Effects of forest management and time on herbaceous species dynamics in the western Upper Peninsula of Michigan

The herbaceous layer is a dynamic layer in a forest ecosystem which often contains the highest species richness in northern temperate forests. Few long-term studies exist in northern hardwood forests with consistent management practices to observe herbaceous species dynamics. The Ford Forest (Michigan Technological University) reached its 50th year of management during the winter of 2008-2009. Herbaceous species were sampled during the summers pre- and post-harvest. Distinct herbaceous communities developed in the 13-cm diameter-limit treatment and the uncut control. After the harvest, the diameter-limit treatments had herbaceous communities more similar to the 13-cm diameter-limit treatment than the uncut control; the herbaceous layer contained more exotic and early successional species. Fifty years of continuous management changed the herbaceous community especially in the diameter-limit treatments. Sites used in the development of habitat classification systems based on the presence and absence of certain herbaceous species can also be used to monitor vegetation change over time. The Guide to Forest Communities and Habitat Types of Michigan was developed to aid forest managers in understanding the potential productivity of a stand, and often aid in the development of ecologically-based forest management practices. Subsets of plots used to create the Western Upper Peninsula Guide were resampled after 10 years. During the resampling, both spring and summer vegetation were sampled and earthworm populations were estimated through liquid extraction. Spring sampling observed important spring ephemerals missed during summer sampling. More exotic species were present during the summer 2010 sampling than the summer 2000 sampling. Invasive European earthworms were also observed at all sample locations in all habitat types; earthworm densities increased with increasing habitat richness. To ensure the accuracy of the guide book, plots should be monitored to see how herbaceous communities are changing. These plots also offer unique opportunities to monitor for invasive species and the effects of a changing climate.

Optimization of ethanol production by yeasts from lignocellulosic feedstocks

Ethanol from lignocellulosic feedstocks is not currently competitive with corn-based ethanol in terms of yields and commercial feasibility. Through optimization of the pretreatment and fermentation steps this could change. The overall goal of this study was to evaluate, characterize, and optimize ethanol production from lignocellulosic feedstocks by the yeasts Saccharomyces cerevisiae (strain Ethanol Red, ER) and Pichia stipitis CBS 6054. Through a series of fermentations and growth studies, P. stipitis CBS 6054 and S. cerevisiae (ER) were evaluated on their ability to produce ethanol from both single substrate (xylose and glucose) and mixed substrate (five sugars present in hemicellulose) fermentations. The yeasts were also evaluated on their ability to produce ethanol from dilute acid pretreated hydrolysate and enzymatic hydrolysate. Hardwood (aspen), softwood (balsam), and herbaceous (switchgrass) hydrolysates were also tested to determine the effect of the source of the feedstock. P. stipitis produced ethanol from 66-98% of the theoretical yield throughout the fermentation studies completed over the course of this work. S. cerevisiae (ER) was determined to not be ideal for dilute acid pretreated lignocellulose because it was not able to utilize all the sugars found in hemicellulose. S. cerevisiae (ER) was instead used to optimize enzymatic pretreated lignocellulose that contained only glucose monomers. It was able to produce ethanol from enzymatically pretreated hydrolysate but the sugar level was so low (>3 g/L) that it would not be commercially feasible. Two lignocellulosic degradation products, furfural and acetic acid, were evaluated for whether or not they had an inhibitory effect on biomass production, substrate utilization, and ethanol production by P. stipitis and S. cerevisiae (ER). It was determined that inhibition is directly related to the concentration of the inhibitor and the organism. The final phase for this thesis focused on adapting P. stipitis CBS 6054 to toxic compounds present in dilute acid pretreated hydrolysate through directed evolution. Cultures were transferred to increasing concentrations of dilute acid pretreated hydrolysate in the fermentation media. The adapted strains’ fermentation capabilities were tested against the unadapted parent strain at each hydrolysate concentration. The fermentation capabilities of the adapted strain were significantly improved over the unadapted parentstrain. On media containing 60% hydrolysate the adapted strain yielded 0.30 g_ethanol/g_sugar ± 0.033 (g/g) and the unadapted parent strain yielded 0.11 g/g ±0.028. The culture has been successfully adapted to growth on media containing 65%, 70%, 75%, and 80% hydrolysate but with below optimal ethanol yields (0.14-0.19 g/g). Cell recycle could be a viable option for improving ethanol yields in these cases. A study was conducted to determine the optimal media for production of ethanol from xylose and mixed substrate fermentations by P. stipitis. Growth, substrate utilization, and ethanol production were the three factors used to evaluate the media. The three media tested were Yeast Peptone (YP), Yeast Nitrogen Base (YNB), and Corn Steep Liquor (CSL). The ethanol yields (g/g) for each medium are as follows: YP - 0.40-0.42, YNB -0.28-.030, and CSL - 0.44-.051. The results show that media containing CSL result in slightly higher ethanol yields then other fermentation media. P. stipitis was successfully adapted to dilute acid pretreated aspen hydrolysate in increasing concentrations in order to produce higher ethanol yields compared to the unadapted parent strain. S. cerevisiae (ER) produced ethanol from enzymatic pretreated cellulose containing low concentrations of glucose (1-3g/L). These results show that fermentations of lignocellulosic feedstocks can be optimized based on the substrate and organism for increased ethanol yields.

