Stability of commercial glucanase and ß-glucosidase preparations under hydrolysis conditions.

The cost of enzymes makes enzymatic hydrolysis one of the most expensive steps in the production of lignocellulosic ethanol. Diverse studies have used commercial enzyme cocktails assuming that change in total protein concentration during hydrolysis was solely due to adsorption of endo- and exoglucanases onto the substrate. Given the sensitivity of enzymes and proteins to media conditions this assumption was tested by evaluating and modeling the protein concentration of commercial cocktails at hydrolysis conditions. In the absence of solid substrate, the total protein concentration of a mixture of Celluclast 1.5 L and Novozyme 188 decreased by as much as 45% at 50 °C after 4 days. The individual cocktails as well as a mixture of both were stable at 20 °C. At 50 °C, the protein concentration of Celluclast 1.5 was relatively constant but Novozyme 188 decreased by as much as 77%. It was hypothesized that Novozyme 188 proteins suffer a structural change at 50 °C which leads to protein aggregation and precipitation. Lyophilized ß-glucosidase (P-ß-glucosidase) at 50 °C exhibited an aggregation rate which was successfully modeled using first order kinetics (R (2) = 0.97). By incorporating the possible presence of chaperone proteins in Novozyme 188, the protein aggregation observed for this cocktail was successfully modeled (R (2) = 0.96). To accurately model the increasing protein stability observed at high cocktail loadings, the model was modified to include the presence of additives in the cocktail (R (2) = 0.98). By combining the measurement of total protein concentration with the proposed Novozyme 188 protein aggregation model, the endo- and exoglucanases concentration in the solid and liquid phases during hydrolysis can be more accurately determined. This methodology can be applied to various systems leading to optimization of enzyme loading by minimizing the excess of endo- and exoglucanases. In addition, the monitoring of endo- and exoglucanases concentrations can be used to build mass balances of enzyme recycling processes and to techno-economically evaluate the viability of enzyme recycling.

Methods for mitigation of bio-oil extract toxicity.

Levoglucosan (1,6-anhydro-beta-d-glucopyranose) and other anhydrosugars can be produced in significant quantities during fast pyrolysis of lignocellulosic material. Levoglucosan can be extracted and hydrolyzed to produce fermentable glucose, however co-extraction of fermentation inhibitors can reduce ethanol yields. This work was aimed at evaluating various methods for mitigating the toxicity of bio-oil aqueous extract. Among the detoxification techniques tested, it was found that overliming and solvent extraction were able to improve the fermentability of bio-oil hydrolyzates. Overliming was able to increase the yield of ethanol from bio-oil hydrolyzate by 0.19+/-0.01 (g ethanol/g glucose) at 50% volume hydrolyzate and 0.45+/-0.05 (g ethanol/g glucose) at 40% volume hydrolyzate. A number of extractants were examined and the best solvent was tri-n-octylamine with co-solvent 1-octanol. It was able to selectively (100% glucose retention) remove at least 90+/-6.8% of acetic acid, which was the targeted inhibitor in bio-oil hydrolyzate. This increased the ethanol yield by 0.24 (g ethanol/g glucose) at 40% volume of hydrolyzate. In addition, a technique called adaptive evolution of yeasts was applied, which was capable of increasing the ethanol yield by up to 39% when compared with the unadapted parental strains.

Extraction and hydrolysis of levoglucosan from pyrolysis oil.

