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.

Ethanol Production from Hardboard Manufacturing Process Wastewater: Methods for Improving Product Yields from Hemicellulose Hydrolysates

Waste effluents from the forest products industry are sources of lignocellulosic biomass that can be converted to ethanol by yeast after pretreatment. However, the challenge of improving ethanol yields from a mixed pentose and hexose fermentation of a potentially inhibitory hydrolysate still remains. Hardboard manufacturing process wastewater (HPW) was evaluated at a potential feedstream for lignocellulosic ethanol production by native xylose-fermenting yeast. After screening of xylose-fermenting yeasts, Scheffersomyces stipitis CBS 6054 was selected as the ideal organism for conversion of the HPW hydrolysate material. The individual and synergistic effects of inhibitory compounds present in the hydrolysate were evaluated using response surface methodology. It was concluded that organic acids have an additive negative effect on fermentations. Fermentation conditions were also optimized in terms of aeration and pH. Methods for improving productivity and achieving higher ethanol yields were investigated. Adaptation to the conditions present in the hydrolysate through repeated cell sub-culturing was used. The objectives of this present study were to adapt S. stipitis CBS6054 to a dilute-acid pretreated lignocellulosic containing waste stream; compare the physiological, metabolic, and proteomic profiles of the adapted strain to its parent; quantify changes in protein expression/regulation, metabolite abundance, and enzyme activity; and determine the biochemical and molecular mechanism of adaptation. The adapted culture showed improvement in both substrate utilization and ethanol yields compared to the unadapted parent strain. The adapted strain also represented a growth phenotype compared to its unadapted parent based on its physiological and proteomic profiles. Several potential targets that could be responsible for strain improvement were identified. These targets could have implications for metabolic engineering of strains for improved ethanol production from lignocellulosic feedstocks. Although this work focuses specifically on the conversion of HPW to ethanol, the methods developed can be used for any feedstock/product systems that employ a microbial conversion step. The benefit of this research is that the organisms will the optimized for a company's specific system.

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.

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.

EXHAUST EMISSIONS OF LOW LEVEL BLEND ALCOHOL FUELS FROM TWO-STROKE AND FOUR-STROKE MARINE ENGINES

The U.S. Renewable Fuel Standard mandates that by 2022, 36 billion gallons of renewable fuels must be produced on a yearly basis. Ethanol production is capped at 15 billion gallons, meaning 21 billion gallons must come from different alternative fuel sources. A viable alternative to reach the remainder of this mandate is iso-butanol. Unlike ethanol, iso-butanol does not phase separate when mixed with water, meaning it can be transported using traditional pipeline methods. Iso-butanol also has a lower oxygen content by mass, meaning it can displace more petroleum while maintaining the same oxygen concentration in the fuel blend. This research focused on studying the effects of low level alcohol fuels on marine engine emissions to assess the possibility of using iso-butanol as a replacement for ethanol. Three marine engines were used in this study, representing a wide range of what is currently in service in the United States. Two four-stroke engine and one two-stroke engine powered boats were tested in the tributaries of the Chesapeake Bay, near Annapolis, Maryland over the course of two rounds of weeklong testing in May and September. The engines were tested using a standard test cycle and emissions were sampled using constant volume sampling techniques. Specific emissions for two-stroke and four-stroke engines were compared to the baseline indolene tests. Because of the nature of the field testing, limited engine parameters were recorded. Therefore, the engine parameters analyzed aside from emissions were the operating relative air-to-fuel ratio and engine speed. Emissions trends from the baseline test to each alcohol fuel for the four-stroke engines were consistent, when analyzing a single round of testing. The same trends were not consistent when comparing separate rounds because of uncontrolled weather conditions and because the four-stroke engines operate without fuel control feedback during full load conditions. Emissions trends from the baseline test to each alcohol fuel for the two-stroke engine were consistent for all rounds of testing. This is due to the fact the engine operates open-loop, and does not provide fueling compensation when fuel composition changes. Changes in emissions with respect to the baseline for iso-butanol were consistent with changes for ethanol. It was determined iso-butanol would make a viable replacement for ethanol.

ASSESSMENT OF ASPEN AND NORTHERN HARDWOODS EXTENT IN THIRTY THREE COUNTIES OF UPPER/LOWER MICHIGAN

The importance of the United States' wood and wood byproducts as biomass feedstocks is increasing as the concern about security and sustainability of global energy production continues to rise. Thus, second generation woody feedstock sources in Michigan, e.g., hybrid poplar and hybrid willow (Populus spp.), are viewed as a potential source of biomass for the proposed biofuel ethanol production plant in Kinross, MI. It is important to gain an understanding of the spatial distribution of current feedstock sources, harvesting accessibility via the transportation infrastructure and land ownerships in order to ensure long-term feedstock extent. This research provides insights into the current extent of aspen and northern hardwoods, and an assessment of potential for expanding the area of these feedstock sources based on pre-European settlement conditions. A geographic information system (GIS) was developed to compile available geospatial data for 33 counties located within 150 miles of the Kinross facility. These include present day and pre-European settlement land use/cover, soils, road infrastructure, and land ownerships. The results suggest that a significant amount of northern hardwoods has been converted to other land use/cover types since European settlement, and the "scattering" of aspen stands has increased. Furthermore, a significant amount of woody biomass is available in close proximity to the existing road network, which can be effectively utilized as feedstock. Potential aspen and northern hardwoods restoration areas are identified in the vicinity of road networks which can be used for future woody feedstock production.

