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.

Hydrothermal Carbons from Hemicellulose-Derived Aqueous Hydrolysis Products as Electrode Materials for Supercapacitors

Acid pretreatment of lignocellulosic biomass, required for bioethanol production, generates large amounts of by-products, such as lignin and hydrolyzed hemicellulose fractions, which have found so far very limited applications. In this work, we demonstrate how the recovered hemicellulose hydrolysis products can be effectively utilized as a precursor for the synthesis of functional carbon materials through hydrothermal carbonization (HTC). The morphology and chemical structure of the synthesized HTC carbons are thoroughly characterized to highlight their similarities with glucose-derived HTC carbons. Furthermore, two routes for introducing porosity within the HTC carbon structure are presented: i) silica nanoparticle hard-templating, which is shown to be a viable method for the synthesis of carbonaceous hollow spheres; and ii) KOH chemical activation. The synthesized activated carbons (ACs) show an extremely high porosity (pore volume≈1.0 cm3 g−1) mostly composed of micropores (90 % of total pore volume). Because of their favorable textural properties, the ACs are further tested as electrodes for supercapacitors, yielding very promising results (300 F g−1 at 250 mA g−1) and confirming the high suitability of KOH-activated HTC carbons derived from spruce and corncob hydrolysis products as materials for electric double layer supercapacitors.

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.

Pretreatment of sugarcane bagasse by acid-catalysed process in aqueous ionic liquid solutions

A biomass pretreatment process was developed using acidified ionic liquid (IL) solutions containing 10-30% water. Pretreatment of sugarcane bagasse at 130°C for 30min by aqueous 1-butyl-3-methylimidazolium chloride (BMIMCl) solution containing 1.2% HCl resulted in a glucan digestibility of 94-100% after 72h of enzymatic hydrolysis. HCl was found to be a more effective catalyst than H(2)SO(4) or FeCl(3). Increasing acid concentration (from 0.4% to 1.2%) and reaction temperature (from 90 to 130°C) increased glucan digestibility. The glucan digestibility of solid residue obtained with the acidified BMIMCl solution that was re-used for three times was >97%. The addition of water to ILs for pretreatment could significantly reduce IL solvent costs and allow for increased biomass loadings, making the pretreatment by ILs a more economic proposition.

Multi-scale structural and chemical analysis of sugarcane bagasse in the process of sequential acid-base pretreatment and ethanol production by Scheffersomyces shehatae and Saccharomyces cerevisiae

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Environmental assessment of residues generated after consecutive acid-base pretreatment of sugarcane bagasse by advanced oxidative process

Abstract Background Biofuels produced from sugarcane bagasse (SB) have shown promising results as a suitable alternative of gasoline. Biofuels provide unique, strategic, environmental and socio-economic benefits. However, production of biofuels from SB has negative impact on environment due to the use of harsh chemicals during pretreatment. Consecutive sulfuric acid-sodium hydroxide pretreatment of SB is an effective process which eventually ameliorates the accessibility of cellulase towards cellulose for the sugars production. Alkaline hydrolysate of SB is black liquor containing high amount of dissolved lignin. Results This work evaluates the environmental impact of residues generated during the consecutive acid-base pretreatment of SB. Advanced oxidative process (AOP) was used based on photo-Fenton reaction mechanism (Fenton Reagent/UV). Experiments were performed in batch mode following factorial design L9 (Taguchi orthogonal array design of experiments), considering the three operation variables: temperature (°C), pH, Fenton Reagent (Fe2+/H2O2) + ultraviolet. Reduction of total phenolics (TP) and total organic carbon (TOC) were responsive variables. Among the tested conditions, experiment 7 (temperature, 35°C; pH, 2.5; Fenton reagent, 144 ml H2O2+153 ml Fe2+; UV, 16W) revealed the maximum reduction in TP (98.65%) and TOC (95.73%). Parameters such as chemical oxygen demand (COD), biochemical oxygen demand (BOD), BOD/COD ratio, color intensity and turbidity also showed a significant change in AOP mediated lignin solution than the native alkaline hydrolysate. Conclusion AOP based on Fenton Reagent/UV reaction mechanism showed efficient removal of TP and TOC from sugarcane bagasse alkaline hydrolysate (lignin solution). To the best of our knowledge, this is the first report on statistical optimization of the removal of TP and TOC from sugarcane bagasse alkaline hydrolysate employing Fenton reagent mediated AOP process.