Assessing success of forest restoration efforts in degraded montane cloud forests in southern Mexico

Montane cloud forests are home to great biodiversity. However, non-sustainable anthropogenic activities have led to the loss of forest cover in southern Mexico. Increasing conservation, restoration and sustainable use of forest resources prevents the loss of cloud forests. In this study, success of forest restoration was evaluated in a degraded forest of Highlands Chiapas. The goal of this study was to assess the structure and composition of native tree species. We evaluated vegetation composition at three sites that had undergone enrichment plantings. Floristic composition and structure of the herbaceous, seedling, sapling, and overstory layers were measured. A total of sixty-six native tree species were recorded. Enrichment planting was found to have increased tree diversity. Moreover, 54% of the planted species were found in the understory, indicating that they were successfully recruiting. In conclusion, enrichment planting can aid in the conservation of forest cover in degraded areas.

Wolf habitat selection at the territory level : seasonal and interannual variation and influence on reproductive success

Habitat selection has been one of the main research topics in ecology for decades. Nevertheless, many aspects of habitat selection still need to be explored. In particular, previous studies have overlooked the importance of temporal variation in habitat selection and the value of including data on reproductive success in order to describe the best quality habitat for a species. We used data collected from radiocollared wolves in Yellowstone National Park (USA), between 1996 and 2008, to describe wolf habitat selection. In particular, we aimed to identify i) seasonal differences in wolf habitat selection, ii) factors influencing interannual variation in habitat selection, and iii) the effect of habitat selection on wolf reproductive success. We used probability density functions to describe wolf habitat use and habitat coverages to represent the habitat available to wolves. We used regression analysis to connect habitat use with habitat characteristics and habitat selection with reproductive success. Our most relevant result was discovering strong interannual variability in wolf habitat selection. This variability was in part explained by pack identity and differences in litter size and leadership of a pack between two years (summer) and in pack size and precipitation (winter). We also detected some seasonal differences. Wolves selected open habitats, intermediate elevations, intermediate distances from roads, and avoided steep slopes in late winter. They selected areas close to roads and avoided steep slopes in summer. In early winter, wolves selected wetlands, herbaceous and shrub vegetation types, and areas at intermediate elevation and distance from roads. Surprisingly, the habitat characteristics selected by wolves were not useful in predicting reproductive success. We hypothesize that interannual variability in wolf habitat selection may be too strong to detect effects on reproductive success. Moreover, prey availability and competitor pressure may also have an influence on wolf reproductive success, which we did not assess. This project demonstrated how important temporal variation is in shaping patterns of habitat selection. We still believe in the value of running long-term studies, but the effect of temporal variation should always be taken into account.

