Abstract: Pyrethrins, the most economically important natural insecticide, comprise a group of six closely related monoterpene esters. The industrial production is based on their extraction from Chrysanthemum cinerariaefolium (Pyrethrum) capitula. The world production of natural pyrethrins still falls short of global market demand stimulating the research in in vitro production as an alternative to conventional cultivation methods. The different biotechnological alternatives such as callus cultures, shoot and root cultures, plant cell suspension cultures, and bioconversion of precursors by means of enzymatic synthesis or genetically engineered microorganisms, as well as the progress achieved in methods for the identification and quantitation of insecticidal compounds have been reviewed. Although technology for plant cell culture exists, industrial applications have, to date, been limited due to both the low economical viability and technological feasibility at large scale. Bioconversion of readily available precursors looks more attractive, but more research is needed before this technology is used for the industrial production of pyrethrins.

Abstract: Simultaneous isomerisation and fermentation (SIF) of xylose and simultaneous isomerisation and cofermentation (SICF) of a glucose/xylose mixture was carried out by Saccharomyces cerevisiae in the presence of xylose isomerase. The SIF of 50 g l−1 xylose gave an ethanol concentration and metabolic yield of 7.5 g l−1 and 0.36 g (g xylose consumed)−1. These parameters improved to 13.4 g l−1 and 0.40 respectively, when borate was added to the medium. The SICF of a mixture of 50 g l−1 glucose and 50 g l−1 xylose gave an ethanol concentration and metabolic yield of 29.8 g l−1 and 0.42 respectively, in the presence of borate. Temperature modulation from 30 °C to 35 °C during fermentation further enhanced the above parameters to 39 g l−1 and 0.45 respectively. The approach was extended to the bioconversion of sugars present in a real lignocellulose hydrolysate (peanut-shell hydrolysate) to ethanol, with a fairly good yield.

Abstract: Biocatalysis harnesses the catalytic potential of enzymes to produce building blocks and end-products for the pharmaceutical and chemical industry. Located at the interface between fermentation processes and petrol-based chemistry, biotransformation processes broaden the toolbox for bioconversion of organic compounds to functionalized products. BIOCATALYSIS AND BIOTRANSFORMATION CONNECT PETROL-BASED CHEMICALS AND RENEWABLE CARBON SOURCES Improvement of synthetic routes according to the guidelines of green chemistry requires increased reaction specificities, reduction of solvent use and emissions, and the use of renewable resources. At first glance, these measures primarily aim at protection of the environment. However, green chemistry also makes economical sense, due to considerable savings on wastewater treatment, energy use, and organic carbon resources. The latter aspects bear directly on the problems associated with our decreasing fossil organic carbon stocks. A recently published analysis of the life-time of the organic carbon feed stocks indicates that the maximal production rates of crude oil will be reached in the next 10 to 20 years (1). Alternative stocks such as Canada's oil tars might be available by then (2), but an increase of the oil price in the next few decades is to be anticipated. Alternative energy sources and new sources for building blocks for chemi- cal synthesis have to be developed. At this point it should be emphasized that a significant share of today's oil production is needed for synthetic chemistry. The EU uses 57 MMT (approx. 8% of the total consumption of the EU) as organic carbon feedstock (3). A key technology to facilitate and smooth the necessary transition is the use of renewable re- sources for the production of chemicals using biocatalytic processes. These processes can be subdi- vided into biotransformation reactions in which a reaction precursor, renewable or petrol-based, is converted to the desired product, and fermentations that use the carbon source for de novo product synthesis, usually from a renewable carbon source (Fig. 1). Fermentation processes are commonly used for transformation of renewable carbon to typical "biological" compounds such as alcohols, ketones, vitamins, antibiotics, or amino acids, which are metabolites or dead-end products in the production organism. As a matter of fact, the range of fermen- tation products is much broader than commonly realized. Compounds usually perceived as non-natural such as, for example, indigo, catechol, hexanoic acid, or poly-( b)-hydroxyalkanoates can be produced from renewable carbon as well. However, the biochemical and genetic engineering that is required for the development of mature processes from early observations on the lab-scale is demanding. Therefore, the total number of compounds that are produced by fermentation today is still relatively limited. Biocatalytic processes employ a different strategy. Precursor molecules are fed to the biocatalyst, which transforms them to the desired compound by a limited number of functionalizing steps (usually one). Carbon and energy required for production of the biocatalyst commonly come from a different, easily metabolizable carbon source such as a sugar. Here, the range of products is not limited by the metabolism of the biocatalyst: non-natural (xenobiotic) precursor molecules can be efficiently trans-