Fermentable sugar obtained from lignocellulosic material exhibits great potential as a renewable feedstock for the production of bio-ethanol. One potentially viable source of fermentable sugars is pyrolysis oil, commonly called bio-oil. Depending on the type of lignocellulosic material and the operating conditions used for pyrolysis, bio-oil can contain upwards of 10 wt% of 1,6-anhydro-beta-D-glucopyranose (levoglucosan, LG), an anhydrosugar that can be hydrolyzed to glucose. This research investigated the extraction of levoglucosan from pyrolysis oil via phase separation, the acid-hydrolysis of the levoglucosan into glucose, and the subsequent fermentation of this hydrolysate into ethanol. Optimal selection of water-to-oil ratio, temperature and contact time yielded an aqueous phase containing a levoglucosan concentration of up to 87 g/L, a yield of 7.8 wt% of the bio-oil. Hydrolysis conditions of 125 degrees C, 44 min and 0.5 M H(2)SO(4) resulted in a maximum glucose yield of 216% (when based on original levoglucosan), inferring other precursors of glucose were present in the aqueous phase. The aqueous phase contained solutes which inhibited fermentation, however, up to 20% hydrolysate solutions were efficiently fermented (yield=0.46 g EtOH/g glucose; productivity=0.55 g/L h) using high yeast inoculums (1 g/L in flask) and micro-aerophilic conditions.

A kinetic model for production of glucose by hydrolysis of levoglucosan and cellobiosan from pyrolysis oil.

Anhydro sugars, produced during wood pyrolysis, can by hydrolyzed to sugars under acidic conditions. The acid hydrolysis of two common anhydro sugars in wood pyrolysis oils, levoglucosan (1,6-anhydro-beta-D-glucopyranose) and cellobiosan (beta-D-glucopyranosyl-(1-->4)-1,6-anhydro-D-glucopyranose), was investigated. Levoglucosan hydrolysis to glucose follows a first-order reaction, with an activation energy of 114 kJ mol(-1). For cellobiosan hydrolysis, 44% of the cellobiosan is hydrolyzed initially via the beta-(1-->4) glycosidic bond to form levoglucosan and glucose. The remaining cellobiosan is hydrolyzed initially at the 1,6 anhydro bond to form cellobiose. Both reactions are first order with respect to cellobiosan, with an activation energy of 99 kJ mol(-1). The intermediate levoglucosan and cellobiose are hydrolyzed to glucose.

Heterotrophic bacterial activities and treatment performance of surface flow constructed wetlands receiving woodwaste leachate.

Heterotrophic activities were investigated by measuring 3H-leucine incorporation to bacterial protein and 14C-glucose turnover in surface flow constructed wetlands receiving woodwaste leachate. No significant longitudinal variation was found in heterotrophic activities of bacterioplankton. An open wetland, a vegetated wetland, and a fertilized vegetated wetland were used to examine the effects of vegetation and ammonium nitrate amendment. There was not a significant difference in treatment performance among the three wetlands, except for a significant pH increase and more efficient volatile fatty acids removal in the fertilized wetland. The fertilized wetland had the highest leucine incorporation rate and shortest glucose turnover time accompanied by the lowest glucose mineralization percentage, followed by the open wetland, then the vegetated wetland. Planktonic and sedimentary bacteria contributed to the majority of the total heterotrophic activities; epiphytic bacteria played a minor role. Heterotrophic activities were influenced by the availability of nutrient, electron acceptor, and organic substrate.

Xylose fermentation by genetically modified Saccharomyces cerevisiae 259ST in spent sulfite liquor.

Spent sulfite pulping liquor (SSL) is a high-organic content byproduct of acid bisulfite pulp manufacture which is fermented to make industrial ethanol. SSL is typically concentrated to 240 g/l (22% w/w) total solids prior to fermentation, and contains up to 24 g/l xylose and 30 g/l hexose sugars, depending upon the wood species used. The xylose present in SSL is difficult to ferment using natural xylose-fermenting yeast strains due to the presence of inhibitory compounds, such as organic acids. Using sequential batch shake flask experiments, Saccharomyces cerevisiae 259ST, which had been genetically modified to ferment xylose, was compared with the parent strain, 259A, and an SSL adapted strain, T2, for ethanol production during SSL fermentation. With an initial SSL pH of 6, without nutrient addition or SSL pretreatment, the ethanol yield ranged from 0.32 to 0.42 g ethanol/g total sugar for 259ST, compared to 0.15-0.32 g ethanol/g total sugar for non-xylose fermenting strains. For most fermentations, minimal amounts of xylitol (<1 g/l) were produced, and glycerol yields were approximately 0.12 g glycerol/g sugar consumed. By using 259ST for SSL fermentation up to 130% more ethanol can be produced compared to fermentations using non-xylose fermenting yeast.

