Scott W. Pryor

Combined effect of pelleting and pretreatment on enzymatic hydrolysis of switchgrass

Switchgrass was pelleted to evaluate the effect of densification on acidic and alkaline pretreatment efficacy. Bulk density and durability of pellets were 724 kg/m(3) and 95%, respectively. Ground switchgrass (D(90) = 21.7 mm) was further ground to a fine power (D(90) = 0.5mm) in the pellet mill prior to densification. This grinding increased enzymatic hydrolyzate glucose yields of non-pretreated materials by 210%. Pelleting had no adverse impact on dilute acid pretreatment efficacy. Grinding and pelleting increased hydrolyzate glucose yields of switchgrass pretreated by soaking in aqueous ammonia (SAA) by 37%. Xylose yields from SAA-pretreated switchgrass pellets were 42% higher than those from the original biomass. Increases in sugar yields from SAA-pretreated pelleted biomass are attributed to grinding and heating of biomass during the pelleting process. Potential transportation, storage, and handling benefits of biomass pelleting may be achieved without negatively affecting the downstream processing steps of pretreatment or enzymatic hydrolysis.

Highly Efficient Phase Boundary Biocatalysis with Enzymogel Nanoparticles

The enzymogel nanoparticle made of a magnetic core and polymer brush shell demonstrates a novel type of remote controlled phase-boundary biocatalysis that involves remotely directed binding to and engulfing insoluble substrates, high mobility, and stability of the catalytic centers. The mobile enzymes reside in the polymer brush scaffold and shuttle between the enzymogel interior and surface of the engulfed substrate in the bioconversion process. Biocatalytic activity of the mobile enzymes is preserved in the enzymogel while the brush-like architecture favors the efficient interfacial interaction when the enzymogel spreads over the substrate and extends substantially the reaction area as compared with rigid particles.

Production and characterization of epoxidized canola oil.

Epoxidized canola oil may be well suited to the partial replacement of petroleum products in composite matrices; however, a process is needed to obtain this material from canola oil at sufficient conversion and scale to assess product properties. Therefore, canola oil was epoxidized in a solvent-free process with a heterogeneous catalyst; a fractional factorial design was used to determine the impact of processing conditions and their two-factor interactions on epoxy group content of epoxidized canola oil. The studied parameters were: molar ratio of acetic acid to unsaturation, molar ratio of hydrogen peroxide to unsaturation, concentration of hydrogen peroxide, concentration of catalyst, and temperature. Epoxidized canola oil with up to 98.5% conversion was produced. The parameters molar ratio of acetic acid to unsaturation, concentration of hydrogen peroxide, temperature, and their interactions were found to be significant in the defined design space. Process conditions that achieved the highest conversion were scaled to 300 g to compare the conversion, production yield, and rheological and melting properties of products of the epoxidation of both canola and soybean oil with and without solvent. Epoxidized canola oil crystallized at room temperature; at 40°C it was shear-thinning with an apparent viscosity of 140 to 150 mPa·s. Elimination of solvent in the epoxidation process decreased the yield 10% but did not reduce the conversion to epoxy groups. Therefore, the scaled-up, solvent-free process is proposed as a green alternative for sufficient epoxidized canola oil to test composite applications.

Effects of Protein Separation Conditions on the Functional and Thermal Properties of Canola Protein Isolates

UNLABELLED Canola meal protein isolates were prepared from defatted canola meal flour using alkaline solubilization and acid precipitation. A central composite design was used to model 2nd-order response surfaces for the protein yield and the functional properties of protein isolates. The solubilization pH and precipitation pH were used as design factors. The models showed that the protein yield and functional properties of isolates, such as water absorption and fat absorption, were sensitive to both solubilization pH and precipitation pH, whereas the emulsification was sensitive to only solubilization pH. Gel electrophoresis analysis of protein fractions gave evidence to the compositional changes between proteins isolated under different conditions. Differences in glass transition temperatures suggest that proteins tend to be more denatured when solubilized at highly alkaline conditions. These conformational and compositional changes due to different protein separation conditions have contributed to the changes in functional properties of protein isolates. PRACTICAL APPLICATION   Protein isolation conditions may be determined primarily through optimization of total protein yield. Improvements in protein functional properties may be achieved with a relatively small sacrifice in yield by altering isolation conditions.

Effect of hemicellulase addition during enzymatic hydrolysis of switchgrass pretreated by soaking in aqueous ammonia.

The focus of this study was to determine the effect of supplementing cellulase with hemicellulase during enzymatic hydrolysis of switchgrass pretreated by soaking in aqueous ammonia (SAA) under a range of conditions. SAA was performed using 15% aqueous ammonia for 8 or 24h at temperatures of 40 or 60°C. The combined effect of cellulase and hemicellulase loadings on glucose yield during enzymatic hydrolysis was modeled for each pretreatment condition. Glucose yields greater than 85% of theoretical were achieved for pretreatment at 40°C for 24h and for 60°C for 8h. Hemicellulase supplementation was not sufficient to achieve these glucose yields at lower severity SAA pretreatment. High severity SAA pretreatment also led to low yields despite improved delignification.

