Dilute acid pretreatment of softwoods

Selective thinning of forests in the western United States will generate a large, sustainable quantity of softwood residues that can be an attractive feedstock for fuel ethanol production. The major species available from thinning of forests in northern California and the eastern Rocky Mountains include white fir (Abies concolor), Douglas fir (Pseudotsuga menziesii), and Ponderosa pine (Pinus ponderosa). Douglas fir chips were soaked in 0.4% sulfuric acid solution, then pretreated with steam at 200 – 230°C for 1 – 5 min. After pretreatment, 90 – 95% of the hemicellulose and as much as 20% of the cellulose was solubilized in water, and 90% of the remaining cellulose can be hydrolyzed to glucose by cellulase enzyme. The prehydrolysates, at as high as 10% total solid concentration, can be readily fermented by the unadapted yeastSaccharomyces cerevisiae D₅A.     

Continuous pH monitoring during pretreatment of yellow poplar wood sawdust by pressure cooking in water

Yellow poplar wood sawdust consists of 41% cellulose and 19% hemicellulose. The goal of pressure cooking this material in water is to hydrate the more chemically resistive regions of cellulose in order to enhance enzymatic conversion to glucose. Pretreatment can generate organic acids through acid-catalyzed degradation of monosaccharides formed because of acids released from the biomass material or the inherent acidity of the water at temperatures above 160°C. The resulting acids will further promote the acid-catalyzed degradation of monomers that cause both a reduction in the yield and the formation of fermentation inhibitors such as hydroxymethyl furfural and furfural. A continuous pH-monitoring system was developed to help characterize the trends in pH during pretreatment and to assist in the development of a base (2.0 M KOH) addition profile to help keep the pH within a specified range in order to reduce any catalytic degradation and the formation of any monosac-charide degradation products during pretreatment. The results of this work are discussed.     

Dilute-acid pretreatment of two short-rotation herbaceous crops

Applied Biochemistry and Biotechnology -     

Coordinating California’s efforts to promote waste to alcohol production

Alcohol fuels produced from biomass can improve air quality, enhance energy security, create employment opportunities, and reduce waste disposal problems. Opportunities in California exist to produce alcohols from waste streams from various sectors of the economy. Government agencies have promoted waste-to-alcohol activities, but efforts have been inconsistent and intermittent. Often these efforts have been hindered by contradictory but mandate-driven policies. A prudent approach to coordinate statewide efforts includes the development of an integrated statewide policy to examine barriers that impede private sector business efforts to produce alcohols from biomass. A multi-agency task force to promote research, development, commercialization, and marketing efforts for biomass-produced alcohols is desirable. The views and opinions contained in this document do not necessarily reflect those of the California Energy Commission, its staff, management, or the State of California.     

Kinetics of thermochemical pretreatment of lignocellulosic materials

The results of an experimental study of the acid hydrolysis of hardwood are presented in the form of values for the three parameters, activation energy, power on the acid concentration, and pre-exponen-tial factor, of the first order kinetic constants for each of the following reaction participants: xylan remaining, glucan remaining, xylose formed, and xylose decomposed. These are used as a base for a quantitative theory to predict the temperature, time, and acid concentrations needed for effective pretreatment of the substrate for subsequent enzymatic hydrolysis of the glucan. This theory is based on the assumption that successful pretreatment requires >90% removal of the xylan, <10% removal of the glucan, and >80% xylose yield. This theory is compared with selected published data.     