Effect of oxygen delignification operating parameters on downstream enzymatic hydrolysis of softwood substrates.

Enhanced oxygen delignification of softwood pulp samples (taken upstream and downstream of a commercial oxygen delignification unit) improved the initial rate of enzymatic saccharification and overall yield of monomeric sugars by 62-82% and 76-80%, respectively. Laboratory-scale experiments were used to examine the effect of a broad range of operating parameters (temperature, time, caustic concentration, and oxygen partial pressure) on the effectiveness of oxygen delignification. Using empirical models, kappa number (residual lignin content) was found to effectively predict final conversion to monomeric sugars. Application of oxygen delignification to sulfite mill knots resulted in smaller (20-25%) reduction in lignin content. However, using a combination of oxygen delignification and particle size reduction, up to 80% of the carbohydrate in the reject knots could be converted to fermentable sugars.

Combined biological and ozone treatment of log yard run-off.

Batch biological treatment of log yard run-off reduced biochemical oxygen demand (BOD), chemical oxygen demand (COD) and tannin and lignin (TL) concentration by 99%, 80%, and 90%, respectively. Acute (Microtox) toxicity was decreased over treatment, from an initial EC50 of 1.83% to a value of 50.4% after 48 h of treatment. Kinetics of biodegradation were determined using respirometry and fitted using the Monod and Tessier model. For the Monod model the maximum substrate uptake rate, and Ks values determined were 0.0038 mg BOD/mgVSS min, and 1.4 mg/L, respectively. The efficacy of ozone as a pre- and post- biological treatment stage was also assessed. During ozone pretreatment, TL concentration and acute toxicity were rapidly reduced by 70% and 71%, respectively. Pre-ozonation reduced BOD and COD concentration by < 10%, however a larger fraction of residual COD was non biodegradable after ozonation. Biologically treated effluent was subjected to ozonation to determine whether further improvements in effluent quality could be achieved. A reduction in COD and TL concentration was observed during ozonation, however no further improvement in toxicity was observed. Ozonation increased BOD by 38%, due to conversion of COD to BOD.

Catabolite Inactivation in the Methylotrophic Yeast Pichia pastoris.

Inactivation of the alcohol oxidase enzyme system of Pichia pastoris, during the whole-cell bioconversion of ethanol to acetaldehyde, was due to catabolite inactivation. Electron microscopy showed that methanol-grown cells contained peroxisomes but were devoid of these microbodies after the bioconversion. Acetaldehyde in the presence of O(2) was the effector of catabolite inactivation. The process was initiated by the appearance of free acetaldehyde, and was characterized by an increase in the level of cyclic AMP, that coincided with a rapid 55% drop in alcohol oxidase activity. Further enzyme inactivation, believed to be due to proteolytic degradation, then proceeded at a constant but slower rate and was complete 21 h after acetaldehyde appearance. The rate of catabolite inactivation was dependent on acetaldehyde concentration up to 0.14 mM. It was temperature dependent and occurred within 24 h at 37 degrees C and by 6 days at 15 degrees C but not at 3 degrees C. Alcohol oxidase activity was psychrotolerant, with only a 17% decrease in initial specific activity over a temperature drop from 37 to 3 degrees C. In contrast, protease activity was inhibited at temperatures below 15 degrees C. When the bioconversion was run at 3 degrees C, catabolite inactivation was prevented. In the presence of 3 M Tris hydrochloride buffer, 123 g of acetaldehyde per liter was produced at 3 degrees C, compared with 58 g/liter at 30 degrees C. By using 0.5 M Tris in a cyclic-batch procedure, 140.6 g of acetaldehyde was produced.