Management impacts on Carex assiniboinensis and Cardamine concatenata on the Ottawa National Forest

Monitoring of herbaceous plants on the Ottawa National Forest (ONF) is used to understand the impact of forest management on understory composition and site conditions. In their planning, national forests are required to take into account management impacts on diversity and ecosystem health. The effect of management on understory species is dependent on various factors, including the intensity of disturbance and the biology of the plant. In the first study in this report, a population of Carex assiniboinensis, a Michigan state threatened species, was monitored for seven seasons including before logging commenced, in order to determine the sedge’s response to a single-tree selection harvest. Analyses provided insights for management of C. assiniboinensis at the stand level over the short-term. In the second study in this report, the use of the cutleaf toothwort (Cardamine concatenata) as a Management Indicator Species on the ONF was reviewed. Data were analyzed to determine the suitability of using C. concatenata to monitor impacts of forest management on site conditions. The various factors that affect understory species population dynamics illuminated the challenges of using indicator species to monitor site conditions. Insights from the study provide a greater understanding of management impacts on understory species across the Ottawa National Forest.

Land use/cover change using Remote Sensing and Geographic Information Systems : Pic Macaya National Park, Haiti

A post classification change detection technique based on a hybrid classification approach (unsupervised and supervised) was applied to Landsat Thematic Mapper (TM), Landsat Enhanced Thematic Plus (ETM+), and ASTER images acquired in 1987, 2000 and 2004 respectively to map land use/cover changes in the Pic Macaya National Park in the southern region of Haiti. Each image was classified individually into six land use/cover classes: built-up, agriculture, herbaceous, open pine forest, mixed forest, and barren land using unsupervised ISODATA and maximum likelihood supervised classifiers with the aid of field collected ground truth data collected in the field. Ground truth information, collected in the field in December 2007, and including equalized stratified random points which were visual interpreted were used to assess the accuracy of the classification results. The overall accuracy of the land classification for each image was respectively: 1987 (82%), 2000 (82%), 2004 (87%). A post classification change detection technique was used to produce change images for 1987 to 2000, 1987 to 2004, and 2000 to 2004. It was found that significant changes in the land use/cover occurred over the 17- year period. The results showed increases in built up (from 10% to 17%) and herbaceous (from 5% to 14%) areas between 1987 and 2004. The increase of herbaceous was mostly caused by the abandonment of exhausted agriculture lands. At the same time, open pine forest and mixed forest areas lost (75%) and (83%) of their area to other land use/cover types. Open pine forest (from 20% to 14%) and mixed forest (from18 to 12%) were transformed into agriculture area or barren land. This study illustrated the continuing deforestation, land degradation and soil erosion in the region, which in turn is leading to decrease in vegetative cover. The study also showed the importance of Remote Sensing (RS) and Geographic Information System (GIS) technologies to estimate timely changes in the land use/cover, and to evaluate their causes in order to design an ecological based management plan for the park.

The influence of concurrent disturbances on plant community dynamics in northern hemlock-hardwood forests

Throughout the Upper Great Lakes region, alterations to historic disturbance regimes have influenced plant community dynamics in hemlock-hardwood forests. Several important mesic forest species, eastern hemlock (Tsuga canadensis), yellow birch (Betula alleghaniensis), eastern white pine (Pinus strobus), and Canada yew (Taxus canadensis), are in decline due to exploitive logging practices used at the turn of the 20th century and the wave of intense fires that followed. Continued regeneration and recruitment failure is attributed to contemporary forest management practices and overbrowsing by white-tailed deer (Odocoileus virginianus). Therefore, I examined the influence of two concurrent disturbances, overstory removal and herbivory, on plant community dynamics in two hemlock-hardwood forests. I measured the post-disturbance regeneration response (herbaceous and woody species) inside and outside of deer exclosures in 20 artificial canopy gaps (50 – 450 m2) and monitored survival and growth for hundreds of planted seedlings. The results of this research show that interacting disturbances can play a large role in shaping plant community composition and structure in hemlock-hardwood forests. White-tailed deer herbivory homogenized the post-disturbance plant communities across the experimental gradient of gap areas, essentially making species compositions in small gaps “look like” those in large gaps. Deer browsing also influenced probability of survival for planted Canada yew cuttings; all else being equal an individual was nearly seven times more likely to survive if protected from herbivory (P < 0.001). In contrast, the ability of sugar maple (Acer saccharum) to persist under high levels of herbivory and respond rapidly to overstory release appears to be related to the presence of stem layering(i.e., portions of below-ground prostrate stem). Layering occurred in 52% of excavated saplings (n = 100) and was significantly associated with increased post-disturbance height growth. Understory light was also important to planted seedling establishment and height growth. Higher levels of direct under-canopy light negatively impacted survival for shade-tolerant hemlock and Canada yew, while an increase in diffuse light was linked to a higher probability of survival for yellow birch and height growth for hemlock and Canada yew. Increases in white pine height growth were also significantly associated with a decrease in canopy cover.

