Abstract: Glyceryl trinitrate (GTN) 2 mM was quantitatively converted into its 1 and 2 mononitrate derivatives by Geotrichum candidum, with consumption of the nitrate ions produced. The conversion proceeded at a rate independent of the addition of either organic carbon or organic nitrogen sources. Eight batches of nitrate ester, which were added every 24 hours, were successfully converted as far as during the bioconversion process GTN concentration did not exceed 2 mM. When those limiting conditions were not observed, dramatic toxivity of GTN was noticed.

Abstract: A new method has been developed for the use of enzymes as catalysts in organic solvents. The enzyme and cofactor are entrapped in a lyotropic liquid crystal which is insoluble in an organic solvent. The biocatalyst is significantly stabilized in this biphasic reaction system and product recovery is facilitated.

Abstract: Kinnow-mandarin waste (peel, pulp and seeds) was assessed for single-cell protein (SCP) production byChaetomium globosum andSporotrichum pulverulentum in shake-flask culture. The maximum protein enrichment (32% and 34%) of the substrate was achieved after 5 and 7 days of incubation by the two organisms, respectively. Of various nitrogen sources, NaNO3 and NH4Cl gave maximum protein enrichment of the substrate byC. globosum andS. pulverulentum, respectively.

Abstract: Hydrogen peroxide production by blue-green algae (cyanobacteria) under photoautotrophic conditions is of great interest as a model system for the bioconversion of solar energy Our experimental system was based on the photosynthetic reduction of molecular oxygen with electrons from water by Anacystis nidulans 1402-1 as the biophotocatalyst and methyl viologen as a redox intermediate It has been demonstrated that the metabolic conditions of the algae in their different growth stages strongly influence the capacity for hydrogen peroxide photoproduction, and so the initial formation rate and net peroxide yield became maximum in the mid-log phase of growth The overall process can be optimized in the presence of certain metabolic inhibitors such as iodoacetamide and p-hydroxymercuribenzoate, as well as by permeabilization of the cellular membrane after drastic temperature changes and by immobilization of the cells in inert supports such as agar and alginate

Abstract: Plant cells of Mucuna pruriens L. entrapped In calcium alginate, calcium pectinate, agarose, or gelatine were able to convert L-tyrosine to L-DOPA, which was released Into the medium. Michaelis-Menten kinetics could be applied on the entrapped cells, based on the measurement of initial rates of L-DOPA production. The calculated apparent affinity constants were comparable with the affinity constants obtained with enzyme preparations. Comparison of the apparent maximum rate of bioconversion of the entrapped cells and the maximum rate of bioconversion of a derived cell homogenate indicated that the systems were not operating optimally. Measurement of the effective diffusion coefficients of L-tyrosine pointed out that this substrate could diffuse freely into the matrices. From the initial rates of bioconversion and the effective diffusion coefficients, the observable modulus was calculated for each system. The obtained values confirmed that the diffusional supply rate of L-tyrosine was not the limiting factor. For oxygen, which was utilized for byconversion as well as for cell respiration, the calculated observable moduli was directed toward strong oxygen transfer limitations. The values found for the oxygen consumption indicated that the entrapped cells remained partly or totally viable in the four matrices tested. Based on the highest viability and the highest rates of bioconversion, it was concluded that alginate-entrapped cells of M. pruriens formed the most suitable biocatalytic system for the production of L-DOPA from L-tyrosinre.

Abstract: One of the next challenges in the use of biocatalysts (enzyme or microbial cells) is the upgrading of biological reactions of oxidoreduction. The oxidoreductases need cofactors that must be regenerated. Practical experience shows that this is most readily achieved by using living cells of microorganisms. Living cells ofSaccharomyces cerevisiae are able to bioconvert vanillin to vanillyl alcohol (1). By working with a two-phase reactor (dodecanol—feeding medium) it has been possible to use higher vanillin concentrations without inhibiting the bioconversion (2). Several parameters, such as, volume ratio of aqueous over organic phase, pH, vanillin concentration seem to influence the bioconversion greatly. Bearing this is mind, two-phase reactors have been set up. Productivities exceeded 5000 g/m3/d. On the other hand,Saccharomyces cerevisiae mutants have been selected as vanillyl alcohol hyperproducers: vanillyl alcohol productivity of the best selected mutant is twice as high as wild-type strain productivity. Their specific behavior has been studied.

