Browsing by Subject "Lignocellulosic biomass"
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Item The genetic architecture of quantitative traits in locally adapted plant ecotypes(2015-08) Milano, Elizabeth Rose; Juenger, Thomas; Kirkpatrick, Mark; Linder, Craig R; Lloyd, Alan; Martin, NolandLocally adapted ecotypes are a common phenomenon generating plant diversity within species, yet we know surprisingly little about the genetic mechanisms that lead to locally adapted traits. The genetic architecture underlying traits can indicate evolutionary history and predict response to selection, with applications in evolutionary ecology, conservation, and crop development. This research broadly investigates the genetic architecture of quantitative traits in paired ecotypes from different plant species. I used multivariate comparative methods and quantitative trait loci (QTL) mapping to quantify genetic correlations and population divergence, between ecologically relevant traits, both at the phenotypic and genotypic level. I tested for adaptive floral trait evolution in a perennial wildflower by comparing differentiation at neutral loci to differentiation in a suite of quantitative floral traits in an Ipomopsis aggregata hybrid zone. I used multivariate comparisons to incorporate the genetic covariance architecture underlying floral display and reward traits, and found a strong signal for divergent selection. Non-neutral divergence for multivariate quantitative traits suggests that selection by pollinators is maintaining a correlation between floral display and reward. In Panicum virgatum, a native perennial grass, I used a genetic mapping population, segregating ecotypic variation, to construct a linkage map, and map QTL for nine ecological traits. Most QTL had intermediate to small effects and clustered on a limited number of linkage groups. I also found over half of the functional allelic effects displayed patterns associated with fixed differences between ecotypes. These results suggest there is considerable standing genetic variation within local populations, as well as between ecotypes for ecologically relevant traits. Lastly, I explored the genetics of plant tissue quality in Panicum hallii, a model lignocellulosic grass system. Cell wall components compose the bulk of lignocellulosic biomass and contribute to the recalcitrance of plant tissue. I characterized the divergence of four major cell wall components between ecotypes, identified 14 QTL, and found half of the QTL localized to a single linkage group. Exploring the genetic architecture of tissue traits in a tractable system will lead to a better understanding of cell wall structure and function as well as provide genomic resources for bioenergy crop improvement.Item Pretreatment and Fermentation of Sugarcane Trash to Carboxylic Acids(2010-01-14) Nachiappan, BalasubramanThe rising price of oil is hurting consumers all over the world. There is growing interest in producing biofuels from non-food crops, such as sugarcane trash. Lignocellulosic biomass (e.g., sugarcane trash) is an abundant, inexpensive, and renewable resource. The patented MixAlco process is a cost-effective solution, which does not require sterility or the addition of expensive enzymes to convert lignocellulosic biomass to transportation fuels and valuable chemicals. In this study, the MixAlco process was used to convert sugarcane trash to carboxylic acids under thermophilic conditions. Lime-treated sugarcane trash (80%) and chicken manure (20%) was used as the feedstock in rotary 1-L fermentors. Ammonium bicarbonate buffer was used to mitigate the effects of product (carboxylic acid) inhibition. Marine inoculum was used because of the high adaptability of the mixed culture of microorganisms present. Iodoform solution was added to inhibit methanogenesis. Preliminary batch studies over a 20-day period produced 19.7 g/L of carboxylic acids. Sugarcane trash had the highest average yield (0.31 g total acid/g VS fed) and highest average conversion (0.70 g VS digested/g VS fed) among the three substrates compared. Countercurrent fermentations were performed at various volatile solid loading rates (VSLR) and liquid residence times (LRT). The highest acid productivity of 1.40 g/(L?d) was at a total acid concentration of 29.9 g/L. The highest conversion and yield were 0.64 g VS digested/g VS fed and 0.36 g total acid/g VS fed, respectively. The continuum particle distribution model (CPDM) was