Planting and production of switchgrass (Panicum virgatum L.) as a bioenergy crop in Michigan's Upper Peninsula

Switchgrass (Panicum virgatum L.) is a perennial grass holding great promise as a biofuel resource. While Michigan’s Upper Peninsula has an appropriate land base and climatic conditions, there is little research exploring the possibilities of switchgrass production. The overall objectives of this research were to investigate switchgrass establishment in the northern edge of its distribution through: investigating the effects of competition on the germination and establishment of switchgrass through the developmental and competitive characteristics of Cave-in-Rock switchgrass and large crabgrass (Digitaria sanguinalis L.) in Michigan’s Upper Peninsula; and, determining the optimum planting depths and timing for switchgrass in Michigan’s Upper Peninsula. For the competition study, a randomized complete block design was installed June 2009 at two locations in Michigan’s Upper Peninsula. Four treatments (0, 1, 4, and 8 plants/m2) of crabgrass were planted with one switchgrass plant. There was a significant difference between switchgrass biomass produced in year one, as a function of crabgrass weed pressure. There was no significant difference between the switchgrass biomass produced in year two versus previous crabgrass weed pressure. There is a significant difference between switchgrass biomass produced in year one and two. For the depth and timing study, a completely randomized design was installed at two locations in Michigan’s Upper Peninsula on seven planting dates (three fall 2009, and four spring 2010); 25 seeds were planted 2 cm apart along 0.5 m rows at depths of: 0.6 cm, 1.3 cm, and 1.9 cm. Emergence and biomass yields were compared by planting date, and depths. A greenhouse seeding experiment was established using the same planting depths and parameters as the field study. The number of seedlings was tallied daily for 30 days. There was a significant difference in survivorship between the fall and spring planting dates, with the spring being more successful. Of the four spring planting dates, there was a significant difference between May and June in emergence and biomass yield. June planting dates had the most percent emergence and total survivorship. There is no significant difference between planting switchgrass at depths of 0.6 cm, 1.3 cm, and 1.9 cm. In conclusion, switchgrass showed no signs of a legacy effect of competition from year one, on biomass production. Overall, an antagonistic effect on switchgrass biomass yield during the establishment period has been observed as a result of increasing competing weed pressure. When planting switchgrass in Michigan’s Upper Peninsula, it should be done in the spring, within the first two weeks of June, at any depth ranging from 0.6 cm to 1.9 cm.

Kinetic characterization for pretreatment of timber varieties and switchgrass using diluted acid hydrolysis

