L. F. Chen

Cellulose hydrolysis using zinc chloride as a solvent and catalyst

Cellulose gel with < 10% of crystallinity was prepared by treatment of microcrystalline cellulose, Avicel, with zinc chloride solution at a ratio of zinc chloride to cellulose from 1.5 to 18 (w/w). The presence of zinc ions in the cellulose gels enhanced the rate of hydrolysis and glucose yield. The evidence obtained from X-ray diffraction, iodine absorption experiments; and Nuclear Magnetic Resonance spectra analysis suggested the presence of zinc-cellulose complex after Avicel was treated with zinc chloride. Zinc-cellulose complex was more susceptible to hydrolysis than amorphous cellulose. Under the experimental condition, cellulose gels with zinc ions were hyrolyzed to glucose with 95% theoretical yield and a concentration of 14% (w/v) by cellulases within 20 h. The same gel was hydrolyzed by acid to glucose with 91.5% yield and a concentration of 13.4% (w/v).

Acid hydrolysis of cellulose in zinc chloride solution.

The efficient conversion of cellulosic materials to ethanol has been hindered by the low yield of sugars, the high energy consumption in pretreatment processes, and the difficulty of recycling the pretreatment agents. Zinc chloride may provide an alternative for pretreating biomass prior to the hydrolysis of cellulose. The formation of a zinc-cellulose complex during the pretreatment of cellulose improves the yield of glucose in both the enzymatic and acid hydrolysis of cellulose. Low-temperature acid hydrolysis of cellulose in zinc chloride solution is carried out in two stages, a liquefaction stage and a saccharification stage. Because of the formation of zinc-cellulose complex in the first stage, the required amount of acid in the second stage has been decreased significantly. In 67% zinc chloride solution, a 99.5% yield of soluble sugars has been obtained at 70°C and 0.5M acid concentration. The ratio of zinc chloride to cellulose has been reduced from 4.5 to 1.5, and the yield of soluble sugars is kept above 80%. The rate of hydrolysis is affected by the ratio of zinc chloride to cellulose, acid concentration, and temperature.

The effect of cell density on the production of xylitol from D-xylose by yeast

The rate of xylitol production from D-xylose increased with increasing yeast cell density. The optimal temperature for xylitol production is 36‡ C, and the optimal pH range is from 4.0 to 6.0. At high initial yeast cell concentration of 26 mg/mL, 210 g/L of xylitol was produced from 260 g/L of D-xylose after 96 h of incubation with an indicated yield of 81% of the theoretical value.

D-Xylose fermentation to ethanol bySchizosaccharomycespombe cloned with xylose isomerase gene

SummarySchizosaccharomycespombe cloned with the xylose isomerase gene from E. coli is able to grow on YNB and YMP broths containing xylose as the sole carbon source. This yeast can ferment D-xylose to ethanol directly; however, the ethanol production rate and the yield were dependent on the nitrogen source. With the YMP broth as a nitrogen source, the final ethanol concentration can reach 3.7% (w/v), and the ethanol yield was 80% of the theoretical value based on the amount of xylose that was metabolized. The ethanol production is slow, and the xylitol production is still very active; apparently, the limiting step is the isomerization of xylose to xylulose.

Enzymatic hydrolysis of corn starch after extraction of corn oil with ethanol

Simultaneous corn oil extraction and alcohol dehydration, a solvent corn-milling process developed in our laboratory, was tested on a pilot scale to recover corn oil. Over 92% of the corn oil was extracted by 95% ethanol with a liquid-to-solid ratio of 0.75 at 65°C. Following the oil extraction, 95% of zein fraction was also extracted from the defatted ground corn by 65% ethanol at 65°C with a liquid-to-solid ratio of 1.0 to produce extracted ground corn. After corn oil and zein extraction by ethanol, the ability of the amylases to hydrolyze the starch fraction in extracted ground corn was studied. Results showed the starch fraction can be hydrolyzed to glucose by α-amylase and glucoamylase without separatio of fiber and residual protein. The glucose yield in hydrolysis was a function of temperature, enzyme concentration, and solid-to-liquid ratio. With a two-step heating hydrolysis, corn starch was converted to glucose with a 97.2% yield from extracted ground corn. After filtration and washing of the hydrolyzed mass, the final glucose concentration was 24.3% glucose (w/v).