CHARACTERIZING AND IMPROVING PRODUCTION OF FERMENTABLE SUGARS AND CO-PRODUCTS FROM A FOREST PRODUCT INDUSTRY WASTEWATER STREAM

Hardboard processing wastewater was evaluated as a feedstock in a bio refinery co-located with the hardboard facility for the production of fuel grade ethanol. A thorough characterization was conducted on the wastewater and the composition changes of which during the process in the bio refinery were tracked. It was determined that the wastewater had a low solid content (1.4%), and hemicellulose was the main component in the solid, accounting for up to 70%. Acid pretreatment alone can hydrolyze the majority of the hemicellulose as well as oligomers, and over 50% of the monomer sugars generated were xylose. The percentage of lignin remained in the liquid increased after acid pretreatment. The characterization results showed that hardboard processing wastewater is a feasible feedstock for the production of ethanol. The optimum conditions to hydrolyze hemicellulose into fermentable sugars were evaluated with a two-stage experiment, which includes acid pretreatment and enzymatic hydrolysis. The experimental data were fitted into second order regression models and Response Surface Methodology (RSM) was employed. The results of the experiment showed that for this type of feedstock enzymatic hydrolysis is not that necessary. In order to reach a comparatively high total sugar concentration (over 45g/l) and low furfural concentration (less than 0.5g/l), the optimum conditions were reached when acid concentration was between 1.41 to 1.81%, and reaction time was 48 to 76 minutes. The two products produced from the bio refinery were compared with traditional products, petroleum gasoline and traditional potassium acetate, in the perspective of sustainability, with greenhouse gas (GHG) emission as an indicator. Three allocation methods, system expansion, mass allocation and market value allocation methods were employed in this assessment. It was determined that the life cycle GHG emissions of ethanol were -27.1, 20.8 and 16 g CO2 eq/MJ, respectively, in the three allocation methods, whereas that of petroleum gasoline is 90 g CO2 eq/MJ. The life cycle GHG emissions of potassium acetate in mass allocation and market value allocation method were 555.7 and 716.0 g CO2 eq/kg, whereas that of traditional potassium acetate is 1020 g CO2/kg.

Sustainable Energy Production in the United States: Life Cycle Assessment of Biofuels and Bioenergy

The United States of America is making great efforts to transform the renewable and abundant biomass resources into cost-competitive, high-performance biofuels, bioproducts, and biopower. This is the key to increase domestic production of transportation fuels and renewable energy, and reduce greenhouse gas and other pollutant emissions. This dissertation focuses specifically on assessing the life cycle environmental impacts of biofuels and bioenergy produced from renewable feedstocks, such as lignocellulosic biomass, renewable oils and fats. The first part of the dissertation presents the life cycle greenhouse gas (GHG) emissions and energy demands of renewable diesel (RD) and hydroprocessed jet fuels (HRJ). The feedstocks include soybean, camelina, field pennycress, jatropha, algae, tallow and etc. Results show that RD and HRJ produced from these feedstocks reduce GHG emissions by over 50% compared to comparably performing petroleum fuels. Fossil energy requirements are also significantly reduced. The second part of this dissertation discusses the life cycle GHG emissions, energy demands and other environmental aspects of pyrolysis oil as well as pyrolysis oil derived biofuels and bioenergy. The feedstocks include waste materials such as sawmill residues, logging residues, sugarcane bagasse and corn stover, and short rotation forestry feedstocks such as hybrid poplar and willow. These LCA results show that as much as 98% GHG emission savings is possible relative to a petroleum heavy fuel oil. Life cycle GHG savings of 77 to 99% were estimated for power generation from pyrolysis oil combustion relative to fossil fuels combustion for electricity, depending on the biomass feedstock and combustion technologies used. Transportation fuels hydroprocessed from pyrolysis oil show over 60% of GHG reductions compared to petroleum gasoline and diesel. The energy required to produce pyrolysis oil and pyrolysis oil derived biofuels and bioelectricity are mainly from renewable biomass, as opposed to fossil energy. Other environmental benefits include human health, ecosystem quality and fossil resources. The third part of the dissertation addresses the direct land use change (dLUC) impact of forest based biofuels and bioenergy. An intensive harvest of aspen in Michigan is investigated to understand the GHG mitigation with biofuels and bioenergy production. The study shows that the intensive harvest of aspen in MI compared to business as usual (BAU) harvesting can produce 18.5 billion gallons of ethanol to blend with gasoline for the transport sector over the next 250 years, or 32.2 billion gallons of bio-oil by the fast pyrolysis process, which can be combusted to generate electricity or upgraded to gasoline and diesel. Intensive harvesting of these forests can result in carbon loss initially in the aspen forest, but eventually accumulates more carbon in the ecosystem, which translates to a CO2 credit from the dLUC impact. Time required for the forest-based biofuels to reach carbon neutrality is approximately 60 years. The last part of the dissertation describes the use of depolymerization model as a tool to understand the kinetic behavior of hemicellulose hydrolysis under dilute acid conditions. Experiments are carried out to measure the concentrations of xylose and xylooligomers during dilute acid hydrolysis of aspen. The experiment data are used to fine tune the parameters of the depolymerization model. The results show that the depolymerization model successfully predicts the xylose monomer profile in the reaction, however, it overestimates the concentrations of xylooligomers.