Sheldon J. B. Duff

Effect of media composition and growth conditions on production of cellulase and β-glucosidase by a mixed fungal fermentation

The effect of growth temperature, medium composition and pH were examined in shake-flask-scale studies to determine the optimum conditions for cellulase production in mixed cultures of Trichoderma reesei Rut C30 and Aspergillus phoenicis. The optimum temperature and pH were 27°C and pH 4.6. Decreases in medium complexity and cost were achieved through reductions in the concentration of Ca, Mg, and K salts. Ten litre fermentations were implemented to study the kinetics of substrate utilization and product formation. The pH at which the fermentation was controlled appeared to be the critical parameter for batch growth of mixed cultures of Trichoderma reesei Rut C30 and Aspergillus phoenicis.

Cellulase and beta-glucosidase production by mexied culture of trichoderma reesei rut C30 and aspergillus phoenicis

SummaryWhen lignocellulosic materials are hydrolysed using cellulase fromTrichodermareesei, cellobiose accumulates due to a deficiency in β-glucosidase activity of the enzyme complex. Cellobiose decreases the rate and extent of hydrolysis through feedback inhibition of exo β-1,4-glucanase. Cellulase produced through mixed culture ofTrichoderma andAspergillus showed increased β-glucosidase activity and greatly improved hydrolytic potential.

Induction and stability of alcohol oxidase in the methylotrophic yeast Pichia pastoris

SummaryAlcohol oxidase biosynthesis was induced when Pichia pastoris was grown in a medium containing methanol as the sole carbon and energy source. Specific activity was highest during the logarithmic phase of growth (1.22 g acetaldehyde produced/g cell dry wt. per hour), and declined steadily thereafter. The addition of 0.1% (w/v) yeast extract to the methanol growth medium promoted higher biomass production, increased alcohol oxidase specific activity, and contributed to increased enzyme stability under use conditions. When P. pastoris was used for wholecell bioconversions, 30.2 g of ethanol were oxidized to 28 g acetaldehyde in 12 h, at a carbon recovery of 97%. Acetaldehyde concentrations in excess of 1 M were achieved when the concentration of the TRIS buffer, used to chemically trap the acetaldehyde, was increased to 1 M.

Bio-oxidation of aliphatic and aromatic high molecular weight alcohols by Pichia pastoris alcohol oxidase

SummaryThe alcohol-oxidase-mediated oxidation of hexanol to hexanal was conducted by whole cells of Pichia pastoris in a biphasic reaction medium consisting of 3% water and 97% (v/v) water-saturated hexane. At substrate levels of ca. 10 g/l, hexanal was produced at a rate of 0.2 g/g cell dry wt. per hour with product yields and carbon recoveries of 96% or greater. Although the substrate range of P. pastoris alcohol oxidase has been documented as C1–C5 aliphatic alcohols and benzyl alcohol, the use of a biphasic organic reaction medium showed that this enzyme can also oxidize higher molecular weight aliphatic alcohols of C6–C11, as well as the aromatic alcohols phenethyl alcohol and 3-phenyl-1-propanol. The ability of alcohol oxidase to oxidize low-water-soluble alcohols greatly extends the utility of this enzyme.

Evaluation of the hydrolytic potential of a crude cellulase from mixed cultivation of Trichoderma reesei and Aspergillus phoenicis

Mixed cultivation of Trichoderma reesei Rut C30 and Aspergillus phoenicis produced a cellulase complex which showed enhanced activity against cellulose. Initial rates of glucose production were improved up to twofold over cellulase from Trichoderma alone. For the mixed culture cellulase, the pH optimum was found to be slightly different when measured on the basis of glucose production rate (4.5–4.75), than if measured on the basis of production of total reducing sugars (4.0–4.25). The initial rate of glucose and reducing sugar production increased with temperature over the range tested (40–60°C). Stability of individual enzyme component activities were tested at 50 and 60°C. β-Glucosidase (cellobiase or β-d-glucoside glucohydrolase, EC 3.2.1.21) activity was most labile at elevated temperatures (t12 = 10 h at 60°C and 48 h at 50°C). The mixed culture enzyme was more resistant to end product inhibition by glucose than the Trichoderma enzyme. Hydrolysis of steam exploded aspen wood (SEAW) by the mixed culture cellulase was markedly improved over that achieved by the Trichoderma cellulase.

Effect of hydraulic and organic overloading on thermophilic downflow stationary fixed film (DSFF) reactor

SummaryTests to determine the stability of thermophilic DSFF reactors using bean blanching waste as substrate, showed them to become unstable at 30 to 40 kg/m3/day whether hydraulically or organically overloaded. This level is significantly less than mesophilic reactors could handle (90 kg/m3/day).

Factors affecting the yeast-mediated conversion of ethanol to acetaldehyde in batch reactors

Abstract Whole cells of Pichia pastoris can be used to catalyse the oxidation of ethanol to acetaldehyde. The main product of the oxidation, acetaldehyde, is a competitive inhibitor of alcohol oxidase ( K 1 = 2.6 g l −1 ). When included in the reaction fluid, Tris (hydroxymethyl methylamine) is able to bind acetaldehyde and markedly improve reaction yields. Hydrogen peroxide, which is produced during the reaction, is very efficiently degraded by catalase present within the whole cells. Initial reaction rates can be increased by more than an order of magnitude through the addition of H 2 O 2 . A model equation that describes the effect of the concentrations of oxygen, Tris buffer, ethanol, and biomass is described.

Biological upgrading of dilute ethanol streams to acetaldehyde for low-temperature recovery☆

Abstract A whole-cell process, using the highly active alcohol oxidase enzyme system of methylotrophic yeasts, was developed for the biological upgrading of dilute ethanol streams to the more valuable, more easily recoverable product, acetaldehyde. Of five methylotrophic yeasts, Pichia pastoris exhibited the highest level of oxidative activity (1·22 g acetaldehyde g cells dry wt −1 h −1 ). Under optimized conditions, 95% of a dilute 18 g l −1 ethanol solution was oxidized to acetaldehyde at 100% of theoretical yield. The alcohol oxidase enzyme system of P. pastoris was psychrotolerant, and showed only a 32·5% decrease in activity when the bioconversion temperature was lowered from 30 to 3°C. Under practical applied conditions, dilute ethanol solutions ranging from 0·5 to 3·0% (w/v) were converted to acetaldehyde at process efficiencies of 73 to 61%, respectively. Acetaldehyde readily evaporated from reaction media at 22°C, offering an attractive alternative to the expense of ethanol distillation.

Effect of effluent recirculation on start-up and steady state operation of the downflow stationary fixed film (DSFF) reactor

SummaryIn this study, the effect of effluent recirculation on the start-up and steady state operation of the DSFF is reported. Recirculation had little effect on start-up; however, the maximum loading rate which could be applied to the reactor was higher at higher rates of effluent recirculation.

Treatment of fish processing waste using the downflow stationary fixed film (DSFF) reactor

SummaryThe DSFF reactor has been shown to be capable of treating a wide variety of wastes. In this study, a high protein fish processing waste was treated at several influent concentrations. Chemical oxygen demand (COD) removal efficiencies of up to 90% were achieved at loading rates in excess of 10 kg COD/m3/day.