used to predict acid concentration at various VSLR and LRT. The average error in between the predicted and experimental acid concentration and conversion were 4.62% and 1.42%, respectively. The effectiveness of several pretreatment methods was evaluated using the CPDM method. The best-performing method was short-term, no-wash, oxidative lime pretreatment with ball milling. At an industrial-scale solids loading of 300 g VS/L liquid, the CPDM ?map? predicts a total acid concentration of 64.0 g/L at LRT of 30 days, VSLR of 7 g/(L?d), and conversion of 57%. Also high conversion of 76% and high acid concentration of 52 g/L are achieved at a VSLR of 4 g/(L?d) and LRT of 30 days.Item Production of Bioethanol and Biobutanol Using Genetically Engineered(2010-12) Ryu, Sh; Karim, M. Nazmul; Khare, Rajesh; San Francisco, Michael; Vaughn, Mark W.In this dissertation, the different Escherichia coli strains were engineered for producing bioethanol from the lignocellulosic biomass, and biobutanol from glucose. The recombinant whole-cell biocatalyst was developed by expressing three cellulases from Clostridium cellulolyticum: endoglucanase (Cel5A), exoglucanase (Cel9E), and β-glucosidase (BGL), onto the Escherichia coli LY01 cell surface (LY01/pRE1H-AEB). The above-mentioned cellulases were expressed by fusing with the anchor protein PgsA. The developed whole cell biocatalyst was used for single-step ethanol fermentation using the phosphoric acid swollen cellulose (PASC), and the dilute acid pretreated corn stover. The ethanol production was 3.59 ± 0.15 g/L using 10 g/L of phosphoric acid swollen cellulose (PASC) which corresponds to 95.4 ± 0.15% of the theoretical yield. The ethanol production was 0.30 ± 0.02 g/L when the cellulosic fraction of the dilute sulfuric acid pretreated corn stover (PCS) equivalent of 1 g/L glucose, was fermented for 84 h. Total 0.71 ± 0.12 g/L ethanol was obtained in 48 h when the fermentation of PCS were performed in the simultaneous saccharification and co-fermentation (SSCF) process using the hemicellulosic (1 g/L total soluble sugar) as well as cellulosic (1 g/L glucose equivalent) parts of PCS. In a control experiment, 0.48 g/L ethanol was obtained from 1 g/L of hemicellulosic PCS. It was concluded that the whole cell biocatalyst could convert both cellulosic and hemicellulosic substrate into ethanol in a single reactor. A new recombinant E. coli (KO11) was constructed by expressing the extracellularly secreted as well as the cell surface displayed endoglucanase. The expression vector pHR5A was constructed with the (EAAAK)5 peptide linker between the pgsA and the endoglucanase cel5A genes. Due to the auto-cleavage characteristic of the (EAAAK)5 peptide linker, Cel5A was partially detached from the PgsA anchor protein during the fermentation. The enzymatic hydrolysis efficiency of the recombinant KO11/pHR5A was investigated with various pure insoluble cellulosic substrates such as PASC, Solka-Floc 200NF, and Avicel.. Compared to the control strain, KO11/pH5A, with the recombinant strain, KO11/pHR5A, the saccharification efficiency was increased by 37%, 29%, and 17%, with substrates PACS, Solka-Floc 200NF, and Avicel, respectively. Ethanol production, supplemented by Novozyme 188 (commercial β-glucosidase), was found to be greater with the KO11/pHR5A than with the KO11/pH5A over 48 h fermentation. It was concluded that the extracellularly secreted single enzyme component enhanced the saccharification efficiency. For the butanol production, Escherichia coli BL21 was engineered. The mutant E. coli BM322 was constructed by disrupting phosphotransacetylase (pta), lactate dehydrogenase A (ldhA), bifunctional acetaldehyde/ethanol dehydrogenase (adhEB), fumarate reductase (frdBC), and pyruvate oxidase B (poxB) genes. Additionally, the transcriptional regulator FNR protein gene was also deleted. The recombinant plasmid pBUOH was constructed by cloning the key enzyme coding genes that are involved in the butanol production pathway in Clostridium acetobutylicum. These genes include: thiolase (thl), 3-hydroxybutyryl-CoA dehydrogenase (hbd), crotonase (crt), butyryl-CoA dehydrogenase (bcd), electron transfer flavoprotein (etfAB), and the bifunctional butyraldehyde/butanol dehydrogenase (adhEC). The recombinant E. coli BM322/pBUOH produced 1.2 g/L butanol with 10.2 g/L lactate as a by-product for 60 h. The obtained butanol yield and productivity was 0.07 g butanol/g glucose and 0.04 g/L•h, respectively.