Abstract: Alginate-entrapped cells of Mucuna pruriens L. hydroxylate L-tyrosine, tyramine, para-hydroxyphenylpropionic acid, and para-hydroxyphenylacetic acid to their corresponding catechols, which were released into the incubation medium. Michaelis-Menten kinetics was applied for each bioconversion. The apparent affinity constants were comparable with the affinity constants obtained with a homogenate directly prepared from the cells used for entrapment and with a derived partly purified phenoloxidase. The values found for the apparent maximum rates of bioconversion of the entrapped cells were ca. 50% of the values of the maximum rates of bioconversion of the cell homogenate, indicating that the entrapped cell system was not operating optimally. The effective diffusivities of the substrates and products were measured with alginate-entrapped, inactivated cells. From the five inactivation methods tested, glutaric aldehyde treatment was chosen as the general procedure. Calculated effective diffusivities for the monophenols and catechols demonstrated that these compounds could diffuse freely into and out of the beads. For each bioconversion, the observable modulus was calculated from the initial rate of bioconversion and the effective diffusivity of the substrate. The resulting values indicated that the diffusional supply rate of the substrates was not the limiting factor, except for the conversion of tyramine for which a modulus higher than one was obtained. Analogously, the observable moduli were calculated for oxygen, which was utilized for bioconversion and cell respiration, and these values pointed towards strong oxygen limitation in all cases. The bioconversion rates of the entrapped cells increased with decreasing cell aggregate size. Therefore, it was concluded that direct cell-matrix contact determined the amount of phenoloxidase involved in the bioconversions. The bioconversion rate on a protein basis was constant with enhancement of the bead charge and thus, in spite of limitations, the mixing conditions as such were relatively optimal. In conclusion, the nonoptimal efficiency of the plant cell system studied was caused by oxygen limitation and a partial phenoloxidase participation, but not by mass transfer limitations for substrates and products with the exception of the conversion of tyramine into dopamine.

Abstract: Biotransformation of benzaldehyde and vanillin by growing cells of Saccharomyces cerevisiae was performed in an aqueous medium containing either α- or β-cyclodextrin at the same molar concentration as the substrate. The yeast fermentative activity, as reflected by CO2 evolution, and bioconversion to the corresponding alcohols were both faster and greater in the presence of the cyclic dextrins. Clearly, cyclodextrins were shown to significantly alleviate the inhibitory effects of the aromatic aldehydes.

Abstract: The bioconversion activity of calcium alginate-immobilized Clostridium acetobutylicum ATCC 824 was investigated in a continuous reactor system utilizing a defined feed medium which did not support cell growth. The changes in biocatalytic activity with time were studied at different pH values as well as when different metabolites (butyric, acetic, and acetoacetic acids) were present in the feed stream. Although the nongrowing cells were metabolically active, the product distribution was shifted from solvent production to acidogenesis. Overall activity losses occurred due to cell lysis, sporulation, and the effects of nitrogen level on macromolecular turnover. These effects were minimized under some operating conditions (e.g., pH 6), resulting in significantly longer productivity lifetimes.

Abstract: The enzymatic hydrolysis of plant carbohydrate polymers is discussed with particular emphasis on lignocellulose. The polysaccharides include starch, inulin, cellulose and the hemicelluloses, i.e., the heteroxylans and glucomannans. Commercial operations exist for the enzymatic hydrolysis of starch and its fermentation into chemicals such as ethanol. Enzymatic hydrolysis of lignocellulose is more complex and the enzymes are rather expensive to produce, which currently precludes the commercial processing of lignocellulosic materials. The bioconversion of lignocellulose consists of 4 process steps: pretreatment, enzyme production, enzymatic saccharification and fermentation. Except for the last step, each of these process steps is discussed. The discussion is highlighted with examples of lignocellulosic waste materials (e.g., sugar cane and a hardwood and softwood sawdust) which are of potential use in a bioconversion process for providing sugar hydrolysates that can serve as fermentation substrates.

