William D. Murray

Microbiological and Enzymatic Production of Flavor and Fragrance Chemicals

(1989). Microbiological and Enzymatic Production of Flavor and Fragrance Chemicals. Critical Reviews in Biotechnology: Vol. 9, No. 2, pp. 105-169.

Bacteroides cellulosolvens sp. nov., a cellulolytic species from sewage sludge.

A new mesophilic, cellulolytic species of Bacteroides was isolated from a methanogenic cellulose enrichment culture of municipal sewage sludge and is described. This species ferments only cellulose and cellobiose. The fermentation products are acetic acid. CO2, H2, ethanol, and small amounts of lactic acid. The deoxyribonucleic acid base composition is 43 mol% guanine plus cytosine. The name Bacteroides cellulosolvens is proposed. Type strain WM2 is deposited in the National Research Council of Canada culture collection as strain NRCC 2944.

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.

Clostridium saccharolyticum sp. nov., a Saccharolytic Species from Sewage Sludge

A new species of Clostridium isolated from a methanogenic cellulose-enrichment culture of sewage sludge is described. The colonies produced by these bacteria were white, circular, and convex with smooth margins. The cells were straight, spindle-shaped rods, 0.6 by 3.0 μm in size. They were gram negative and nonmotile, and they formed round, terminal spores. A wide variety of carbohydrates was fermented by this mesophilic anaerobe. The major fermentation products were acetic acid, hydrogen, carbon dioxide, and ethanol. The deoxyribonucleic acid base composition was 28 mol% guanine plus cytosine. The name Clostridium saccharolyticum is proposed for this new species on the basis of its broad saccharolytic activity. The type strain of C. saccharolyticum is WM1 (= NRC 2533).

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.

A generally applicable technique for the extraction of restrictable DNA from methanogenic bacteria

Abstract We present a method, which is generally applicable to a wide variety of methanogenic bacteria, for the extraction of restrictable genomic DNA. Lysis involves treatment of frozen-thawed cells with pronase, dithiothreitol and sodium dodecyl sulphate, followed by recovery of the DNA by ethanol precipitation.

Clostridium lentocellum sp. nov., a cellulolytic species from river sediment containing paper-mill waste*

Summary A new mesophilic cellulolytic species of Clostridium was isolated from an estuarine mud bank of a river receiving paper-mill and domestic effluent. Major fermentation products are carbon dioxide, hydrogen, ethanol, acetic acid and small amounts of lactic acid. The deoxyribonucleic acid base composition is 36 mol% guanine plus cytosine. The name Clostridium lentocellum is proposed. The type strain is RHM5, and it is deposited in the National Collection of Industrial Bacteria, in Aberdeen, Scotland (NCIB no. 11756).

Increased ethanol production and tolerance by a pyruvate-negative mutant of Clostridium saccharolyticum

SummaryTo improve the conversion of hexoses and pentoses to ethanol, a pyruvate-negative (PN) mutant of Clostridium saccharolyticum, having lower acetate kinase activity, was obtained. The PN mutant used more substrate (glucose or xylose) and produced more biomass and ethanol, but less acetic acid. This shift in catabolism raised the ethanol/acetate ratio from 6.7 to 13. The PN mutant converted both glucose and xylose to ethanol at an efficiency of 80% of the theoretical yield as compared to 64% for C. saccharolyticum wild type. This improved production of ethanol was also accompanied by an increased tolerance to ethanol. The PN mutant showed 50% growth inhibition at an ethanol concentration of 6.5% (v/v) as compared to 3.5% for the parent strain.

Single step conversion of cellulose to ethanol by a mesophilic coculture

SummaryA coculture consisting of two mesophilic anaerobes, produced about 0.8 mole of ethanol per mole of cellulose from a variety of cellulosic materials. The non-cellulolytic member of this coculture, Clostridium saccharolyticum sp. nov. converted glucose and xylose to ethanol and acetic acid in ratios over 4 to 1.

Ethanol fermentation of hexose and pentose wood sugars produced by hydrogen-fluoride solvolysis of aspen chips

SummaryHexose and pentose sugars, produced by hydrogen-fluoride solvolysis of aspen wood chips, were totally consumed in a coculture fermentation by Zymomonas mobilis and a mutant of Clostridium saccharolyticum. Z. mobilis converted the glucose to ethanol, while the mutant, which was improved in both ethanol production and tolerance, converted the xylose component to ethanol. A high conversion efficiency of wood sugars to ethanol was obtained, and the cells after the fermentation were successfully used for cell recycle.