Abstract: Resting cells of a locally isolated strain of Aspergillus niger caused the bioconversion of alpha pinene to verbenone. The formation of verbenone was raised from trace amounts (under screening conditions) to 3.28 mg/100 ml (equivalent to a molar yield of 16.5% conversion of the substrate) by amending the cultivation medium for the fungus. The optimal conditions were: 6 g/100 ml for the glucose concentration, a pH of 7.0, an alpha pinene concentration of 20 mg/100 ml, and a 6-h incubation period for the reaction.

Abstract: A residual mud sample from the marigold flower dehydration process was screened and 19 putative colonies were isolated for their ability to degrade lutein in a chemically defined medium supplemented with marigold flower flour as a carbon source. Among the colonies isolated, two generated volatile compounds in fermentation and one was chosen for further study for its ability to produce a strong tobacco smell. This colony contained two microorganisms, identified as Geotrichum sp. and Bacillus sp. The aroma production requires the presence of both microorganisms and lutein. Using gas chromatography coupled to mass spectrometry (GC/MS), four compounds were identified: 7,8-dihydro- β-ionol, β-ionone, 7,8-dihydro-β-ionone, and 3-hydroxy-β-ionone, in proportions of 84.2%, 9.4%, 3.5%, and 2.9%, respectively.

Abstract: The continuous bioconversion of xylose-containing solutions (obtained by acid hydrolysis of barley bran) into xylitol was carried out using the yeast Debaryomyces hansenii under microaerophilic conditions with or without cell recycle. In fermentations without cell recycle, the volumetric productivities ranged from 0.11–0.6 g l−1 h−1 were obtained for dilution rates of 0.008–0.088 h−1. In experiments performed with cell recycle after membrane separation, the optimum xylitol productivity (2.53 g l−1 h−1) was reached at a dilution rate of 0.284 h−1.

Abstract: The bioconversion of xylose into ethanol with the yeast Pichia stipitis CBS 5773 is inhibited when 20 g/L of ethanol are present in the fermentation broth. In order to avoid this limitation, the fermentation was carried out with simultaneous recovery of product by CO(2) stripping. The fermentation was also improved by attaching a side-arm to the main body of a classical gas-lift loop fermentor. This side-arm increases the liquid circulation, mass transfer, and gas distribution, reducing the amount of oxygen in the inlet gas necessary to perform the fermentation of xylose under microaerobic conditions (K(L)a approximately 16 h(-1)). The continuous stripping of ethanol from the fermentation broth in this new bioreactor system allowed the consumption of higher xylose concentrations than using Erlenmeyer shaker flasks, improved significantly the process productivity and provided a clean ethanol solution by using an ice-cooled condenser system. Finally, a fed-batch fermentation was carried out with a K(L)a = 15.8 h(-1). Starting with 248.2 g of xylose, 237.6 g of xylose was consumed to produce 88.1 g of ethanol which represents 72.6% of the theoretical yield (47.2 g/L of ethanol was recovered in the condenser, while 9.6 g/L remained in the fermentation broth).

Abstract: The present invention relates to a new microbial process for the preparation of the compound of formula (I) from a compound of general formula (II) wherein R+ stands for an alkali metal or ammonium ion, by the submerged cultivation of the strain able to 6β-hydroxylate a compound of formula (II) in aerobic fermentation and by the separation and purification of the product of formula (I) formed in the course of the bioconversion, which comprises cultivating a strain of the genera Micromonospora able to 6β-hydroxylate a compound of the general formula (II), wherein R+ is as defined above, on a nutrient medium containing assimilable carbon and nitrogen sources and mineral salts at 25-32 °C, thereafter feeding the substrate to be transformed into the developed culture, then fermenting the substrate until the end of bioconversion, then separating the compound of formula (I) from the culture broth and, if desired, purifying the same.

