Robert P. Chambers

Ethanol fermentation of red oak acid prehydrolysate by the yeast Pichia stipitis CBS 5776

Abstract Xylose, the dominant sugar in red oak acid prehydrolysate, was fermented to ethanol. In batch cultures of xylose concentrations ranging from 10 to 100 g l −1 , Pichia stipitis CBS 5776 yielded 0.50–0.40 g ethanol per g xylose consumed. Model compounds of inhibitors generated in the acid prehydrolysis of red oak hemicelluloses, lignin and extractives, hindered the fermentation. Recycled yeasts and treatments with molecular sieve or mixed bed ionresins facilitated the ethanol fermentation of red oak acid prehydrolysate. A maximal ethanol concentration of 9.9 g l −1 was obtained from an acid prehydrolysate containing 21.7 g l −1 of xylose. Fermentation inhibitors derived from red oak lignin and extractives were identified.

Red oak wood derived inhibitors in the ethanol fermentation of xylose byPichia stipitis CBS 5776

SummaryXylose, the predominant sugar in red oak prehydrolysate, is fermented to ethanol byPichia stipitis CBS 5776. Toxic model compounds derived from red oak hemicelluloses, lignin, and extractives inhibited the fermentation. Treatment of the prehydrolysate with molecular sieve and mixed bed ion resins facilitated the ethanol fermentation giving about 10 g/l ethanol from 32 g/l initial xylose. Fermentation inhibitors derived from red oak lignin and extractives were identified.

Characterization of a Ca-alginate-immobilized Trametes versicolor bioreactor for decolorization and AOX reduction of paper mill effluents

Abstract The reduction of color and adsorbable organic halides (AOX) from the kraft bleach plant effluents was investigated using a laboratory-scale fungal bioreactor. The fungal bioreactor, containing calcium-alginate-entrapped white-rot fungus Trametes versicolor , was developed and evaluated for continuous color and AOX reduction. A color reduction percentage of 69% (±6%) and AOX reduction percentage of 58% (±6%) were achieved using caustic stage effluent as bioreactor feed at a residence time of 1 day. Retention time, inlet liquor concentration and fungal bead size were investigated for their effect on biodegradation. Percentage reductions of color and AOX ranging from 61%(±5%) to 72%(±2%) and 54%(±4%) to 49%(±2%), respectively, were observed using combined (caustic and acidic stages) effluent as bioreactor feed. Scanning electron microscopy studies showed that the immobilized mycelia was localized to a limited thickness near the outer surface of the polymer bead.

Lignin and extractives derived inhibitors in the 2,3-butanediol fermentation of mannose-rich prehydrolysates

SummaryMannose, the predominant sugar in southern pine water prehydrolysates, has been fermented to 2,3-butanediol by Klebsiella pneumoniae AU-1-d3. Lignin derivatives and extractives soluble in the water prehydrolysates, however, hindered the butanediol fermentation. Treatments with sequential lime-sulfuric acid or mixed bed ion resins facilitated the butanediol fermentation of the water prehydrolysates. Fermentation inhibitors derived from southern pine lignin and extractives were identified.

Delignification of an unbleached hardwood kraft pulp by Phanerochaete chrysosporium

SummaryCulture conditions affecting lignin degradation of an unbleached hardwood kraft pulp by Phanerochaete chrysosporium have been examined. Optimum pH and temperature for lignin degradation (about 33%) were 3.5 and 38°C, respectively. Optimum fungal growth was at a pH of 4.5 and a temperature of around 32°C. Addition of exogeneous glucose to the cultures lessened the degradation of pulp carbohydrates. Lignin degradation was stimulated by oxygen atmosphere and non-agitated cultures. Increased surface to volume ratio (decreased culture depth) enhanced lignin degradation (about 56% at a depth of 1.2 cm). Finally, the correlations: pulp yield vs. residual glucose, ligninase activity vs. mycelium, and extent of delignification vs. residual extracellular H2O2 were discussed in light of recent findings of ligninases responsible for ligninolysis.

Continuous decolorization and AOX reduction of bleach plant effluents by free and immobilized Trametes versicolor

Abstract Continuous decolorization of bleach plant effluents was studied using white‐rot fungus, Trametes (Coriolus) versicolor. The experiments were carried out using free and immobilized fungus. In the free cell system, 56–77% continuous decolorization and 41–69% AOX reduction were achieved with a retention time of 24 hours. Treatment of bleach plant effluents with the same fungus encapsulated in calcium alginate polymer beads resulted in 76–83% decolorization and 43–59% AOX reduction. These are the highest levels achieved in a continuous bioreactor system using Trametes (Coriolus) versicolor.

Hollow Fiber-Entrapped Liver Microsomes: A Potential Extracorporeal Drug Detoxifier

We are attempting to develop an extracorporeal hollow fiber drug detoxifier (EDD) based on the NADPH-dependent, multienzyme, drug hydroxylation complex present in liver endoplasmic reticulum and isolatable in particulate form as microsomes (MS). In addition to its potential use as an EDD this system serves as a model for the use of membrane-bound, cofactor-requiring, multienzyme complexes in industrial as well as medical applications. This work also may contribute toward the development of liver assist devices as well as provide for production of drug metabolites for subsequent therapeutic screening.

