Being the most abundant carbon-containing terrestrial biopolymer, lignocellulose serves as one of the best candidate feedstocks for biofuel production. The current cost-ineffective method for lignocellulose pretreatment is one of the major barriers that hinder the development of biofuel production. This leads to an exploration in the potential application of lignocellulolytic enzymes in this biorefinery process. Taking advantage of the strong activity of ligninolytic enzymes in L. edodes, we aimed at cloning and heterologously expressing these enzymes. The present project applied a yeast expression system, Pichia pastoris, as a laboratory-scale platform for heterologous expression of one of our target ligninolytic enzymes, manganese peroxidase (MnP). We successfully cloned and expressed recombinant MnP. Its enzymatic activity was the highest when grown in the presence of hemoglobin. Our long-term goal is to establish a platform for the large-scale production of recombinant lignocellulotyic enzymes at low-cost, which would strengthen their application in biofuel production. / The shitake mushroom, Lentinula edodes, is one of the most commonly consumed edible mushrooms in Asian countries. It is a saprophyte that naturally colonizes dead wood. As a member of wood-decaying white rot basidiomycete, L. edodes is able to depolymerize lignin and hydrolyze wood polysaccharides. However, the enzymatic mechanism for its lignocellulolytic system is poorly understood. Examination on the L. edodes genome and transcriptome revealed a unique lignocellulolytic system. L. edodes has a diverse enzymatic arsenal for lignin degradation. The enzymes include laccase, manganese peroxidase, cellobiose dehydrogenase and various lignin degrading auxiliary enzymes. When compared to another white rot fungus Phanerochaete chrysosporium, L. edodes possesses more hemicellulase- and pectinase-coding genes, and fewer genes encoding cellulases, suggesting that it preferentially attacks non-cellulosic polysaccharides. The transcription analysis on genes related to antioxidative mechanisms also offers insights to the oxidative stress encountered by mycelium during the free radical-mediated lignin degradation. / Kwok, Sze Wai. / Adviser: Hoi-Shan Kwan. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 141-160). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

In the world today there is a massive dependency on fossil fuels as they are currently used to provide around 80% of the world’s energy. This is hugely detrimental to the environment and is a major contributory factor in climate change. Biofuel is a renewable energy source that is already being used to lessen some of the fossil fuel dependency. 2nd generation biofuels, by using non-food parts of plants, circumvent the food vs fuel argument, and by using farming waste or surplus can also avoid changing land use problems. Additionally liquid biofuels can use existing infrastructure for storage and delivery, and also fit into current lifestyles. Cost-effective 2nd generation biofuel production is directly affected by the presence of the polymer lignin in plant biomass, as it has been shown to impede enzymatic sugar release (saccharification) that is used for biofuel production. The work undertaken in this project developed a high-throughput methodology for the assessment of straw lignin content and composition across a large population of elite varieties in the economically important cereal crop, barley. Saccharification yield was also measured across the same population along with a number of other agronomically important traits, such as thousand-grain weight, biomass, mechanical stem properties and height. The data provided by these measurements allowed correlations between traits to be identified and their strength gauged. Genome wide association studies (GWAS) were also carried out and identified influential regions of the genome for each trait. The results revealed varying levels of association between measured traits and lignin content and monomeric constituents. Importantly a negative connection was shown between lignin content and saccharification yield, with lignin content being responsible for approximately 1/5th of the variation seen. Interestingly there was no correlation between lignin content and mechanical stem properties, an important factor in the agronomically important trait, lodging. GWAS results revealed a number of genomic regions that were influential across several traits indicating regions that would be difficult to separate through breeding due to their close proximities. However, unique QTL were identified for saccharification yield and lignin content providing candidates for breeding or genetic manipulation to improve the crop for biofuel production.