Molecular cloning of theEscherichiacoli gene encoding xylose isomerase

SummaryA 2.4 Kb DNA fragment restricted from a Clarke-Carbon ColEl plasmid, pLC32-9, containing the xylose isomerase gene has been inserted into the PstI site of pDB248, a shuttle plasmid between the bacteriumE.coli and the fission yeast,Schizosaccharomycespombe. This recombinant plasmid, pDB248-XI, can genetically complement xylose isomerase deficientE.coli strains and xylose isomerase gene can be expressed inSchizosaccharomycespombe.

Xylan hydrolysis in zinc chloride solution

Xylan is the major component of hemicellulose, which consists of up to one-third of the lignocellulosic biomass. When the zinc chloride solution was used as a pretreatment agent to facilitate cellulose hydrolysis, hemicellulose was hydrolyzed during the pretreatment stage. In this study, xylan was used as a model to study the hydrolysis of hemicellulose in zinc chloride solution. The degradation of xylose that is released from xylan was reduced by the formation of zinc-xylose complex. The xylose yield was >90% (w/w) at 70°C. The yield and rate of hydrolysis were a function of temperature and the concentration of zinc chloride. The ratio of zinc chloride can be decreased from 9 to 1.3 (w/w). At this ratio, 76% of xylose yield was obtained. When wheat straw was pretreated with a concentrated zinc chloride solution, the hemicellulose hydrolysate contained only xylose and trace amounts of arabinose and oligosaccharides. With this approach, the hemicellulose hydrolysate can be separated from cellulose residue, which would be hydrolyzed subsequently to glucose by acid or enzymes to produce glucose. This production scheme provided a method to produce glucose and xylose in different streams, which can be fermented in separated fermenters.

Patents and literature

Protein engineering and site-directed mutagenesis is becoming immensely important in both fundamental studies and commercial applications involving proteins and enzymes in biocatalysis. Protein engineering has become a powerful tool to help biochemists and molecular enzymologists elucidate structure-function relationships in enzymic active sites, to understand the intricacies of protein folding and denaturation, and to alter the selectivity of enzymatic catalysis. Commercial applications of engineered enzymes are being developed to increase protein stability, widen or narrow substrate specificity, and to develop novel approaches for use of enzymes in organic synthesis, drug design, and clinical applications. In addition to protein engineering, novel expression systems have been designed to prepare large quantities of genetically engineered proteins. Recent US patents and scientific literature on protein engineering, site-directed mutagenesis, and protein expression systems related to protein engineering are surveyed. Patent abstracts are summarized individually and a list of literature references are given.

Ethanol Fermentation in a Tower Fermenter Using Self-Aggregating Saccharomyces uvarum Bioreactor Performance

A self-aggregating strain ofSaccharomyces uvarum (U4) was used as a biocatalyst to carry out continuous ethanol fermentation in a tower fermentor equipped with a cell separator. Cell aggregates (2–3 mm) formed a stable packed bed in the fermentor, and the cell separator retained yeast cells effectively. Corn steep liquor was used as a nitrogen source for the fermentation of corn syrup and black strap molasses. An ethanol productivity of 54 g/L/h was reached using corn syrup at a dilution rate of 0.7/h, and sugar concentration in the feed was 15% (w/v). For molasses fermentation, an ethanol productivity of 22 g/L/h was obtained at a dilution rate of 0.7/h, and sugar concentration in the feed was 12.5% (w/v). Ethanol yields obtained from tower fermentation are higher than those obtained from flask fermentation (96% for corn syrup fermentation and 92% for molasses fermentation). No significant loss in fermentation activity was observed after 3 mo of operation.

Prediction of Bed Height in a Self-Aggregating Yeast Ethanol Tower Fermenter

A packed bed region and a mixed region were observed in an ethanol tower fermenter packed with flocs of self-aggregating yeast. Sizes of yeast flocs were 2–3 mm and 0.2–0.3 mm in diameter in the packed bed region and the mixed region, respectively. Three major factors were found to affect the height of the packed bed region significantly. They were dilution rate, nutrient limitation, and hydrodynamic limitation. An empirical method was proposed using these three factors to predict the height of the packed bed region in the fermenter.