In recent years, growing attention has been devoted to the use of lignocellulosic biomass as a feedstock to produce renewable carbohydrates as a source of energy products, including liquid alternatives to fossil fuels. The benefits of developing woody biomass to ethanol technology are to increase the long-term national energy security, reduce fossil energy consumption, lower greenhouse gas emissions, use renewable rather than depletable resources, and create local jobs. Currently, research is driven by the need to reduce the cost of biomass-ethanol production. One of the preferred methods is to thermochemically pretreat the biomass material and subsequently, enzymatically hydrolyze the pretreated material to fermentable sugars that can then be converted to ethanol using specialized microorganisms. The goals of pretreatment are to remove the hemicellulose fraction from other biomass components, reduce bioconversion time, enhance enzymatic conversion of the cellulose fraction, and, hopefully, obtain a higher ethanol yield. The primary goal of this research is to obtain kinetic detailed data for dilute acid hydrolysis for several timber species from the Upper Peninsula of Michigan and switchgrass. These results will be used to identify optimum reaction conditions to maximize production of fermentable sugars and minimize production of non-fermentable byproducts. The structural carbohydrate analysis of the biomass species used in this project was performed using the procedure proposed by National Renewable Energy Laboratory (NREL). Subsequently, dilute acid-catalyzed hydrolysis of biomass, including aspen, basswood, balsam, red maple, and switchgrass, was studied at various temperatures, acid concentrations, and particle sizes in a 1-L well-mixed batch reactor (Parr Instruments, ii Model 4571). 25 g of biomass and 500 mL of diluted acid solution were added into a 1-L glass liner, and then put into the reactor. During the experiment, 5 mL samples were taken starting at 100°C at 3 min intervals until reaching the targeted temperature (160, 175, or 190°C), followed by 4 samples after achieving the desired temperature. The collected samples were then cooled in an ice bath immediately to stop the reaction. The cooled samples were filtered using 0.2 μm MILLIPORE membrane filter to remove suspended solids. The filtered samples were then analyzed using High Performance Liquid Chromatography (HPLC) with a Bio-Rad Aminex HPX-87P column, and refractive index detection to measure monomeric and polymeric sugars plus degradation byproducts. A first order reaction model was assumed and the kinetic parameters such as activation energy and pre-exponential factor from Arrhenius equation were obtained from a match between the model and experimental data. The reaction temperature increases linearly after 40 minutes during experiments. Xylose and other sugars were formed from hemicellulose hydrolysis over this heat up period until a maximum concentration was reached at the time near when the targeted temperature was reached. However, negligible amount of xylose byproducts and small concentrations of other soluble sugars, such as mannose, arabinose, and galactose were detected during this initial heat up period. Very little cellulose hydrolysis yielding glucose was observed during the initial heat up period. On the other hand, later in the reaction during the constant temperature period xylose was degraded to furfural. Glucose production from cellulose was increased during this constant temperature period at later time points in the reaction. The kinetic coefficient governing the generation of xylose from hemicellulose and the generation of furfural from xylose presented a coherent dependence on both temperature and acid concentration. However, no effect was observed in the particle size. There were three types of biomass used in this project; hardwood (aspen, basswood, and red maple), softwood (balsam), and a herbaceous crop (switchgrass). The activation energies and the pre-exponential factors of the timber species and switchgrass were in a range of 49 - 180 kJ/mol and from 7.5x104 - 2.6x1020 min-1, respectively, for the xylose formation model. In addition, for xylose degradation, the activation energies and the preexponential factors ranged from 130 - 170 kJ/mol and from 6.8x1013 - 3.7x1017 min-1, respectively. The results compare favorably with the literature values given by Ranganathan et al, 1985. Overall, up to 92 % of the xylose was able to generate from the dilute acid hydrolysis in this project.

Regulation of woody plant development by gibberellin catabolic and signaling genes

Gibberellin (GA) is a growth promoting hormone implicated in regulating a diversity of plant processes. This dissertation examines the role of GA metabolic and signaling genes in woody plant growth and development. Transgenic modifications, expression analysis, physiological/biochemical assays, biometric measurements and histological analysis were used to understand the regulatory roles these genes play in the model woody plant, Populus. Our results highlight the importance of GA regulatory genes in woody perennial growth, including: phenology, wood formation, phenotypic plasticity, and growth/survival under field conditions. We characterize two putative Populus orthologs of the SHORT INTERNODES (SHI) gene from Arabidopsis, a negative regulator of GA signaling. RNAi-mediated suppression of Populus SHI-like genes increased several growth-related traits, including extent of xylem proliferation, in a dose-dependent manner. Three Populus genes, sharing sequence homology to the positive regulator of GA signaling gene PHOTOPERIOD-RESPONSIVE 1 (PHOR1) from Solanum, are up-regulated in GA-deficient and insensitive plants suggesting a conserved role in GA signaling. We demonstrate that Populus PHOR1-like genes have overlapping and divergent function(s). Two PHOR1-like genes are highly expressed in roots, predominantly affect root growth (e.g., morphology, starch quantity and gravitropism), and induced by short-days (SD). The other PHOR1-like gene is ubiquitously expressed with a generalized function in root and shoot development. The effects of GA catabolic and signaling genes on important traits (e.g., adaptive and productivity traits) were studied in a multi-year field trial. Transgenics overexpressing GA 2-oxidase (GA2ox) and DELLA genes showed tremendous variation in growth, form, foliage, and phenology (i.e., vegetative and reproductive). Observed gradients in trait modifications were correlated to transgene expression levels, in a manner suggesting a dose-dependent relationship. We explore GA2ox and DELLA genes involvement in mediating growth responses to immediate short-term drought stress, and SD photoperiods, signaling prolonged periods of stress (e.g., winter bud dormancy). GA2ox and DELLA genes show substantial up-regulation in response to drought and SDs. Transgenics overexpressing homologs of these genes subjected to drought and SD photoperiods show hypersensitive growth restraint and increased stress resistances. These results suggest growth cessation (i.e., dormancy) in response to adverse conditions is mediated by GA regulatory genes.