Luo, Xiao. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 118-134). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.iv / Abbreviations --- p.v / Contents --- p.vi / List of Figures --- p.ix / List of Tables --- p.xii / Chapter Chapter 1 : --- Literature Review --- p.1 / Chapter 1.1 --- Lentinula edodes --- p.1 / Chapter 1.1.1 --- Introduction and taxonomy --- p.1 / Chapter 1.1.2 --- Nutritional values and medical values --- p.2 / Chapter 1.2 --- Life cycle and morphology --- p.5 / Chapter 1.3 --- Lignocellulolytic system in wood-rotting fungi --- p.9 / Chapter 1.3.1 --- Structures of lignin --- p.9 / Chapter 1.3.2 --- Wood-rotting fungi --- p.11 / Chapter 1.3.3 --- Lignin degradation by white rot fungi --- p.12 / Chapter 1.3.4 --- Ligninolytic enzymes --- p.16 / Chapter 1.3.4.1 --- Lignin peroxidase --- p.16 / Chapter 1.3.4.2 --- Maganese peroxide --- p.16 / Chapter 1.3.4.3 --- Laccases --- p.19 / Chapter 1.3.5 --- Potential Industrial application of liglinolytic enzymes --- p.22 / Chapter 1.3.6 --- Ligninolytic enzymes in L. edodes --- p.23 / Chapter 1.4 --- Expression systems for fungal ligninolytic enzymes --- p.24 / Chapter 1.5 --- Aim of this project --- p.27 / Chapter 1.6 --- Long-term significance --- p.28 / Chapter Chapter 2: --- Sequence analysis of ligninolytic enzymes from Lentinula edodes --- p.29 / Chapter 2.1 --- Introduction --- p.29 / Chapter 2.2 --- Materials and methods --- p.32 / Chapter 2.2.1 --- Phylogenetic study and signal peptide prediction of the decuced ligninolytic enzymes --- p.32 / Chapter 2.2.2 --- Comparison ligninolytic enzymes of L. edodes and other basidiomycetes fungi --- p.32 / Chapter 2.3 --- Results --- p.34 / Chapter 2.3.1 --- Protein sequence analysis and signature sequences identification of L. edodes laccases --- p.34 / Chapter 2.3.2 --- Protein sequence analysis of L. edodes manganese peroxidases --- p.34 / Chapter 2.3.3 --- Phylogenetic study of ligninolytic genes from L.edodes --- p.35 / Chapter 2.4 --- Disscussion --- p.52 / Chapter Chapter 3: --- Transcription profiling of ligninolytic enzymes from Lentinula edodes --- p.56 / Chapter 3.1 --- Introduction --- p.56 / Chapter 3.2 --- Materials and Methods --- p.61 / Chapter 3.2.1 --- Strain cultivation --- p.61 / Chapter 3.2.2 --- "RNA extraction, mRNA isolation and cDNA synthesis" --- p.63 / Chapter 3.2.3 --- RNA Quality Estimation --- p.64 / Chapter 3.2.4 --- cDNA synthesis --- p.65 / Chapter 3.2.5 --- Primer verification --- p.66 / Chapter 3.2.6 --- Quantitative RT-PCR --- p.66 / Chapter 3.3 --- Results --- p.70 / Chapter 3.3.1 --- RNA quality estimation --- p.70 / Chapter 3.3.2 --- Quantification real time PCR --- p.70 / Chapter 3.3.3 --- Transcriptional profiling of laccases during the development of L edodes --- p.70 / Chapter 3.3.4 --- Transcriptional profiling of MnPs during the development of L edodes --- p.71 / Chapter 3.3.5 --- Transcript level analysis of laccases from in mycelia grown on lignocelluloses medium and non -lignocelluloses medium --- p.71 / Chapter 3.3.6 --- Transcript level analysis of MnPs in mycelia grown on lignocelluloses medium and non -lignocelluloses medium --- p.72 / Chapter 3.3.7 --- Differential expression of laccases from L. edodes grownin lignocelluloses medium during mycelia stage --- p.72 / Chapter 3.3.8 --- Differential expression of laccases from L. edodes grownin lignocelluloses medium during mycelia stage --- p.72 / Chapter 3.4 --- Discussion --- p.87 / Chapter 3.4.1 --- Transcriptional profiling of laccases and MnPs during four developmental stages --- p.87 / Chapter 3.4.2 --- Transcriptional profiling of laccases and MnPs in mycelium grown in lignocelluloses and non-lignocelluloses medium --- p.88 / Chapter 3.4.3 --- Temporal differential expression of laccases and manganese peroxidases --- p.90 / Chapter 3.5 --- Conclusion --- p.92 / Chapter Chapter 4: --- "Cloning and heterologous expression of Lentinula edodes laccase, lac1B, in yeast Pichia pastoris" --- p.93 / Chapter 4.1 --- Introduction --- p.93 / Chapter 4.2 --- Materials and Methods --- p.95 / Chapter 4.2.1 --- Strain cultivation --- p.95 / Chapter 4.2.2 --- First strand cDNA synthesis --- p.95 / Chapter 4.2.3 --- Construction of cDNA library --- p.95 / Chapter 4.2.4 --- Signal peptide prediction of Iac1 B --- p.96 / Chapter 4.2.5 --- Cloning of native laccase into Pichia pastoris expression vector --- p.96 / Chapter 4.2.6 --- Screening for positive colonies --- p.97 / Chapter 4.2.7 --- Construction of pool of recombinant vector --- p.97 / Chapter 4.2.8 --- Transformation of P. pastoris --- p.98 / Chapter 4.2.9 --- Screening for expression cassette into Pichia pastoris --- p.98 / Chapter 4.2.10 --- Enzyme Activity assay --- p.99 / Chapter 4.2.11 --- SDS-PAGE --- p.100 / Chapter 4.3 --- Results --- p.103 / Chapter 4.3.1 --- Screening for positive colonies with recombinant vector in TOP10 --- p.103 / Chapter 4.3.2 --- Screening for expression cassette from transform ants of P pastoris --- p.103 / Chapter 4.3.3 --- Enzyme activity assay --- p.103 / Chapter 4.3.4 --- SDS-PAGE --- p.104 / Chapter 4.4 --- Disscussion --- p.109 / Chapter Chapter 5: --- Concluding Remarks --- p.111 / Reference --- p.118