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.

Laboratory and pilot scale pretreatment of sugarcane bagasse by acidified aqueous glycerol solutions

Pretreatment of sugarcane bagasse with acidified aqueous glycerol solution was evaluated at both laboratory and pilot scales. Laboratory scale pretreatment (4.00 g dry mass in 40.00 g liquid) with glycerol solutions containing ≤ 20 wt% water and 1.2 wt% HCl at 130 °C for 60 min resulted in biomass having glucan digestibilities of ≥ 88%. Comparable glucan enzymatic digestibility of 90% was achieved with bagasse pretreated at pilot scale (10 kg dry mass in 60 kg liquid) using a glycerol solution containing 0.4 wt% HCl and 17 wt% water at 130 °C for 15 min. We attribute more efficient pretreatment at pilot scale (despite shorter reaction time and reduced acid content) to improved mixing and heat transfer in a horizontal reactor. Pretreatment of sugarcane bagasse with acid-catalysed glycerol solutions likely produces glycerol-glycosides, which together with hydrolysed lignin are potential substrates for the production of biopolymers.

Effects of pH on pretreatment of sugarcane bagasse using aqueous imidazolium ionic liquids

Pretreatments of sugarcane bagasse for saccharification using different acid-catalysed imidazolium IL solutions (containing 20% water) at 130 °C for 30 min were investigated. At the same solution pH, pretreatment effectiveness in terms of glucan digestibility, delignification and xylan removal were similar for aqueous 1-butyl-3-methylimidazolium methane sulfonate (BMIMCH3SO3), 1-butyl-3-methylimidazolium methyl sulfate (BMIMCH3SO4), 1-ethyl-3-methylimidazolium chloride (EMIMCl) and 1-butyl-3-methylimidazolium chloride (BMIMCl). Decreasing solution pH of aqueous IL systems from 6.0 to 0.4 increased bagasse delignification and xylan removal, and as a result, improved glucan digestibility. The glucan digestibilities for bagasse samples pretreated by IL solutions with pH ≤ 0.9 were > 90% after 72 h of enzymatic hydrolysis. Without pH adjustment, the effectiveness of these aqueous IL solutions (except BMIMCH3SO3 because of its low natural pH of 0.9) to deconstruct the biomass was poor and the glucan digestibilities of pretreated bagasse samples were < 20%. These results show that pretreatment effectiveness of aqueous imidazolium ILs can simply be estimated from solution pH rather than hydrogen bond basicity (β) of the IL solution.

Effect of pretreatment on saccharification of sugarcane bagasse by complex and simple enzyme mixtures

Saccharification of sugarcane bagasse pretreated at the pilot-scale with different processes (in combination with steam-explosion) was evaluated. Maximum glucan conversion with Celluclast 1.5 L (15–25 FPU/g glucan) was in the following order: glycerol/HCl > HCl > H2SO4 > NaOH, with the glycerol system achieving ∼100% conversion. Surprisingly, the NaOH substrate achieved optimum saccharification with only 8 FPU/g glucan. Glucan conversions (3.6–6%) obtained with mixtures of endo-1,4-β-glucanase (EG) and β-glucosidase (βG) for the NaOH substrate were 2–6 times that of acid substrates. However, glucan conversions (15–60%) obtained with mixtures of cellobiohydrolase (CBH I) and βG on acidified glycerol substrate were 10–30% higher than those obtained for NaOH and acid substrates. The susceptibility of the substrates to enzymatic saccharification was explained by their physical and chemical attributes. Acidified glycerol pretreatment offers the opportunity to simplify the complexity of enzyme mixtures required for saccharification of lignocellulosics.

