Hydrothermal liquefaction is a thermochemical technique for obtaining crude bio-oil from lignocellulosic biomass with moderate temperature and pressure. The crude bio-oil can then be upgraded to various biofuels and bioproducts. Hydrothermal liquefaction is amenable to use of biomass feedstocks that have high-moisture. The overall goal of this research is to demonstrate the effectiveness of white rot fungus (WRF) as a pretreatment option in the production of bio-oil from switchgrass through hydrothermal liquefaction. If WRF is an effective pretreatment, it could be a cost-effective option for commercialization, allowing hydrothermal liquefaction to be used on an industrial scale to produce high quality bio-oil capable of replacing some of the fossil fuel liquids used today. This thesis specifically focuses on the investigation of the effects of particle size and culture time on lignin degradation using Phanerochaete chrysosporium as a pretreatment method on switchgrass. In addition, the conversion efficiency of WRF treated switchgrass was compared to that of torrefied switchgrass and untreated switchgrass after the pyrolysis conversion process. The results indicate that WRF outperforms torrefaction as a pretreatment method for the conversion of sugar-based components, thus may be an attractive alternative for fermentation conversion processes, but probably not for thermochemical processes.

Graduation date: 2006

Victoria blight of oats is caused by the fungus Cochhobolus victoriae. This fungus is pathogenic due to its ability to produce the host-selective toxin victorin. Previously, a 100-kD protein that binds victorin in vivo only in susceptible genotypes was identified as the P protein of the glycine decarboxylase complex (GDC). Victorin is a potent in vivo inhibitor of GDC. Leaf slices pretreated with victorin displayed an effective Victorin inhibited the concentration for 50% inhibition (EC������) of 81 ��M for GDA. glycine-bicarbonate exchange reaction in vitro with an EC������ of 23 ��M. We also identified a 15-kD mitochondrial protein in susceptible and resistant genotypes that hound victorin. Amino acid sequence analysis indicated this protein is the H protein component of the GDC. Thus, victorin specifically binds to two components of the GDC. Victorin had no detectable effect on GDC in isolated mitochondria, apparently due to the inability of isolated mitochondria to import victorin. The interaction of victorin with the GDC may be central to victorin's mode of action. Supporting this observation is the finding that CO��� gives partial protection against victorin. Elevated CO��� is known to ameliorate the effect of GDC inhibition. Victorin treated plants incubated in the light develop more severe symptoms than dark-incubated plants. Victorin appears to induce a plant-wide signal transduction cascade, resulting in diverse effects. Victorin induces specific proteolytic cleavage of the Rubisco large subunit (LSU). Leaf slices incubated with victorin for 4 hours in the dark accumulate a form of LSU which is cleaved after the N-terminal lysine 14. LSU cleavage in leaf slices is prevented by the protease inhibitors E-64 and calpeptin. LaCl��� prevents this cleavage of LSU and LaCl��� also confers complete protection against victorin at the whole plant level. Victorin also causes lipid peroxidation as measured by MDA accumulation. DNA laddering is seen in leaves after 3 hr treatment with toxin. The ethylene inhibitors AOA and STS give significant protection against victorin at the whole plant level, and also prevent LSU cleavage. / Graduation date: 1998