A billion tons per year of genetically modified corn residues are soil incorporated having both direct and indirect effects on the belowground environment, soil carbon (C) sequestration, and nutrient cycling. If Bt genetic modification has non-target effects on corn stover structural/non-structural carbohydrate and nitrogen (N) concentrations, then the degradation rate of Bt-corn stover may be different than that of non-Bt isolines, possibly influencing soil C storage and N mineralization. Thus, this research focused primarily on the comparison of C and N mineralization of corn stover in soil as affected by Bt-trait, plant portion, water-availability and HFC-trait; and secondarily on the existence of Bt-related variations in the chemical structure of corn residues that might affect the degradation rate of stover in soil and consequently the soil C and N dynamics. A laboratory experiment was conducted under non-limiting N conditions with stover of Bt/non-Bt isogenic pairs of two varieties, a ?high fermentable corn? (HFC) line harvested at Snook, Texas and a non-HFC corn line harvested at the irrigated field of Snook and the non-irrigated field of College Station, Texas. The stover was partitioned into three plant portions, incorporated into a Weswood soil and incubated during 223 days. Results showed that the differences observed in the degradation in soil of Bt vs. non-Bt corn stover were dependent on environmental conditions (irrigated vs. non-irrigated settings) and hybrid variety (HFC vs. non-HFC hybrid lines). The structural composition of corn plants was affected by the Bt-trait, HFC-trait, irrigation and their interactions. Variations in the biomass fractions of the initial stover of Bt and non-Bt hybrids had minimum to non-impact on soil C and N concentrations measured at the end of the 223-day incubation period. Lignin concentration was affected by a Bt-trait*variety interaction. There were no significant differences in lignin concentration between non-Bt/Bt-corn derived stovers of the non-HFC variety irrespective of irrigation regime but Bt-hybrids of the HFC variety contained more than twice as much lignin as the non-Bt isogenic plants. The effects of higher lignin concentration on C mineralization rate appeared to be offset by an increased lignin degradability inherent in HFC-trait. Overall, results indicated that the cultivation of Bt-modified maize lines is not likely to have significant effects on soil C or N dynamics compared with the cropping of non-Bt hybrids.

Under alkaline conditions hydrogen peroxide can be used either as a 1ignin-degrading or a 1ignin-preserving bleaching agent. If heavy metal ions are present and/or silicate is absent in the reaction medium, hydrogen peroxide decomposes via hydroxyl radicals and superoxide ions to oxygen and water. These decomposition products are able to react for example with phenolic lignin structures and thereby cause a partial degradation of lignin. In such a system peroxide could act as a bleaching and delignifying agent at the same time and these properties can be utilized for the bleaching of chemical pulps.In order to elucidate the factors which influence the degradation of phenolic structures by oxidation with alkaline hydrogen peroxide the lignin model compounds-methylsyringyl alcohol was studied.By determining the first order reaction rate constants for the oxidation, the main results which were obtained indicate that phenolic lignin structures can be efficiently degraded especially if:A. The pH in the bleaching liquor is close to the pK -valueàfor hydrogen peroxide.B. The ionic strength in the bleaching medium is as high as possible.C. A fixed amount of heavy metal ions (manganese) is added to the bleaching liquor.In the presence of silicate and diethylentriaminepenta-acetic acid (DTPA) hydrogen peroxide is stabilized against decomposition. Under these conditions alkaline hydrogen peroxide is able to react only with lignin units containing conjugated carbonyl groups such as quinone, aryl-oe-carbonyl and cinnamaldehyd structures, leading to an elimination of the chromophoric structures without any substantial dissolution of lignin. In this part of work we have elucidated the kinetic behavior and the reaction products from lignin model compounds of the aryl-of- carbonyl and cinnamaldehyde types.1,2-Diarylpropan-1,3,-diol structures constitute an important building unit in native lignins. We have demonstrated that under hydrogen peroxide bleaching conditions the model compound 2,3--bis(4-hydroxy-3-methoxyphenyl)-3-ethoxy-propanol was converted to stilbenes, ûe. structures which when present in pulps may contribute to a rapid yellowing. The results obtained with model compounds under simulated lignin retaining bleaching conditions demonstrate that there are possibilities to improve the bleaching of mechanical pulps with hydrogen peroxide if:A. The remaining heavy metal ions complexed with DTPA are present in their lowest valence states.B. The concentration of hydroperoxy ions can be maintained at a high level at the lowest possible pH-value. / digitalisering@umu