PHYLOGENETIC ANALYSES, GENE DOSAGE EFFECT AND SELECTION IN MEMBERS OF THE LATERAL BOUNDARY DOMAIN (LBD) GENES TRANSCRIPTION FACTOR FAMILY

Wood formation is an economically and environmentally important process and has played a significant role in the evolution of terrestrial plants. Despite its significance, the molecular underpinnings of the process are still poorly understood. We have previously shown that four Lateral Boundary Domain (LBD) transcription factors have important roles in the regulation of wood formation with two (LBD1 and LBD4) involved in secondary phloem and ray cell development and two (LBD15 and LBD18) in secondary xylem formation. Here, we used comparative phylogenetic analyses to test potential roles of the four LBD genes in the evolution of woodiness. We studied the copy number and variation in DNA and amino acid sequences of the four LBDs in a wide range of woody and herbaceous plant taxa with fully sequenced and annotated genomes. LBD1 showed the highest gene copy number across the studied species, and LBD1 gene copy number was strongly and significantly correlated with the level of ray seriation. The lianas, cucumber and grape, with multiseriate ray cells showed the highest gene copy number (12 and 11, respectively). Because lianas’ growth habit requires significant twisting and bending, the less lignified ray parenchyma cells likely facilitate stem flexibility and maintenance of xylem conductivity. We further demonstrate conservation of amino acids in the LBD18 protein sequences that are specific to woody taxa. Neutrality tests showed evidence for strong purifying selection on these gene regions across various orders, indicating adaptive convergent evolution of LBD18. Structural modeling demonstrates that the conserved amino acids have a significant impact on the tertiary protein structure and thus are likely of significant functional importance.

Spatial and temporal patterns in ungulate-ecosystem interactions

Ungulates are important components of a variety of ecosystems worldwide. This dissertation integrates aspects of ungulate and forest ecology to increase our understanding of how they work together in ways that are of interest to natural resource managers, educators, and those who are simply curious about nature. Although animal ecology and ecosystem ecology are often studied separately, one of the general goals of this dissertation is to examine how they interact across spatial and temporal scales. Forest ecosystems are heterogeneous across a range of scales. Spatial and temporal habitat use patterns of forest ungulates tend to be congregated in patches where food and/or cover are readily available. Ungulates interact with ecosystem processes by selectively foraging on plants and excreting waste products in concentrated patches. Positive feedbacks may develop where these activities increase the value of habitat through soil fertilization or the alteration of plant chemistry and architecture. Heterogeneity in ecosystem processes and plant community structure, observed at both stand and local scales, may be the integrated outcome of feedbacks between ungulate behavior and abiotic resource gradients. The first chapter of this dissertation briefly discusses pertinent background information on ungulate ecology, with a focus on white-tailed deer (Odocoileus virginianus) in the Upper Great Lakes region and moose (Alces acles) in Isle Royale National Park, Michigan, USA. The second chapter demonstrates why ecological context is important for studying ungulate ecology in forest ecosystems. Excluding deer from eastern hemlock (Tsuga canadensis) stands, which deer use primarily as winter cover, resulted in less spatial complexity in soil reactive nitrogen and greater complexity in diffuse light compared to unfenced stands. The spatial patterning of herbaceous-layer cover was more similar to nitrogen where deer were present, and was a combination of nitrogen and light within deer exclosures. This relationship depends on the seasonal timing of deer habitat use because deer fertilize the soil during winter, but leave during the growing season. The third chapter draws upon an eight-year, 39-stand data set of deer fecal pellet counts in hemlock stands to estimate the amount of nitrogen that deer are depositing in hemlock stands each winter. In stands of high winter deer use, deer-excreted nitrogen inputs consistently exceeded those of atmospheric deposition at the stand scale. At the neighborhood scale, deer-excreted nitrogen was often in excess of atmospheric deposition due to the patchy distribution of deer habitat use. Spatial patterns in habitat use were consistent over the eight-year study at both stand and neighborhood scales. The fourth chapter explores how foraging selectivity by moose interacts with an abiotic resource gradient to influence forest structure and composition. Soil depth on Isle Royale varies from east to west according to glacial history. Fir saplings growing in deeper soils on the west side are generally more palatable forage for moose (lower foliar C:N) than those growing in shallower soils on the east side. Therefore, saplings growing in better conditions are less likely to reach the canopy due to moose browsing, and fir is a smaller overstory component on the west side. Lastly, chapter five focuses on issues surrounding eastern hemlock regeneration failure, which is a habitat type that is important to many wildlife species. Increasing hemlock on the landscape is complicated by several factors including disturbance regime and climate change, in addition to the influence of deer.