A knowledge of molecular properties and structure of heat-stable enzymes is important for the understanding of basic principles governing thermo stability of proteins and evolution of life at high temperatures. Information on functional characteristics of thermo stable enzymes is necessary also for improving existing biotechnologies and developing new ones. Because of these reasons enzymes from thermophilic organisms are being exploited. In this context, amylolytic enzymes represent a useful choice for investigation from both basic and applied points of view. a-Amylase and glucoamylase hydrolyse starch into oligosaccharides and glucose, respectively. In the present study a thermophilic fungus, Thermomyces lanuginosus, was selected as a source of thermostable starch-degrading enzymes. The main objectives of this research were to understand the physicochemical properties, mechanism of starch utilization by T. lanuginosus and effect of heat, on amylo1ytic enzymes. Purification of amylolytic enzymes A strain of T, lanuginosus, IlSc 91, isolated from a manure heap in our laboratory was found to produce higher levels of extra cellular amylolytic enzymes than strains obtained from culture collection in U.S.A, and Europe. This strain produced 4 units of glucoamylase and 40 units of a-amylase per ml of culture filtrate when grown on 2% starch at 50 C. Culture filtrate was used as the starting material for purification of these enzymes. Glucoamylase and a-amylase were purified by ultrafiltration and a combination of ion exchange and gel-filtration chromatography 93- and 112-fold with 30 and 41% recovery, respectively; Homogeneity of purified enzymes was established by the criteria of native- PAGE, SDS-PAGE, gel-filtration on HPLC and N-terminal amino acid analysis. Some of the physicochernical properties of these enzymes were studied. Physicochemical characteristics Glucoernylase is a monomeric glycoprotien (carbohydrate content 11 %, w/w) and has a molecular weight, of 45 kDa. It produces only glucose from starch. Km and Vmax, for soluble potato starch are 0.04 mg ml-1 and 666 p mole glucose min-1 mg protein-1, respectively. The enzyme is optimally active at 70 C at pH 6.0. Its activation energy is 14.0 kCal mole-1. It has melting temperature of 73 C. Molar extinction coefficient of glucoamylase is 5.5 x 104 mole-1 cm-l. It is stable at 60°C for > 7h. The enzyme is rich in alanine, serine and aspartate/ asparagine. Glucoamylase contains alanine as the N-terminal amino acid. It does not contain cysteine. Purified a-amylase is a homodimeric protein of 40 kDa and contains 5% (w/w) carbohydrate. It liberates oligosaccharides from starch with maltose being the principle product of hydrolysis. The Km for soluble starch is 2.5 mg ml-1. A high Vmax, of 8000 mg starch min-1 mg protein-1 was found. The enzyme is optimally active at 65°C at pH 5.6. The activation energy is 10.9 kCal mole-1. At 50DC, which is the optimal temperature of growth of T. lanuginosus, purified a-amylase is completely stable for over 6 h. Ca2+ increases the melting temperature of a-amylase from 66°C to 73°C. a-Amylase requires Ca2+ for its activity and structural stabi1it.y The molar extinction coefficient of the enzyme is 4.7 x 10' mole-1 cm-1 a- Amylase is rich in aspartate / asparagine, glutamatme /glutamine, alanine, glycine and leucine. It does not contain cysteine. a- Amylase contains alanine as the N-t.ermina1 amino acid. Hydrolysis of starch by a-amylase and glucoamylase Experiments were done to understand the role of a-amylase and glucoamylase in the utilization of starch by T. lanuginosus. Crude and purified amylase preparations hydrolyse raw potato starch slightly more efficiently than soluble potato starch. The extent of starch hydrolysis by a mixture of a-amylase and glucoamylase is equal to that by culture filtrate containing the same amount of enzyme activities, Electrophoresis of crude culture filtrate proteins on native-PAGE and activity staining on gel showed the presence of one species each of a-amylase and glucoamylase. This suggests that in T. lanuginosus hydrolysis of starch is mediated by one species each of extracellular a-amylase and glucoamylase. The hydrolysis of starch by a mixture of a-amylase and glucoamylase is equal to the arithmetic sum of hydrolysis by individual enzyme showing that the enzymes do not act synergistically. a-Amylase is the main starch depolymerizing enzyme. Conversion of starch into glucose by glucoamylase does not require the presence of a-amylase. Starch is hydralysed to a maximum of 72 and 97% by glucoamylase and a-amylase, respectively. Effect of heat on a-amylase The effect, of heat, on a-amylase and glucoamylase was studied with the view to obtain information on the thermal inactivation of these proteins. Five-min heat treatment of the native a-amylase (40 kDa) results in the specific conversion of all protein molecules into partially active (approximately 50% residual activity) and SDS-undissociable dimer of 45 kDa. a-Amylase (45 kDa) after 5-min heat treatment. is partially active and can he rendered completely active by incubation at 37°C for 3 h. This altered form of a-amylase is not due to the formation of disulfide linkage in protein because the enzyme does not contain cysteine and b mercaptoethanol does not prevent heat-induced structural change. Heat, treatment, for 20 min or more results in further structural changes which result in the irreversible inactivation of the enzyme. Prolonged heating (>40 min) probably causes the degradation of protein. Reactivation of 20-min heat-inactivated a-amylase occurs specifically at 37°C or 50°C within 3 h but not at lower temperatures (0°C or 4°C). Native-PAGE analysis of the native and 20-min heated-reactivated a-amylase shows that the reactivated sample is comprised of two protein species of different charge and/or mass. Activity staining shows that only one of these protein band is active and it has electrophoretic mobility identical to that of the native enzyme. Native and the active fraction of 20-min heated-reactivated a-amylase possess similar specific activity. This suggests that it is cat8alytmically and perhaps structurally similar to the native enzyme. The native and the reactivated a-amylase are resist ant to trypsin digestion. However, heat- inactivated a-amylase is degraded into low molecular weight, peptides. These observation suggest that heat-inactivated a-amylase is partially unfolded, Unlike the native, the heat-treated (94"C, 5 min) a-amylase can not be stained with AgN03 while both forms can be stained with Coomassie brilliant blue R and by Schiff's base. On the basis of these observations a tentative model was proposed for the effect of heat on a-amylase (Fig.) Staining by + + Staining by AgNO3, + + Staining by Schiff 's base + + Sensitivity to trypsin + + Figure : Schematic represent ation of heat-induced changes in a-amylase