Xylanase recovery by ethanol and Na2SO4 precipitation

Xylans are the major components of the hemicellulosic fraction of lignocellulosic biomass and their hydrolysis can be obtained using xylanases fromPenicillium janthinellum. In this work, sugarcane bagasse hemicellulosic hydrolysate was used as the substrate for producing xylanase. The precipitation of these enzymes was studied using ethanol and Na₂SO₄ as precipitating agents. Ethanol precipitation experiments were performed batchwise in concentrations ranging from 10 to 80%, pH 4.0 to 7.0, at 4áC. The concentrations used in the precipitations with Na₂SO₄ were from 5 to 60% at pH 5.5 and 25áC. Solubility curves as a function of xylanase activity and total protein for both precipitating agents were made. According to the results, Na₂SO₄ is not appropriate for precipitating xylanases in this medium since at salt concentrations higher than 25%, the enzyme was denaturated and at this concentration less than 80% of the enzyme and total protein were precipitated. Because of differences in xylanase and total protein solubility, a fractionated precipitation using ethanol can be performed, since with 40% ethanol, 49% of the total protein was precipitated and more than 95% of the enzyme was kept in solution. On the other hand approx 100% of the xylanases were recovered by precipitation after adding 80% ethanol.     

Fuel ethanol production from corn fiber current status and technical prospects

Corn fiber, which consists of about 20% starch, 14% cellulose, and 35% hemicellulose, has the potential to serve as a low cost feedstock for production of fuel ethanol. Currently, the use of corn fiber to produce fuel ethanol faces significant technical and economic challenges. Its success depends largely on the development of environmentally friendly pretreatment procedures, highly effective enzyme systems for conversion of pretreated corn fiber to fermentable sugars, and efficient microorganisms to convert multiple sugars to ethanol. Several promising pretreatment and enzymatic processes for conversion of corn fiber cellulose, hemicellulose, and remaining starch to fermentable sugars were evaluated. These hydrolyzates were then examined for ethanol production in bioreactors, using genetically modified bacteria and yeast. Several novel enzymes were also developed for use in pretreated corn fiber saccharification. Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Effect of pretreatment of molasses and posttreatment of fermented broth in industrial production of ethanol

The aim of preclarification is to minimize sludge going to yeast separators. This purpose is partially fulfilled. However, it has been measured during the plant trial runs that preclarification does not noticeably improve fermentation. The aim of postclarification is to minimize sludge going to distillation. This purpose is well served as noted from the fact that cycle run of distillation columns using postclarification is three times longer (9–12 mo) as compared to the normal one (3–4 mo).     

The role of ester groups in resistance of plant cell wall polysaccharides to enzymatic hydrolysis

Xylan backbones in native plant cell walls are extensively acety-lated. Previously, no direct investigations as to their role in cellulolytic enzyme resistance have been done, though indirect results point to their importance. An in vitro deesterification of aspen wood and wheat straw has been completed using hydroxylamine solutions. Yields of 90% acetyl ester removal for both materials have been accomplished, with little disruption of other fractions (i.e., lignin). Apparently, as the xylan becomes increasingly deacetylated, it becomes 5–7 times more digestible. This renders the cellulose fraction more accessible, and 2–3 times more digestible. This effect levels off near an acetyl removal of 75%, where other resistances become limiting.     

Effect of surfactants and zeolites on simultaneous saccharification and fermentation of steam-exploded poplar biomass to ethanol

In this work, the effect of the addition of different concentrations of Tween-80 and three different zeolite-like products on enzymatic hydrolysis, ethanol fermentation, and simultaneous saccharification and fermentation (SSF) process has been investigated. The ability of these products to enhance the effectiveness of the SSF process to ethanol of steam-exploded poplar biomass using the thermotolerant strainKluyveromyces marxianus EMS-26 has been tested. Tween-80 (0.4 g/L) increased enzymatic hydrolysis yield by 20% when compared to results obtained in hydrolysis in absence of the additive. Zeolite-like products (ZESEP-56 and ZECER-56) (2.5 g/L) improved rates of conversion and ethanol yields in the fermentation of liquid fraction recovered from steam-exploded poplar. The periods required for the completion of fermentation were approx 10 h in the presence of zeolite-like products and 24 h in the absence of additives. The probable mode of action is through lowered levels of inhibitory substances because of adsorption by the additive.     