Membrane-Immobilized Liver Microsome Drug Detoxifier

The common occurrence of poisoning due to drug overdose has led to an increasing need for a safe, simple, effective, and inexpensive means of removing such drugs from the blood in the emergency treatment of drug-overdose victims. Various devices and techniques based on extracorporeal blood processing have been investigated. Physical techniques include hemodialysis (Kennedy et al., 1969), charcoal adsorption (Widdop et al.,1975; Chang et al., 1973), and ion-exchange adsorption (Rosenbaum et al., 1971; Medd et al., 1974). Biological techniques include liver perfusion (McDermott and Norman, 1971), liver slice perfusion (Koshino et al., 1975), perfusion over hepatic cell suspensions in direct contact with the blood (Eisman and Soyer, 1971; Soyer et al., 1973), and perfusion through hollow fibers containing cultured hepatic cells on the outer surface of the fibers (Wolf and Munkelt, 1975). It is well accepted that the biological system responsible for detoxification of a variety of drugs is located in the smooth endoplasmic reticulum of hepatic cells which can be isolated as microsomes by cell fractionation. The microsomes contain a complex mixture of enzymes, including cytochrome P-450, which requires the coenzyme NADPH and molecular oxygen for drug detoxification. In general these detoxification enzymes modify toxins by increasing their polarity and consequently their aqueous solubility, thus decreasing their permeation into tissues (Mandel, 1971) and promoting excretion. The chemical modifications catalyzed by the microsomal drug detoxification enzymes include, among others, hydroxylation, demethylation, and conjugation reactions. Several of the microsomal detoxification enzymes have been purified (Lu and Levin, 1974; Brunner, 1975; Ziegler and Mitchell, 1972) and at least two have been immobilized by covalent bonding to insoluble particles (Brunner, 1975; Parikh et al., 1976; Sofer et al.,1975). One approach for utilization of these enzymes in an extracorporeal drug detoxification system is to purify the individual enzymes and then reconstitute the multienzyme complex by binding to insoluble particles. An alternative approach, the one we have chosen, is to utilize the isolated microsomes themselves. This method has the advantage of simplicity and lower cost. More importantly, in contrast to the former approach, it assures that all the microsomal enzymes are present in proportions and molecular arrangement closely resembling the in vivo state. We have used an extracorporeal hollow fiber-based enzyme reactor in an attempt to develop a therapeutic technique suitable for treatment of drug and poison detoxification. Since all the protein components of the system are impermeable to the hollow fiber membranes, adverse immunological reactions will be avoided. In addition, the safety of blood processing using hollow fiber devices has been well established in recent years by extensive use in hemodialysis.

Rapid Purification of Aldose Reductase from Candida utilis

Aldose reductase (Alditol: NADP oxidoreductase EC 1.1.1.21) is an enzyme in the polyol pathway that reduces glucose to sorbitol. This enzyme, first characterized by Hers,’ has been obtained from mammalian and microbial s o ~ r c e s . ~ ~ In the present paper we report a rapid purification of the enzyme by the use of adsorption (in the presence of high concentrations of ammonium sulfate),”-’* dye-ligand,”-l4 and ion-exchange chromatography. Freeze-dried Candida u t i h (16.5 g) was autolyzed overnight a t room temperature in 50 ml 1 M sodium acetate, pH 4.6, containing 0.5 mM 2-mercaptoethanol, and centrifuged a t 27,000 g for 15 min. The pellet was reextracted by another 50 ml of acetate buffer and recentrifuged. The combined supernatant was adjusted to pH 6.2 with 1 M sodium bicarbonate and diluted to 0.53 M acetate. The extract was brought to 45% saturation with ammonium sulfate and centrifuged. The supernatant was brought to 60% saturation and applied to a Fractogel TSK HW-65F (EM Science, Gibbstown, NJ) column (3 cm x 6.5 cm) previously equilibrated with 0.53 M sodium acetate, pH 6.2, containing 5 mM 2-mercaptoethanol and 60% saturated with ammonium sulfate. The column was washed with the same acetate buffer (60% saturated ammonium sulfate) until the absorbance of the eluate was less than 0.1. Aldose reductase was eluted by a decreasing (60 to 0% saturation) gradient in the acetate buffer. Enzyme-containing fractions were pooled and applied to a column ( I .4 cm x 16 cm) of Matrex Gel Red A (Amicon Corp., Danvers, MA) in 0.01 M potassium phosphate, pH 7.2, containing 5 mM 2-mercaptoethanol. The column was washed with the equilibration buffer until the 280 nm absorbance of the eluate was less than 0.05. Aldose reductase was recovered by elution with a 0 to 0.4 mM NADPH gradient in this pH 7.2 buffer. The combined fractions with aldose reductase activity were applied directly to a Fractogel TSK DEAE-650s (1.2 cm x 28 cm) column in 0.01 M potassium phosphate, pH 7.2, containing 5 mM 2-mercaptoethanol. Four ml fractions of eluate were collected at a flow rate of about 0.7 ml per min. As shown in FIGURE 1, contaminating proteins were removed by washing the column with the phosphate buffer, and then by a 0.082 M KC1 in buffer. The enzyme was recovered by a 0.082 M to 0.105 M KCI gradient elution in the pH 7.2 buffer. The results of this purification from Candida utilis are shown in TABLE 1. Depending on the scale of purification, the enzyme can be obtained in 2 to 3 days. Aldose reductase is purified 85-fold with a 15% yield. The homogeneity of the purified enzyme was indicated by the presence of a single protein band in disc gel electrophore-