Thesis (M.S.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 84-89). Also available on the World Wide Web.

Thesis (M.S.)--Oregon State University, 2002. / Typescript (photocopy). Includes bibliographical references. Also available on the World Wide Web.

Background and Aims Sexual deception is a species-specific pollination strategy commonly found in Orchidaceae. Sexually deceptive orchids lure male insect pollinators by mimicking the sex pheromones and/or appearance of female insects, which elicit copulatory behaviour with the flower by the male insects. This specialised pollination strategy has recently been found in a Pterostylis species in Australia. Pterostylis orchids also occur in New Zealand, although very few studies have been done on this genus, and no such specialised insect pollination strategy has been documented in New Zealand. Methods I investigated the breeding system and pollinators of three Pterostylis spp. to determine whether sexual deception may be operating in P. oliveri, P. irsoniana and P. venosa growing in native beech forests in Arthur's Pass. We also investigated the floral headspace volatiles of P. oliveri to determine which compounds are present, and which may be responsible for pollinator attraction. Key Results Breeding system experiments suggest that P. oliveri and P. irsoniana are self compatible, but exclusively dependent on insects for pollination. Only male fungus gnats (Diptera: Mycetophilidae) were found carrying pollinia attached to their thoraxes in traps set up over the flowers. Insect identification and ITS DNA analysis of the pollinia showed that each orchid species was pollinated by a specific fungus gnat species; Mycetophila latifascia males found with pollen of P. oliveri; Morganiella fusca males found with pollen of P. irsoniana; and Tetragoneura sp. males found with pollen of P. venosa. Field tests of an unidentified compound found in headspace volatiles of P. oliveri did not attract any Mycetophila latifascia males. Conclusions These results indicate that pollination via sexual deception may be operating in these three Pterostylis spp. However, further floral volatile analyses are required to confirm whether the flowers emit volatile compounds that resemble the sex pheromones of the specific pollinators.

Mycorrhizal fungi are a major component of agricultural systems and play a key role in plant nutrition. Little is known about the effects of tillage practices and manuring on arbuscular mycorrhizal fungi (AMF). The purpose of this study was to evaluate the effect of soil disturbance on winter survival, development and distribution of AMF in soil and on plant nutrient uptake and productivity. This research was conducted in long-term corn plots in two soils and under controlled conditions. / A growth chamber study with field soil demonstrated that most of the fungal hyphae with mycorrhizal plants were mycorrhizal rather than saprophytic. This result was extrapolated to subsequent experiments. Soil disturbance reduced corn nutrient uptake and growth by disrupting the AMF hyphal network. Similarly, fallow periods reduced density of AMF hyphae, leading to reduced mineral nutrients uptake and plant growth. Soil disturbance was also found to severely reduce winter survival of AMF hyphae in agricultural soil. AM hyphae could survive the winter in soil, even when they were not attached to roots. Their survival however, was improved when they remained attached to roots. / Under field conditions, indigenous AMF were more abundant in no-till soil, less abundant under reduced tillage and least abundant under conventional tillage. Under all tillage systems, most of AMF hyphae were located in the top 15 cm of the soil profile suggesting that deep plowing could result in dilution of AMF propagules in the seeding zone. There was a seasonal variation in the abundance of hyphae in soil. Soil hyphae and root colonization declined after the silking stage of corn. Hyphal abundance decreased further over the winter, to reach their lowest level in the spring. / The spatial distribution of fungal hyphae in the field was not homogenous. Hyphal density was maximal directly under the corn rows and decreased linearly up to the mid-row. Marked seasonal variations in hyphal densities were observed on the row but fluctuations at mid-row were not significant suggesting that little AMF hyphae were ever present between the rows. Liquid dairy manure had little effect on the abundance of hyphae and spores.

