Bruce E. Dale

The ammonia freeze explosion (AFEX) process - A practical lignocellulose pretreatment

The Ammonia Freeze Explosion (AFEX) process treats lignocellulose with high-pressure liquid ammonia, and then explosively releases the pressure. The combined chemical effect (cellulose decrystallization) and physical effect (increased accessible surface area) dramatically increase lignocellulose susceptibility to enzymatic attack. There are many adjustable parameters in the AFEX process: ammonia loading, water loading, temperature, time, blowdown pressure, and number of treatments. The effect of these parameters on enzymatic susceptibility was explored for three materials: Coastal bermudagrass, bagasse, and newspaper. Nearly quantitative sugar yields were demonstrated for Coastal bermudagrass and bagasse, using a very low enzyme loading (5 IU/g). Newspaper proved to be much more resistant to enzymatic hydrolysis.

Pretreatment of lignocellulosic municipal solid waste by ammonia fiber explosion (AFEX)

The Ammonia Fiber Explosion (AFEX) process treats lignocellulose with high-pressure liquid ammonia and then explosively releases the pressure. The combined chemical effect (cellulose decrystallization) and physical effect (increased accessible surface area) dramatically increase lignocellulose susceptibility to enzymatic attack. For example, bagasse digestibility is increased 5.5 times and that of kenaf core is increased 11 times using extracellular cellulases fromTrichoderma reesei. In this study, we applied the AFEX process to mixed municipal solid waste (MSW) and individual components (e.g., softwood newspaper, kenaf newspaper, copy paper, paper towels, cereal boxes, paper bags, corrugated boxes, magazines, and waxed paper). Softwood newspaper proved to be the most difficult component to digest because of its high lignin content. A combination of oxidative lignin cleavage and AFEX was required to increase softwood newspaper digestibility substantially, whereas AFEX alone was able to make kenaf newspaper digestible. Because most MSW components have been substantially delignified in the paper-making process, AFEX only marginally increased their digestibility.

Enzymatic Hydrolysis of High-Moisture Corn Fiber Pretreated by AFEX and Recovery and Recycling of the Enzyme Complex

Corn fiber is a grain-processing residue containing significant amounts of cellulose, hemicellulose, and starch, which is collected in facilities where fuel ethanol is currently manufactured. Preliminary research has shown that corn fiber (30% moisture dry weight basis [dwb]) responds well to ammonia-fiber explosion (AFEX) pretreatment. However, an important AFEX pretreatment variable that has not been adequately explored for corn fiber is sample moisture. In the present investigation, we determined the best AFEX operating conditions for pretreatment of corn fiber at high moisture content (150% moisture dwb). The optimized AFEX treatment conditions are defined in terms of the moisture content, particle size, ammonia to biomass ratio, temperature, and residence time using the response of the pretreated biomass to enzymatic hydrolysis as an indicator. Approximate optimal-pretreatment conditions for unground corn fiber containing 150% (dwb) moisture were found to be: temperature, 90‡C; ammonia: dry corn fiber mass ratio, 1:1; and residence time 30 min (average reactor pressure under these conditions was 200 pounds per square inch [psig]). Enzymatic hydrolysis of the treated corn fiber was performed with three different enzyme combinations. More than 80% of the theoretical sugar yield was obtained during enzymatic hydrolysis using the best enzyme combination after pretreatment of corn fiber under the optimized conditions previously described. A simple process for enzyme recovery and reuse to hydrolyze multiple portions of AFEX-treated corn fiber by one portion of enzyme preparation is demonstrated. Using this process, five batches of fresh substrate (at a concentration of 5% w/v) were successfully hydrolyzed by repeated recovery and reuse of one portion of enzyme preparation, with the addition of a small portion of fresh enzyme in each subsequent recycling step.

Ethanol production from enzymatic hydrolysates of AFEX-treated coastal bermudagrass and switchgrass

Switchgrass and coastal bermudagrass were pretreated by ammonia fiber explosion (AFEX), and the treated materials hydrolyzed using 5 IU cellulase/g substrate. Resulting sugar solutions (2–3%, w/v) were fermented with recombinantKlebsiella oxytoca. Glucose was rapidly and completely fermented to ethanol, whereas xylose fermentation was slower and less complete. At higher sugar concentrations (≈8%) glucose fermentation continued, but xylose fermentation almost ceased. Protein extraction somewhat enhanced ethanol production from coastal bermudagrass. Improved fermentation technologies and media appear necessary for practical mixed-sugar lignocellulosic hydrolyzates.