Abstract: The subject of the invention is a process for the production of xylitol from D-xylose by the action of the strain Candida parapsilosis ATCC28474, characterised in that it comprises the steps consisting in: 1. cultivating an inoculum of 2 to 5 x 10 cells/ml of C. parapsilosis ATCC28474, under aerobic conditions, at a temperature of 25 to 35 DEG C, for the time required for the consumption of the sugar, at a pH maintained in the range of 3.8 to 5.4, in a fermenter containing: either a synthetic medium comprising 30 to 100 g/l of D-xylose, 2 to 10 g/l of KH2PO4, 1 to 5 g/l of (NH4)2SO4, and 0.1 to 1 g/l of MgSO4, 7H2O, or a hydrolysate of plant raw materials comprising 50 to 80 g/l of D-xylose, in the presence of 0.5 to 3 g/l of yeast extract, under aeration and stirring conditions ensuring a supply of oxygen such that the bioconversion of the D-xylose to xylitol is brought about, without allowing the reuse of the xylitol by the yeast, and 2. isolating the xylitol from the culture medium. Application to the industrial biosynthesis of xylitol.

Abstract: Disclosed and claimed are DNA gene segments, biologically functional plasmids and recombinant plasmids, and microorganism host cells containing such plasmids, all of which contain toluene monooxygenase genes from Pseudomonas mendocina KR-1 and which are useful in a method for the microbial bioconversion of selected phenyl compounds to selected phenolic compounds. In particular, the method is useful for making p-hydroxyphenylacetic acid which is a valuable chemical intermediate in the preparation of certain antibiotics and certain .beta.-adrenergic blocking agents.

Abstract: The level of glutathione S-transferase (GSH0ST) activity was determined in growing cultures and in washed resting cells of Beauveria strains with and without addition of isosorbide dinitrate (ISDN), by following the reaction with o-dinitrobenzene (o-DNB). The level of GSH-ST varied according to the pH changes of the medium and decreased during culture. The enzymatic activity measured with o-DNB did not correlate with ISDN bioconversion carried out either with B. sulfurescens or B. tenella. Immediately after starting incubation of the resting cells with ISDN, the level of GSH-ST activity initially increased, but declined afterwards, whereas the bioconversion process continued and reached 500 mg/l isosorbide 5-mononitrate. When 1-chloro-2,4-dinitrobenzene was used as a substrate for the evaluation of GSH-ST activity using B. tenella, a conjugation product having a UV absorption at 410 nm was formed.