Barley powdery mildew disease is caused by the highly specialised phytopathogenic fungus, Blumeria graminis f.sp. hordei. Disease is spread prolifically by the production of asexual conidia. Following contact with the barley leaf surface, a short, primary germ tube (PGT) emerges, followed by elaboration of a second-formed germ tube, the appressorial germ tube (AGT). This second-formed germ tube elongates, swells and hooks to form an appressorium, which allows direct penetration of the barley cuticle and infection of the host. Infection structure differentiation in B. graminis is a highly regulated and complex process. It demands the coordinated perception of multiple external signals, but little is known about how these signals are integrated and transduced within the fungus. Protein kinase A (PKA) and cAMP signalling are known to play important, but complex, roles during infection structure development. However, signalling via cAMP alone is not sufficient to promote progression through infection structure differentiation. This study describes the characterization of two B. graminis protein kinase C genes, pkc1 and pkc-like. PKC activity was identified in B. graminis protein extracts. Efforts to find an inhibitor specific for B. graminis PKC were unsuccessful. However, phorbol ester, a PKC agonist, invoked both appressorium formation when applied to spores in vivo and PKC activity in protein extracts. In addition, real-time PCR confirmed the differentially regulated transcript profiles of both pkc1 and pkc-like, revealing a peak in transcript levels just prior to PGT emergence for pkc1, and during PGT differentiation for the pkc-like gene. Two mitogen-activated protein (MAP) kinases, mpk1 and mpk2, were characterized. MAP kinase activity was detected in conidial protein extracts. The MAP kinase kinase (MEK) inhibitor, PD98059, inhibited B. graminis germling morphogenesis. However, a MAP kinase agonist failed to show any effect on germling differentiation. In addition, real-time PCR confirmed the differentially regulated transcript profiles of both mpk1 and mpk2, and revealed a peak in transcript levels during appressorial germ tube elongation and swelling for both genes. The "model" phytopathogenic fungus, Magnaporthe grisea, was employed as a "surrogate host" for the functional analysis of the B. graminis MAP kinase gene, mpk1. Firstly, the mpk1 promoter was sequenced and a plasmid construct made comprising the mpk1 gene under the control of the mpk1 promoter. This, and the control construct, that is the M. grisea PMK1 gene under the control of the PMK1 promoter, were transformed into M. grisea Δpmk1. Southern analysis identified transformants for phenotypic studies. These showed that whereas Δpmk1 was complemented by M. grisea PMK1 in the control experiments, B. graminis mpk1 failed to complement Δpmk1. Expression studies showed that there was no expression of mpk1 in an mpk1 transformant.

The defence mechanisms expressed in roots of Pinus sylvestris seedlings challenged with fungal pathogens were investigated, and a comparison was made between the expression of defences in non-mycorrhizal and mycorrhizal seedlings. Papillae were formed by cortical cells of non-mycorrhizal seedlings infected with Cylindrocarpon destructans. Histochemical evidence was obtained for pectic materials comprising an important polysaccharide component of these structures, and for the deposition of polyphenolic compounds also. Proton induced X-ray emission (PIXE) microanalysis indicated that insoluble calcium levels were elevated in papillae relative to normal cell walls. Although papillae appeared important in protecting cortical cells against penetration by fungal hyphae, a primary role for the wall appositions in the resistance of seedlings of Scots pine against root pathogens could not be proven. Although phytoalexins were not detected in the roots of Scots pine seedlings following infection with C. destructans, the mean content of an abietic acid fraction (comprising six compounds, of which only dehydroabietic acid could be positively identified), increased from 5.2 to 9.7mg g^-1 dry weight. This fraction exhibited some antifungal activity. -related proteins induced de novo by infection could not be detected, but several constitutive apoplastic proteins, including some with chitinase activity, appeared to increase in the needles of root-infected seedlings. The formation of ectomycorrhizae with Pisolithus tinctorius, Suillus bovinus and Hebeloma crustuliniforme did not itself induce papilla formation in the roots of P. sylvestris. Evidence was obtained to suggest that the response was suppressed when mycorrhizal seedlings were challenged with C. destructans. Results highly suggestive of the induction of systemic resistance in P. sylvestris seedlings, consequent upon mycorrhizal infection, were obtained. In seedlings grown in vitro the survival rate of mycorrhizal seedlings challenged aerially with Botrytis cinerea was 37.5% compared with 7.1 in seedlings grown gnotobiotically. However, the physiological mechanisms by which this protection was imparted remain to be determined.