Simultaneous saccharification and fermentation of lignocellulose

Simultaneous saccharification and fermentation (SSF) processes for producing ethanol from lignocellulose are capable of improved hydrolysis rates, yields, and product concentrations compared to separate hydrolysis and fermentation (SHF) systems, because the continuous removal of the sugars by the yeasts reduces the end-product inhibition of the enzyme complex. Recent experiments using Genencor 150L cellulase and mixed yeast cultures have produced yields and concentrations of ethanol from cellulose of 80% and 4.5%, respectively. The mixed culture was employed because B.clausenii has the ability to ferment cellobiose (further reducing end-product inhibition), while the brewing yeastS. cerevisiae provides a robust ability to ferment the monomeric sugars. These experimental results are combined with a process model to evaluate the economics of the process and to investigate the effect of alternative processes, conditions, and organisms.     

Shrinking-bed model for percolation process applied to dilute-acid pretreatment/hydrolysis of cellulosic biomass

For many lignocellulosic substrates, hemicellulose is biphasic upon dilute-acid hydrolysis, which led to a modified percolation process employing simulated two-stage reverse-flow. This process has been proven to attain substantially higher sugar yields and concentrations over the conventional single-stage percolation process. The dilute-acid pretreatment of biomass solubilizes the hemicellulose fraction in the solid biomass, leaving less solid biomass in the reactor and reducing the bed. Therefore, a bed-shrinking mathematic kinetic model was developed to describe the two-stage reverse-flow reactor operated for hydrolyzing biphasic substrates, including hemicellulose, in corn cob/stover mixture (CCSM). The simulation indicates that the shrinking-bed operation increases the sugar yield by about 5%, compared to the nonshrinking bed operation in which 1 reactor volume of liquid passes through the reactor (i.e.,t = 1.0). A simulated optimal run further reveals that the fast portion of hemicellulose is almost completely hydrolyzed in the first stage, and the slow portion of hemicellulose is hydrolyzed in the second stage. Under optimal conditions, the bed shrank 27% (a near-maximum value), and a sugar yield over 95% was attained.     

Kinetics of enzymatic hydrolysis of lignocellulosic materials based on surface area of cellulose accessible to enzyme and enzyme adsorption on lignin and cellulose

Comparison of the model with experimental data is currently in progress. It appears that more detailed studies of the adsorption dynamics, not just adsorption equilibrium, are needed.     

Purification and partial characterization of two lectin isoforms fromCratylia mollis mart. (camaratu bean)

Two additional electrophoretically distinct molecular forms, isoforms (iso) 2 and 3, with lectin properties were isolated fromCratylia mollis Mart, seeds (FABACEAE), by extraction with 0.15M NaCl and ammonium sulfate fractionation, followed by chromatography on Sephadex G-75 and Bio-Gel P-200 (iso 2), as well as CM-Cellulose and Sephadex G-75 (iso 3). Both isoforms were human group nonspecific and showed distinct specificity. Polyacrylamide gel electrophoresis resolved iso 2 and 3 in polypeptides of apparent mol wts 60 and 31 kDa, respectively; a distinct isoelectric focusing pattern was obtained for iso 2 and 3, under denaturing and reducing conditions.     

Comparison between experimental and theoretical values of effectiveness factor in cephalosporin C production process with immobilized cells

Cells ofCephalosporium acretnonium ATCC 48272 immobilized in calcium alginate beads were utilized for cephalosporin C production and the results were compared with those obtained with free cells. The experiments were performed with synthetic medium containing glucose and sucrose as carbon and energy sources. Experimental effectiveness factor values were obtained at various cell and dissolved-oxygen concentrations, considering Monod kinetics for the respiration rate, and were compared with the values calculated with zero-order kinetics in spherical bioparticle. The results showed that the assumption of oxygen limitation by diffusion in the bioparticle was correct, and that cephalosporin C production with immobilized cells is perfectly viable, although a slightly lower rate than that obtained in the free cell process was observed.     