Low temperature pretreatment of sugarcane bagasse at atmospheric pressure using mixtures of ethylene carbonate and ethylene glycol

A low temperature lignocellulose pretreatment process was developed using acid-catalysed mixtures of alkylene carbonate and alkylene glycol. Pretreatment of sugarcane bagasse with mixtures of ethylene carbonate (EC) and ethylene glycol (EG) was more effective than that with mixtures of propylene carbonate (PC) and propylene glycol (PG). These mixtures were more effective than the individual components in making bagasse cellulose more amenable to cellulase digestion. Glucan digestibilities of ≥87% could be achieved with a wide range of EC to EG ratios from 9:1 to 1:1 (w/w). Pretreatment of bagasse by the EC/EG mixture with a ratio of 4:1 in the presence of 1.2% H2SO4 at 90 °C for 30 min led to the highest glucan enzymatic digestibility of 93%. The high glucan digestibilities obtained under these acidic conditions were due to (a) the ability of alkylene carbonate to cause significant biomass size reduction, (b) the ability of alkylene glycol to cause biomass defibrillation, (c) the ability of alkylene carbonate and alkylene glycol to remove xylan and lignin, and (d) the magnified above attributes in the mixtures of alkylene carbonate and alkylene glycol.

Auto-inhibition of hydrogen gas evolution on gold in aqueous acid solution

It was demonstrated recently that dramatic changes in the redox behaviour of gold/aqueous solution interfaces may be observed following either cathodic or thermal electrode pretreatment. Further work on the cathodic pretreatment of gold in acid solution revealed that as the activity of the gold surface was increased, its performance as a substrate for hydrogen gas evolution under constant potential conditions deteriorated. The change in activity of the gold atoms at the interface, which was attributed to a hydrogen embrittlement process (the occurrence of the latter was subsequently checked by surface microscopy), was confirmed, as in earlier work, by the appearance of a substantial anodic peak at ca. 0.5 V (RHE) in a post-activation positive sweep. Changes in the catalytic activity of a metal surface reflect the fact that the structure (or topography), thermodynamic activity and electronic properties of a surface are dependent not only on pretreatment but also, in the case of the hydrogen evolution reaction, vary with time during the course of reaction. As will be reported shortly, similar (and often more dramatic) time-dependent behaviour was observed for hydrogen gas evolution on other metal electrodes.

Pretreatment of sugarcane bagasse by acidified aqueous polyol solutions

Pretreatments of sugarcane bagasse by three high boiling-point polyol solutions were compared in acid-catalysed processes. Pretreatments by ethylene glycol (EG) and propylene glycol solutions containing 1.2 % H2SO4 and 10 % water at 130 °C for 30 min removed 89 % lignin from bagasse resulting in a glucan digestibility of 95 % with a cellulase loading of ~20 FPU/g glucan. Pretreatment by glycerol solution under the same conditions removed 57 % lignin with a glucan digestibility of 77 %. Further investigations with EG solutions showed that increases in acid content, pretreatment temperature and time, and decrease in water content improved pretreatment effectiveness. A good linear correlation of glucan digestibility with delignification was observed with R2 = 0.984. Bagasse samples pretreated with EG solutions were characterised by scanning electron microscopy, Fourier transform infrared spectroscopy and X-ray diffraction, which confirmed that improved glucan enzymatic digestibility is mainly due to delignification and defibrillation of bagasse. Pretreatment by acidified EG solutions likely led to the formation of EG-glycosides. Up to 36 % of the total lignin was recovered from pretreatment hydrolysate, which may improve the pretreatment efficiency of recycled EG solution.