the sugars naturally present in lignocellulosic biomass must be converted into biofuels and biochemicals. The need for a microorganism that can utilize all the sugars present in lignocellulosic biomass and to tolerate the inhibitory compounds generated during biomass pretreatment is therefore apparent. One of the yeast which was identified in our labs as being capable of rapid assimilation and catabolism of five and six carbon sugars (arabinose, galactose, glucose, xylose and mannose) is <italic>Rhodotorula mucilaginosa</italic> strain PTD3, an endophytic yeast of hybrid poplar <italic>Populus trichocarpa x deltoids</italic>. PTD3 was found to be capable of producing xylitol from xylose, ethanol from glucose, galactose, and mannose, and arabitol from arabinose. Glucose-acclimated PTD3 produced enhanced yields of xylitol (67% of theoretical yield) from xylose and of ethanol (84, 86, and 94% of theoretical yield, respectively) from glucose, galactose, and mannose. Additionally, this yeast was capable of metabolizing high concentrations of mixed sugars (150 g/L), with high yields of xylitol (61% of theoretical yield) and ethanol (83% of theoretical yield). A 1:1 glucose:xylose ratio with 30 g/L of each during double sugar fermentation did not affect PTD3's ability to produce high yields of xylitol (65% of theoretical yield) and ethanol (92% of theoretical yield). Surprisingly, the highest yields of xylitol (76% of theoretical yield) and ethanol (100% of theoretical yield) were observed during fermentation of sugars present in the lignocellulosic hydrolysate obtained after steam pretreatment of a mixture of hybrid poplar and Douglas fir. Also, PTD3 demonstrated the ability to tolerate higher concentrations of inhibitors during xylitol and ethanol production compared to other yeasts described in the literature. Concentration of up to 5 g/L of furfural stimulated production of xylitol to 77% of theoretical yield (10% higher compared to the control) by PTD3. Xylitol yields produced by this yeast were not affected in a presence of 5-HMF at concentrations of up to 3 g/L. At higher concentrations of furfural and 5-HMF, xylitol and ethanol yields were negatively affected. The higher the concentration of acetic acid present in a media, the higher the ethanol yield approaching 99% of theoretical yield (15% higher compared to the control) was produced by the yeast. At all concentrations of acetic acid tested, xylitol yield was lowered. PTD3 was capable of metabolizing concentrations of 5, 15, and 5 g/L of furfural, 5-HMF, and acetic acid, respectively. The high xylitol and ethanol yields obtained and significant resistance to the toxicity of the inhibitors indicate the great potential of the tested strain as a realistic candidate for industrial scale biofuels and biochemicals production from lignocellulose. After assessing these PTD3's abilities to ferment glucose and xylose and to tolerate the fermentation inhibitors, the next step was to determine the optimum pretreatment conditions which will cause the production of a unique concentration of fermentation inhibitors that would enhance fermentation of xylitol and ethanol by yeast <italic>Candida guilliermondii</italic>. Based on concentrations of inhibitors present, hydrolysates obtained after steam pretreatment of five lignocellulosic feedstocks (mixed wood, hybrid poplar, <italic>Arundo donax</italic>, switchgrass, and sugarcane bagasse) were evaluated in terms of enhanced xylitol and ethanol production. In addition, for deeper understanding of the effect of process inhibitors on the overall xylitol and ethanol yields after fermentation, hybrid poplar feedstock was selected and pretreated at different severity conditions using steam pretreatment. Customizing the steam pretreatment conditions for production of fermentation inhibitors at unique concentration can be used as a novel approach of improving xylose to xylitol and six carbon sugars to ethanol conversion by <italic>Candida guilliermondii</italic>.
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