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The vulnerability of the Arctic to climate change has been realized due to disproportionately large increases in surface air temperatures which are not uniformly distributed over the seasonal cycle. Effects of this temperature shift are widespread in the Arctic but likely include changes to the hydrological cycle and permafrost thaw, which have implications for the mobilization of organic carbon into rivers. The focus of this research was to describe the seasonal variability of the chemical composition of dissolved organic matter (DOM) in the six largest Arctic rivers (Yukon, Mackenzie, Ob, Yenisei, Lena and Kolyma) using optical properties (UV-Vis Absorbance and Fluorescence) and lignin phenol analysis. We also investigated differences between rivers and how watershed characteristics influence DOM composition. Dissolved organic carbon (DOC) concentrations followed the hydrograph with highest concentrations measured during peak river flow. The chemical composition of peak-flow DOM indicates a dominance of freshly leached material with elevated aromaticity, larger molecular weight, and elevated lignin yields relative to base-flow DOM. During peak flow, soils in the watershed are still frozen and snowmelt water follows a lateral flow path to the river channels. As the soils thaw, surface water penetrates deeper into the soil horizons leading to lower DOC concentrations and likely altered composition of DOM due to sorption and microbial degradation processes. The six rivers studied here shared a similar seasonal pattern and chemical composition. There were, however, large differences between rivers in terms of total carbon discharge reflecting the differences in watershed characteristics such as climate, catchment size, river discharge, soil types, and permafrost distribution. The large rivers (Lena, Yenisei), with a greater proportion of permafrost, exported the greatest amount of carbon. The Kolyma and Mackenzie exported the smallest amount of carbon annually, however, the discharge weighted mean DOC concentration was almost 2-fold higher in the Kolyma, again, indicating the importance of continuous permafrost. The quality and quantity of DOM mobilized into Arctic rivers appears to depend on the relative importance of surface run-off and extent of soil percolation. The relative importance of these is ultimately determined by watershed characteristics.

A billion tons per year of genetically modified corn residues are soil incorporated having both direct and indirect effects on the belowground environment, soil carbon (C) sequestration, and nutrient cycling. If Bt genetic modification has non-target effects on corn stover structural/non-structural carbohydrate and nitrogen (N) concentrations, then the degradation rate of Bt-corn stover may be different than that of non-Bt isolines, possibly influencing soil C storage and N mineralization. Thus, this research focused primarily on the comparison of C and N mineralization of corn stover in soil as affected by Bt-trait, plant portion, water-availability and HFC-trait; and secondarily on the existence of Bt-related variations in the chemical structure of corn residues that might affect the degradation rate of stover in soil and consequently the soil C and N dynamics. A laboratory experiment was conducted under non-limiting N conditions with stover of Bt/non-Bt isogenic pairs of two varieties, a ?high fermentable corn? (HFC) line harvested at Snook, Texas and a non-HFC corn line harvested at the irrigated field of Snook and the non-irrigated field of College Station, Texas. The stover was partitioned into three plant portions, incorporated into a Weswood soil and incubated during 223 days. Results showed that the differences observed in the degradation in soil of Bt vs. non-Bt corn stover were dependent on environmental conditions (irrigated vs. non-irrigated settings) and hybrid variety (HFC vs. non-HFC hybrid lines). The structural composition of corn plants was affected by the Bt-trait, HFC-trait, irrigation and their interactions. Variations in the biomass fractions of the initial stover of Bt and non-Bt hybrids had minimum to non-impact on soil C and N concentrations measured at the end of the 223-day incubation period. Lignin concentration was affected by a Bt-trait*variety interaction. There were no significant differences in lignin concentration between non-Bt/Bt-corn derived stovers of the non-HFC variety irrespective of irrigation regime but Bt-hybrids of the HFC variety contained more than twice as much lignin as the non-Bt isogenic plants. The effects of higher lignin concentration on C mineralization rate appeared to be offset by an increased lignin degradability inherent in HFC-trait. Overall, results indicated that the cultivation of Bt-modified maize lines is not likely to have significant effects on soil C or N dynamics compared with the cropping of non-Bt hybrids.