Abstract: The effect of the oxygen transfer coefficient on the production of xylitol by bioconversion of xylose present in sugarcane bagasse hemicellulosic hydrolysate using the yeast Candida guilliermondii was investigated. Continuous cultivation was carried out in a 1.25-L fermentor at 30°C, pH 5.5, 300 rpm, and a dilution rate of 0.03/h, using oxygen transfer coefficients of 10, 20, and 30/h. The results showed that the microbial xylitol production (11 g/L) increased by 108% with the decrease in the oxygen volumetric transfer coefficient from 30 to 20/h. The maximum values of xylitol productivity (0.7 g/[L.h]) and yield (0.58 g/g) were obtained ak a 20/h.

Abstract: We report the isolation of mutant strains of the methylotrophic yeast Hansenula polymorpha that are able to efficiently oxidize ethanol to acetaldehyde in an intact cell system. The oxidation reaction is catalyzed by alcohol oxidase (AOX), a key enzyme in the methanol metabolic pathway that is typically present only in H. polymorpha cells growing on methanol. At least three mutations were introduced in the strains. Two of the mutations resulted in high levels of AOX in glucose-grown cells of the yeast. The third mutation introduced a defect in the cell's normal ability to degrade AOX in response to ethanol, and thus stabilizing the enzyme in the presence of this substrate. Using these strains, conditions for bioconversion of ethanol to acetaldehyde were examined. In addition to pH and buffer concentration, we found that the yield of acetaldehyde was improved by the addition of the proteinase inhibitor phenylmethylsulfonyl fluoride (PMSF) and by permeabilization of the cells with digitonin. Under optimal shake-flask conditions using one of the H. polymorpha mutant strains, conversion of ethanol to acetaldehyde was nearly quantitative.

Abstract: Albonoursin production was greatly enhanced when cyclo (L-Leu-L-Phe) (CFL), a tetrahydro derivative of albonoursin, was added to the 2-day culture of an albonoursin-producing actinomycete, Streptomyces albulus KO-23. The increase in albonoursin production paralleled the amount of CFL added. Furthermore, the resting cells of the strain catalyzed the bioconversion of CFL to albonoursin. The optimum pH and temperature for the conversion were found to be pH 10.0 and 50 degrees C. The feeding experiments and the resting-cell reactions revealed that albonoursin is biosynthesized by dehydrogenation of CFL in the actinomycete. This is the first report for a dehydrogenation of amino acid residues at the alpha,beta-positions in cyclic dipeptides.

Abstract: Microorganisms can be used as catalysts to produce organic compounds in a highly chemo-, regio- and enantioselective manner, and whole cells do not require the costly addition of cofactors for redox reactions. However, bioconversions are slow compared to alternative chemical reactions, and the biocatalyst works at its best in an aqueous medium, while the transformations of interest frequently involve compounds with a low-aqueous solubility and that are toxic to microorganisms. This results in low-volumetric productivity in classical bioreactors. The Continuous Closed-Gas-Loop Bioreactor is described here—a reactor system with high productivity, but without the problems associated with two-phase systems, such as an emulsified product stream and phase toxicity. Its working principle is to recirculate a gas phase through a bioreaction compartment and a saturator/absorber module where the product accumulates as a clear organic solution. A wide range of bioconversions should be possible in this set-up, and proof of concept was established for the epoxidation of 1,7-octadiene to (R)-1,2-epoxyoct-7-ene by a native strain of Pseudomonas oleovorans. This reaction represents a group of terminal alkene epoxidations where the bioconversion substrate does not support growth of the microorganism. Practical results at a 5l-scale are presented for this bioconversion for both batch and continuous operation with respect to the aqueous phase, showing continuous stable epoxidation at productivities >14 μmol min−1 L−1 (U L−1). The results confirm that the metabolism does not allow a simple optimization strategy, because growth and biotransformation substrates compete for the same enzyme sites, and conversely growth on a substrate using this very enzyme system is necessary for longterm bioconversion. Integrated removal of the CO2 formed via the liquid overflow was estimated from theory and verified in experimental work. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 70: 553–563, 2000.