Deficiency of Cellulase Activity Measurements for Enzyme Evaluation

Switchgrass was used as a model feedstock to determine the influence of pretreatment conditions and biomass quality on enzymatic hydrolysis using different enzyme products. Dilute sulfuric acid and soaking in aqueous ammonia pretreatments were used to produce biomass with varied levels of hemicellulose and lignin sheathing. Pretreated switchgrass solids were tested with simple enzymatic hydrolysis and simultaneous saccharification and fermentation (SSF) with three commercial enzyme products: Accellerase 1000 (Genencor), Spezyme CP (Genencor)/Novozyme 188 (Novozymes), and Celluclast/Novozyme 188 (Novozymes). Enzymes were loaded on a common activity basis (FPU/g cellulose and CBU/g cellulose). Despite identical enzyme loadings, glucose yields were significantly different for both acid and alkaline pretreatments but differences diminished as hydrolysis progressed for acid-pretreated biomass. Cellobiose concentrations in Accellerase treatments indicated an initial β-glucosidase limitation that became less significant over time. SSF experiments showed that differences in glucose and ethanol yields could not be attributed to enzyme product inhibition. Yield discrepancies of glucose or ethanol in acid pretreatment, alkaline pretreatment, and acid pretreatment/SSF were as much as 15%, 19%, and 5%. These results indicate that standardized protocols for measuring enzyme activity may not be adequate for assessing activity using pretreated biomass substrates.

Separation and Evaluation of Canola Meal and Protein for Industrial Bioproducts

Methods and processing conditions used in the preparation of canola protein materials play an important role in determining the properties of the ultimate products that they are used for. Canola seeds were screw pressed, milled, defatted and the meal was subjected to alkaline solubilization and acid precipitation in order to isolate the proteins. Maximum solubility and precipitabiltiy of proteins were observed at pH 12.0 and pH 5.0, respectively. Biuret method was used in determining protein concentration of the supernatants at each solubilization and precipitation step. Specific proteins were isolated according to the Osborn sequence. Albumins and globulins consist of the majority of the proteins isolated with this procedure. Functional properties of canola meal such as water and fat absorption, emulsification, whippability and foaming were determined.

Identification of Antifungal Compounds in a Biological Control Product Using a Microplate Inhibition Bioassay

An inhibition assay was developed to quantify the antifungal activity of a solid-state fermentation of Bacillus subtilis. Methanol extracts were tested against a spore solution of the fungal pathogen Fusarium oxysporum f. sp. melonis using a 96-well microplate bioassay. HPLC analysis of extracts showed positive correlation between inhibition and levels of several active lipopeptides produced during fermentation. MS analysis confirmed the presence of the lipopeptides iturin A, fengycin, and surfactin. HPLC fractionation and subsequent bioassays established that the activity of the extracts was largely associated with the fraction containing fengycins. A smaller amount of activity was attributable to the fraction containing iturin A.

Impact of Meal Preparation Method and Extraction Procedure on Canola Protein Yield and Properties

Screw-pressed and cold-milled, solvent-defatted canola meal was processed with a conventional Osborne sequence extraction method to obtain albumin, globulin, prolamin, and glutelin protein fractions. This extraction sequence was altered by isolating globulins prior to albumins, resulting in an increased globulin yield. The difference can be attributed to the presence of salts in canola meal flour, which increase globulin extraction during albumin isolation in the conventional method. An increase in the cumulative protein yield was observed when cold-milled canola meal flour was used instead of screw-pressed flour. Significant differences were also observed in the functional properties (water absorption, fat absorption, and emulsifying activity) of canola protein fractions with respect to both meal preparation method and protein isolation method. Results suggested that milling temperature and isolation method can have significant impacts on protein quality and yield.

Enzymatic Hydrolysis and Fermentation of Sugar Beet Pulp

Pressed sugar beet pulp, a byproduct of the table sugar industry, has potential as a biofuel feedstock that will benefit producers and processors in North Dakota and Minnesota. The goal of this research is to maximize ethanol titers and yields through enzymatic hydrolysis of sugar beet pulp and fermentation of the five- and six- carbon sugars. Hemicellulose and pectin were hydrolyzed and fermented separately from cellulose in order to increase ethanol titers and yields. A commercial pectinase was used to hydrolyze hemicellulose and pectin in the pulp. The resulting solid and liquid streams were either processed sequentially in a serial fermentation or separated and fermented in parallel. The first hydrolyzate stream, containing high concentrations of glucose, arabinose, and galacturonic acid, was fermented using Escherichia coli KO11. The remaining solids had a high cellulose content and were processed via simultaneous saccharification and fermentation using commercial cellulases and Saccharomyces cerevisiae.