Ethanol production from AFEX pretreated corn fiber by recombinant bacteria

SummaryFermentation of an enzymatic hydrolyzate of ammonia fiber explosion (AFEX) pretreated corn fiber (containing a mixture of different sugars including glucose, xylose, arabinose, and galactose) by genetically-engineered Escherichia coli strain SL40 and KO11 and Klebsiella oxytoca strain P2 was investigated under pH-controlled conditions. Both E. coli strains (SL40 and KO11) efficiently utilized most of the sugars contained in the hydrolyzate and produced a maximum of 26.6 and 27.1 g/l ethanol, respectively, equivalent to 90 and 92% of the theoretical yield. Very little difference was observed in cell growth and ethanol production between fermentations of the enzymatic hydrolyzate and mixtures of pure sugars, simulating the hydrolyzate. These results confirm the fermentability of the AFEX-treated corn fiber hydrolyzate by ethanologenic E. coli. K.oxytoca strain P2, on the other hand, showed comparatively poor growth and ethanol production (maximum 20 g/l) from both enzymatic hydrolyzate and simulated sugar mixtures under the same fermentation conditions.

Integrated production of ethanol fuel and protein from coastal bermudagrass

The herbaceous crops that may provide fermentable carbohydrates for production of fuels and chemicals also contain 10–20% protein. Protein coproduction with biomass-derived fuels and chemicals has important advantages: (1) food and fuel production can be integrated, and (2) protein is a high-value product that may significantly improve overall process economics. We report the results of an integrated approach to producing protein and fermentable sugars from one herbaceous species, Coastal Bermudagrass (CBG). The ammonia fiber explosion (AFEX) process makes possible over 90% conversion of cellulose and hemicellulose to simple sugars (about 650 mg reducing sugars/g dry CBG) at 5 IU cellulase/g vs < 20% conversion for untreated CBG. The AFEX treatment also improves protein extraction from CBG; over 80% protein recovery is possible from AFEX-treated CBG vs about 30% recovery from untreated CBG.

Thermodynamic analysis of trinitrotoluene biodegradation and mineralization pathways.

Biodegradation of 2,4,6‐trinitrotoluene (TNT) proceeds through several different metabolic pathways. However, the reaction steps which are considered rate‐controlling have not been fully determined. Glycolysis and other biological pathways contain biochemical reactions which are acutely rate‐limiting due to enzyme control. These rate‐limiting steps also have large negative Gibbs free energy changes. Because xenobiotic compounds such as TNT can be used by biological systems as nitrogen, carbon, and energy sources, it is likely that their degradation pathways also contain acutely rate‐limiting steps. Identification of these rate‐controlling reactions will enhance and better direct genetic engineering techniques to increase specific enzyme levels.

Volatile fatty acid fermentation of AFEX-treated bagasse and newspaper by rumen microorganisms

Abstract The anaerobic digestion of bagasse and newspaper by rumen microorganisms was studied. A one-half replicate of a 25 factorial design was used to evaluate the effect of type of substrate (bagasse and newspaper), AFEX (Ammonia Fiber Explosion) pretreatment, liquid residence time (LRT), solid/liquid residence time ratio (SRT/LRT), and loading rate (LR) on volatile fatty acid (VFA) yield, productivity, and acid composition. A fermentor for the continuous culture of rumen microorganisms, which allows solids to be retained longer than liquid (SRT/LRT> 1), was used. Yields averaged 8.8 mmoles and 5.8 mmoles VFA per g volatile solids (VS) in bagasse and newspaper fermentations, respectively. The propionic acid molar percent was 52% higher in bagasse fermentation (19%) than in newspaper fermentation (12.5%). AFEX pretreatment increased VFA yield by 21% and decreased propionic acid molar percent by 9.1 %. The best reactor performance (i.e., high VFA yield and high VFA productivity) was achieved with AFEX-treated material at a LR of 14.1 g substrate per 1 day, LRT of 11 h, and SRT of 44 h. At these experimental conditions, productivity and yield were 163 mmoles VFA per 1 per day and 0.64 g VFA/g VS in bagasse fermentation, and 81 mmoles per 1 per day and 0.31 g VFA/g VS in newspaper fermentation. The rumen fermentation product yields are significantly higher than the sugar yields from extracellular cellulase/hemicellulase. A promising application of the rumen fermentation is to produce mixed VFA calcium salts which may be used to remove sulfur from coal-fired boilers. It is estimated that these salts may be produced from municipal solid waste for

Genotoxicity profiles and reaction characteristics of potassium polyethylene glycol dehalogenation of wood preserving waste.

119–137/tonne.

Alteration of glucose consumption kinetics with progression of baculovirus infection in Spodoptera frugiperda cells.

Chemical dehalogenation of pentachlorophenol-containing wood preserving waste (EPA K001) with potassium polyethylene glycol (KPEG) was investigated. The effect of treatment on the waste toxicity was examined by monitoring genotoxicity of the organic wastes. The effects of temperature, reaction time, and pentachlorophenol concentration on dehalogenation were also investigated. Samples were collected throughout treatment and sequentially extracted with dichloromethane and methanol. Chemical analysis indicated that KPEG effectively dehalogenated pentachlorophenol in the waste. Mutagenicity and genotoxicity of the organic extracts were evaluated in the Salmonella/microsome and Escherichia coli prophage induction assays. Extracts exhibited significantly higher genotoxic responses when tested with metabolic activation. KPEG dehalogenation generally decreases waste genotoxicity during treatment; however, additional treatment is required for complete detoxification.