Abstract: A process is provided for the bioconversion of a carbon substrate to 1,3-propanediol by a single organism utilizing microorganisms, such as, Citrobacter, Enterobacter, Clostridium, Klebsiella, Aerobacter, Lactobacillus, Aspergillus, Saccharomyces, Zygosaccharomyces, Pichia, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Torulopsis, Methylobacter, Escherichia, Salmonella, Bacillus, Streptomyces and Pseudomonas, containing the genes encoding for an active glycerol or diol dehydratase enzyme by contacting these organisms with a carbon substrate under the appropriate fermentation conditions. Specifically, Citrobacter and, Klebsiella provide the source of exogenous genes for such active dehydratase enzyme.

Abstract: This invention provides a low-cost method of producing .alpha.,.omega.-alkanedicarboxylic acids. Particular bioconversion conditions result in highly efficient conversion of fatty acid, fatty acid ester, or alkane substrates to diacids. Candida tropicalis AR40 or similar yeast strains are grown in a medium containing a carbon source and a nitrogen source at a temperature of 31.degree. C. to 38.degree. C., while additional carbon source is continuously added, until maximum cell growth is attained. Within 0-3 hours of this point, substrate is added to the culture to initiate conversion. An .alpha.,.omega.-alkanedicarboxylic acid made according to this method is also provided.

Abstract: Bioconversion of wheat straw supplemented or not with ammonium nitrate by Lentinus tuber regium (Fr) was investigated by long term solid state fermentation using fungal growth, sclerotium and sporophore production, lignin degradation and the in vitro digestibility as indices of bioconversion capability. Wheat straw supported vigorous fungal growth producing sclerotium and fruiting body initials within 50 days. Lentinus tuber regium demonstrated high lignolytic capability with high lignin degradation (Org. matter/lignin degradation coefficient = 2). Fungal growth mediated specific physicochemical transformation of straw substrate including degradation of lignin and increase of in vitro digestibility of straw substrate, and production of high quality sclerotium and fruiting body. Ammonium nitrate supplement, although, played little or no role in sclerotium and fruiting body initiation, induced very high increase in fungal metabolic rate, sustained greater sclerotium and fruiting body yield, lignin degradation, enhanced lignin degradation coefficient, breakdown of organic matter, and greater efficiency of degraded organics. It however delayed and depressed the increase in the in vitro digestibility of straw substrate. Protein analysis of the sclerotium showed high values of protein with a high quality amino acid profile for humans. Regression analysis showed proportionality between a) lignin degradation and the in vitro digestibility b) degradation of organic matter and sclerotium/fruiting body production c) lignin degradation and degradation of organic matter. The above underscore the capability of Lentinus tuber regium to selectively biodegrade lignin and efficiently mediate conversion of wheat straw to high quality mushroom for humans and, upgraded high quality animal feed.

Abstract: Bioconversion of fumarate to succinate was anaerobically conducted in a synthetic medium containing glycerol as a hydrogen donor and fumarate as a hydrogen acceptor. We investigated the effects of pH, carbon and nitrogen sources, conversion substrate, and other culture conditions on the production of succinate using a newly isolated Enterococcus faecalis RKY1. Addition of a variety of carbonates to the medium significantly increased the rates of production of succinate. The production of succinate and cell growth were relatively satisfactory in the pH range of 7.0- 7.6. By using glycerol as a hydrogen donor, high purity succinate was produced with few byproducts. Yeast extract as a sole nitrogen source was the most effective for producing succinate. As a result, the optimum condition of bioconversion was obtained at a medium containing 20 g/l glycerol, 50 g/l fumarate, 15 g/l yeast extract. 10 g/l K 2 HPO 4 , 1 g/l NaCl, 50 ppm MgCl 2 . 6H 2 O, 10 ppm FeSO 4 . 7H 2 O, and 5 g/l Na 2 CO 3 at pH7.0-7.6. Under the optimum condition, a succinate concentration of 153 g/l was produced in 36 h. The total volumetric production rate and the molar yield of succinate were 4.3 g/l/h and 85%, respectively.