Abstract: The major objective of this thesis was to investigate and improve a direct microbial fermentation process, using the thermophilic anaerobic bacteria Clostridium thermocellum and Clostridium thermohvdrosulfuricum for the production of fuel ethanol in economically significant concentrations, from cellulose and hemicellulose contained in renewable biomass. Two model substrates representative of readily available lignocellulosic materials were selected for this study. These were wheat straw, which is the largest single biomass resource available in Australia, and waste paper, which is a major component of municipal solid wastes. At the onset of the research, proximate analyses of the composition of the two substrates were carried out to assess the potential yield of ethanol. Based on the total carbohydrate (hexose as well as pentose sugars) content of the materials, a theoretical potential yield of ethanol over 500 litres per dry tonne of biomass was estimated for both substrates. The feasibility of effecting biomass conversion to ethanol by direct fermentation of the substrates was then examined. Fermentation characteristics of 9 strains of the potent cellulolytic anaerobe, C. thermocelIum and 5 strains of the saccharolytic ethanoloaen. C. thermohvdrosulfuricum. were investigated on a range of sugars. Cellulose hydrolysis and fermentation by C. thermocellum strains were also studied with alpha-cellulose and the two model substrates. Variations in growth characteristics, extent and rates of substrate utilization, as well as in the stoichiometry of product formation were noted, not only between the two species, but also among the different strains. A stable coculture comprising the most potent strains of the two species could be established, and this culture efficiently fermented crystalline and native cellulosic substrates to produce ethanol at substantially higher yields than could be achieved with cultures of C. thermocellum alone. At 1% (w/v) concentration of wheat straw and newspaper, ethanol yield amounting to 70% of theoretical was obtained with the coculture, compared to 25% of theoretical yield exhibited bv C. thermocellum. The metabolic basis for the enhanced fermentation effectiveness of the coculture system has been discussed. The feasibility of attaining higher ethanol concentrations in the fermentations was investigated next by employing increased substrate concentrations in batch as well as fed-batch mode of operation. The bioconversion efficiency was observed to systematically decrease with increased substrate concentration, and a limiting ethanol concentration for the cocultures appeared to be around 10-12g/l. At the highest substrate loadings used, the yield of ethanol was only 25% of theoretical. Lignaceous components of biomass and inhibition of bacterial growth by products of fermentation, as well as the physical nature of the substrates were determined to be the major factors limiting the effectiveness of the fermentation. The above observations led to further studies involving a comparative evaluation of range of substrate delignification treatments and a systematic program of strain improvement with respect to increased ethanol tolerance and end product selectivity. A selective solvent extraction procedure using an alkaline ethanol solvent yielded the best delignification performance of all the alternatives examined. Up to 70% lignin removal with a loss of less than 10% of the available carbohydrates was obtained with this method. Coculture fermentation of wheatstraw and newspaper pretreated by this procedure showed a four-fold increase in the maximum volumetric degradation rate as well as nearly 100% increase in the overall extent of substrate utilization, compared to untreated material. Studies aimed at improving the fermentation efficacy were undertaken on both species of organisms. Improved ethanol tolerance was achieved through progressive adaptation of parent strains to higher ethanol concentrations in the growth medium. The strains isolated in this work however tended to have a significantly higher yield of the acid products concomitant with their enhanced ethanol productivity. A separate program of mutation and selective isolation of low acid producing cultures eventually resulted in strains which in coculture, fed batch fermentations were able to produce ethanol at concentrations of up to 30g/l at a net ethanol yield exceeding 60% of theoretical, when grown on pretreated wheat straw and newspaper. A relatively reduced yield of ethanol however, was noted on real biomass compared to similar fermentations using pure substrates. This coincided with increased production of acetate with the crude substrates. An analysis of fermentation kinetics for the various experiments revealed that the ethanol/acetate ratio for deregulated strains of C. thermocellum and C. thermohvdrosulfuricum was strongly dependent on the specific growth rate the organisms achieve during fermentation, which, in turn is determined by the substrate hydrolysis and/or consumption rates. The implications of this to future process improvement studies has been briefly discussed.

Abstract: Two isolated basidiomycetes mould cultures Polyporus BH1 and Polyporus BW1 and two standard white rot fungal cultures Pleurotus ostreatus and Polyporus versicolor were cultivated under submerged conditions on whole (untreated) bagasse for protein production. Some factors promoting better bioconversion were studied and selected. NORKRAN's medium at pH 5.0 with 1.0% bagasse of 80 mesh particle size using an appropriate quantity of inoculum, supplementing medium with an amino acid, without addition of any vitamin, etc. gave best results.

Abstract: Some basic data on extractive bioconversion of glucose to lactic acid are presented. Using Lactobacillus delbruckii, lactic acid fermentation is carried out in a two-phase system consisting of an organic (paraffin) and an aqueous phase. Bacteria and nutrient components are kept nearly quantitatively in the aqueous phase, whereas the lactic acid is considerably transferred into the paraffin phase. Influences of different surfactants and anion carriers on the partition coefficients are evaluated. A new concept of continuous fermentation with simultaneous extraction of the inhibiting product by means of an external extraction loop is discussed.