Biochemical Conversions of Lignocellulosic Biomass for Sustainable Fuel-Ethanol Production in the Upper Midwest

Biofuels are an increasingly important component of worldwide energy supply. This research aims to understand the pathways and impacts of biofuels production, and to improve these processes to make them more efficient. In Chapter 2, a life cycle assessment (LCA) is presented for cellulosic ethanol production from five potential feedstocks of regional importance to the upper Midwest - hybrid poplar, hybrid willow, switchgrass, diverse prairie grasses, and logging residues - according to the requirements of Renewable Fuel Standard (RFS). Direct land use change emissions are included for the conversion of abandoned agricultural land to feedstock production, and computer models of the conversion process are used in order to determine the effect of varying biomass composition on overall life cycle impacts. All scenarios analyzed here result in greater than 60% reduction in greenhouse gas emissions relative to petroleum gasoline. Land use change effects were found to contribute significantly to the overall emissions for the first 20 years after plantation establishment. Chapter 3 is an investigation of the effects of biomass mixtures on overall sugar recovery from the combined processes of dilute acid pretreatment and enzymatic hydrolysis. Biomass mixtures studied were aspen, a hardwood species well suited to biochemical processing; balsam, a high-lignin softwood species, and switchgrass, an herbaceous energy crop with high ash content. A matrix of three different dilute acid pretreatment severities and three different enzyme loading levels was used to characterize interactions between pretreatment and enzymatic hydrolysis. Maximum glucose yield for any species was 70% oftheoretical for switchgrass, and maximum xylose yield was 99.7% of theoretical for aspen. Supplemental β-glucosidase increased glucose yield from enzymatic hydrolysis by an average of 15%, and total sugar recoveries for mixtures could be predicted to within 4% by linear interpolation of the pure species results. Chapter 4 is an evaluation of the potential for producing Trichoderma reesei cellulose hydrolases in the Kluyveromyces lactis yeast expression system. The exoglucanases Cel6A and Cel7A, and the endoglucanase Cel7B were inserted separately into the K. lactis and the enzymes were analyzed for activity on various substrates. Recombinant Cel7B was found to be active on carboxymethyl cellulose and Avicel powdered cellulose substrates. Recombinant Cel6A was also found to be active on Avicel. Recombinant Cel7A was produced, but no enzymatic activity was detected on any substrate. Chapter 5 presents a new method for enzyme improvement studies using enzyme co-expression and yeast growth rate measurements as a potential high-throughput expression and screening system in K. lactis yeast. Two different K. lactis strains were evaluated for their usefulness in growth screening studies, one wild-type strain and one strain which has had the main galactose metabolic pathway disabled. Sequential transformation and co-expression of the exoglucanase Cel6A and endoglucanase Cel7B was performed, and improved hydrolysis rates on Avicel were detectable in the cell culture supernatant. Future work should focus on hydrolysis of natural substrates, developing the growth screening method, and utilizing the K. lactis expression system for directed evolution of enzymes.