Abstract: The present invention relates to a new microbial process for the preparation of compound of formula (I) from a compound of general formula (II) wherein R stands for an alkali metal or ammonium ion, by the submerged culture of a strain which is able to 6β-hydroxylate the compound of formula (II) in aerobic fermentation and by the separation and purification of the product of formula (I) formed in the course of the bioconversion. The latter comprises the cultivation of a Micromonospora strain which is able to 6β-hydroxylate a compound of general formula (II) - wherein R is as defined above - at 25-32 °C on a nutrient medium containing available carbon - and nitrogen sources and mineral salts, thereafter feeding the substrate to be transformed into the developing culture, then hydroxilating the substrate until finishing of the bioconversion, then separating the compound of formula (I) from the culture broth and, if desired, purifying the same.

Abstract: A quantitative knowledge of dynamic behavior is necessary to understand, optimize and control a bioprocess, particularly in a continuous culture. The study of dynamics in glycerol fermentation by Klebsiella pneumoniae plays an important role because the bioconversion process possesses multiplicity and oscillation under certain conditions. The transient responses of Klebsiella pneumoniae to dilution rate and glycerol concentration step changes in continuous bioconversion of glycerol to 1,3-propanediol was studied in this paper. The experimental results showed that there are distinguished differences between cell growth and intracellular metabolism under different conditions. The transient behavior depended on the both states before and after a step change of operation conditions. When substrate concentration transfered from a limiting condition to a excessive condition, the residual concentration of glycerol in reactor constantly increased, but ethanol concentration decreased gradually as fermentation time. Simultaneously biomass, 1,3 propanediol and acetate concentration increased firstly and then decreased. A novel concept of "glycerol concentration change rate" was used to modify the growth model under steady-state in this paper. The modified model simulated the transient behavior of glycerol bioconversion in good agreement with experimental results.

Abstract: In a bioconversion study based on utilisation of by-products from the AB- (acetone - butanol) bioprocess a new isolated gram-negative solvent tolerant bacterium was used to convert the AB process residue after removal of the major part of the solvents. The bacterium identified as a representative of the genus Alcaligenes (designated as Alcaligenes sp. G) was capable of growth up to optical densities ranging from 8 to 20 and simultaneously of polyhydroxyalkanoate-(PHA-)accumulation up to 40% per dry weight. A standardised medium based on AB by-products containing 7 g/l of butyrate and 5 g/l of acetate at pH 7.5 was used in our studies for bioconversion into PHAs. Concentrations of 1-butanol, which is known for its membrane damaging properties in microorganisms, were tolerated in the AB by-products medium up to 4 g/l without significant inhibition of cellular growth. No inhibition of growth was observed, when the medium was adjusted to 40 g/l butyrate. Due to the toxicity of the remaining 1-butanol maintenance of sterility is of no high priority during the process. The use of acetate and butyrate from an AB process is expected to provide a higher return-on-investment than the combustion of biogas to help meet energy demands.

Abstract: The biotransformation of alpha-pinene to verbenone by resting cells of a Penicillium sp. was studied with the aim of optimizing culture conditions for production of this commercially important flavour compound. The bioconversion process was influenced by a range of culture and buffer parameters. Potato dextrose broth with supplementation of 1% glucose and 0.025% yeast extract of pH 5.75 and a temp. of 30C were determined as the optimal conditions for culture growth. 200 mg (dry wt.) of fungal biomass with 20 mg of alpha-pinene in 100 ml of 0.05M phosphate (pH 7.0) at 30C and 6 h of incubation were found to be optimal buffer conditions for the biotransformation.