Yeast adaptation on softwood prehydrolysate

Several strains and genera of yeast, includingSaccharomyces cerevisiae D₅A,Pachysolen tannophilus, S. cerevisiae K-l,Brettanomyces custersii, Candida shehatae, andCandida acidothermophilum, are screened for growth on dilute acid-pretreated softwood prehydrolysate. Selected softwood species found in forest underbrush of the western United States, which contain predominantly hexosan hemicellulose, were studied. This phase of the work emphasized debarked Douglas fir. The two best initial isolates were gradually selected for improved growth by adaptation to increasing prehydrolysate concentrations in batch culture, with due consideration of nutrient requirements. Microaerophilic conditions were evaluated to encourage tolerance of pretreatment hydrolysate, as well as ethanol product. Adaptation and simultaneous saccharification and fermentation (SSF) results are used to illustrate improved performance with an adapted strain, compared to the wild type.     

Xylose fermentation

The economic impact of conversion of xylose to ethanol for a wood-to-ethanol plant was examined, and the maximum potential reduction in the price of ethanol from utilization of xylose is estimated to be 0.42 per gallon from a base case price of0.42 per gallon from a base case price of 1.65. The sensitivity of the price of ethanol to the yield, ethanol concentration and rate of the xylose fermentation was also examined, and the price of ethanol is most affected by changes in yield and ethanol concentration, with rate of lesser importance. Current performances of various xylose conversion biocatalysts were analyzed, andC. shehatae andP. stipitis appear to be the best yeasts.     

Mode of action and synergism of cellulases fromPenicillium funiculosum

A 1,4-β-d-glucan cellobiohydrolase (EC 3.2.1.91) and l,4-β-d-glucan glucanohydrolase (EC 3.2.1.4) were purified from the culture filtrates ofPenicillium funiculosum by using preparative isoelectric focusing. Both the enzymes were homogeneous on polyacrylamide gel with and without sodium dodecyl sulphate. The mol wt of the cellobiohydrolase and endoglucanase were 14,400 and 25,000 respectively. The purified enzymes were free of β-glucosidase activity. Acting in isolation, the cellobiohydrolase had little capacity for solubilizing Avicel or Walseth cellulose, but showed increased rates of hydrolysis when combined with endoglucanase. Cellobiose inhibition (50%) was observed in the initial rate of the hydrolysis of Walseth cellulose. It was also observed that cellobiohydrolase initiates the attack on crystalline cellulose. † NCL communication no. 3898.     

Synthesis of Cyclodextrin Glucosyl Transferase byBacillus cereus for the production of cyclodextrins

A potent indigenous bacillus isolate identified asBacillus cereus (RJ-30) was found to produce Cyclodextrin Glucosyl Transferase (CGTase) extracellularly. Process optimization of various fermentation parameters has been established for optimal growth of bacillus and the maximum enzyme synthesis. The organism had the highest specific growth rate (0.7μ) with a generation time of 1 h in glucose containing medium at the conditions of pH 7.0, 37°C at 300 rpm, 1.5 vvm of agitation, and aeration. At these conditions, it exhibited the maximum activity of 54 U/mL at the synthesis rate of 2.7 U/L/h. CGTase was produced from the early exponential growth and peaked during the midsporulating stage of about 16 h thereafter maintained at the same level of 50 U/mL. Saccharides containing media were better inducers than starch, and the influence of carbohydrate substrates has shown that enzyme synthesis is promoted by xylose (65 U/mL) and, more remarkably, by the supplementation of wheat bran extract in glucose medium (106 U/mL). This organism produced CGTase stably in a chemostat culturing over a period of 400 h with a maximum productivity of 5.4 kU/L/h (threefold higher than obtained in batch culturing [1.75 kU/L/h]). Comparatively, CGTase was produced by immobilized cells in a continuous fluidized bed reactor for over approx 360 h, at a relatively high dilution rate of 0.88 h⁻¹ resulting in the productivity of 23.0 kU/L/h.