The impact of pretreatment severity on the structure of CELF lignin and tensile properties of CELF lignin-based PU.

This study reports results of an extensive and comprehensive study of genetic diversity in 12 genes of the innate immune system in a population of eastern India. Genomic variation was assayed in 171 individuals by resequencing ~75 kb of DNA comprising these genes in each individual. Almost half of the 548 DNA variants discovered was novel. DNA sequence comparisons with human and chimpanzee reference sequences revealed evolutionary features indicative of natural selection operating among individuals, who are residents of an area with a high load of microbial and other pathogens. Significant differences in allele and haplotype frequencies of the study population were observed with the HapMap populations. Gene and haplotype diversities were observed to be high. The genetic positioning of the study population among the HapMap populations based on data of the innate immunity genes substantially differed from what has been observed for Indian populations based on data of other genes. The reported range of variation in SNP density in the human genome is one SNP per 1.19 kb (chromosome 22) to one SNP per 2.18 kb (chromosome 19). The SNP density in innate immunity genes observed in this study (>3 SNPs kb −1 ) exceeds the highest density observed for any autosomal chromosome in the human genome. The extensive genomic variation and the distinct haplotype structure of innate immunity genes observed among individuals have possibly resulted from the impact of natural selection.

Woody biomass is not only abundant and sustainably available but also contains more glucan than most agricultural feedstocks. However, its structural strength and density make it more recalcitrant to enzymatic breakdown and limit SSF performance, especially at higher solids loadings needed to achieve desired ethanol titers. Because Co-Solvent Enhanced Lignocellulosic Fractionation (CELF) highly enriches glucan contents in pretreated solids by removing and recovering most of the hemicellulose and lignin, we hypothesized that the greater glucan in SSF at solids loadings that inhibit mixing than possible for other pretreatments would make it possible to achieve higher ethanol titers and yields. Application of a fractal kinetic model to data from enzymatic hydrolysis of CELF pretreated poplar indicated that CELF solids could sustain high cellulase enzyme activity over the entire course of hydrolysis with minimal enzyme deactivation. Model parameters further suggested that increasing enzyme loadings beyond 15 mg protein/g glucan in raw poplar would not increase saccharification performance. Based on these predictions, SSF was applied to solids produced by CELF pretreatment of poplar using Cellic® CTec2 cellulase at a loading of 15 mg-protein/ g-glucan in combination with S. cerevisiae D5A. At CELF solids loadings of 13, 17, and 20 wt%, SSF realized ethanol titers of 60, 78, and 87 g/L and yields of 87, 84, and 79% of theoretical in respective orders of increasing solids loadings. Furthermore, sugar release was sustained throughout SSF, irrespective of solids loading, suggested that yeast ethanol tolerance and metabolic capability and not glucan digestibility limited SSF performance.

Simultaneous Saccharification and Fermentation (SSF) is effective for minimizing sugar inhibition during high solids fermentation of biomass solids to ethanol. However, fungal enzymes used during SSF are optimal between 50–60°C, whereas most fermentative yeast, such as Saccharomyces cerevisiae, do not tolerate temperatures above 37°C. Kluveromyces marxianus variant CBS 6556 is a thermotolerant eukaryote that thrives at 43°C, thus potentially serving as a promising new host for SSF operation in biorefineries. Here, we demonstrate the application of CBS 6556 in SSF configuration to understand its capabilities and limitations as compared to a proven SSF strain, S. cerevisiae D5A. For this study, we first pretreated hardwood poplar chips using Co-Solvent Enhanced Lignocellulosic Fractionation (CELF) to remove lignin and hemicellulose and to produce cellulose-enriched pretreated solids for SSF. Our results demonstrate that although CBS 6556 could not directly outperform D5A, it demonstrated superior growth rates at higher temperatures and higher early stage ethanol productivity. We discovered that CBS 6556’s membrane was particularly sensitive to higher ethanol concentrations causing it to suffer earlier fermentation arrest than D5A. Cross-examination of metabolite data between CBS 6556 and D5A and cell surface imaging suggests that the combined stresses of high ethanol concentrations and temperature to CBS 6556’s cell membrane was a primary factor limiting its ethanol productivity. Hence, we believe K. marxianus to be an excellent host for future genetic engineering efforts to improve membrane robustness in order to achieve higher ethanol productivity and titers, serving as a viable alternative to D5A.

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