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Abstract: d-Carvone or (4S)-(+)-carvone has a caraway/dill odour and is the main constituent of caraway (Carum carvi) and dill (Anethum graveolens) seed oils whilst l-Carvone or (4R)-(−)-carvone has a sweet spearmint odour and is the main constituent of spearmint (Mentha spicata). Carvone is produced by both extraction and purification of essential oils from caraway, dill and spearmint seeds and by chemical and biotechnological synthesis. The several applications of carvone as fragrance and flavour, potato sprouting inhibitor, antimicrobial agent, building block and biochemical environmental indicator, along with its relevancy in the medical field, justify the interest in this monoterpene. [Copyright &y& Elsevier]

Rhodococcus erythropolisDCL14 has the ability to convert the terpene (-)-carveol to the valuable flavour compound (-)-carvone when growing on a wide range of carbon sources. To study the effect of carbon and energy sources such as alkanes, alkanols and terpenes on the biotechnological process, the cellular adaptation at the level of fatty acid composition of the membrane phospholipids and the (-)-carvone production were examined. All tested carbon sources caused a dose-dependent increase in the degree of saturation of the fatty acids. The exception was observed with short-chain alcohols such as methanol and ethanol, to which the cells adapted with a concentration-dependent decrease in the saturation degree of the membrane phospholipids. This influence of the different carbon sources on the rigidity of the cell membrane also had an impact on the (-)-carvone productivity of the strain. [ABSTRACT FROM AUTHOR]

The behaviour of cells of Rhodococcus erythropolis DCL14, Xanthobacter Py2, Arthrobacter simplex and Mycobacterium sp. NRRL B-3805, in biphasic systems containing different organic solvents was evaluated and compared. The data, obtained mainly by fluorescence microscopy and image analysis, was interpreted using principal components analysis (PCA). With this technique, the variability of the data could be summarised in 7 components, representing 75.8% of the variance of the data. Over a third of the variance could be explained by the first two principal components which represent solvent toxicity. Apparently this is the major factor influencing cell behaviour in an organic:aqueous system. However, factors such as substrate concentration, cell adaptation ability (resulting in morphological changes and aggregation or separation of cells) and membrane composition (specific to each strain) also play an important role in cell resistance to solvent toxicity. The results regarding cell shape indicate that loss of viability occurs, in the tested bacterial strains, after incorporation of molecules of solvent in the cellular membrane. This should result in an increase in membrane fluidity, and thus, in an alteration of cell shape. The ability to form "self-defence" clusters was observed to be different amongst the four strains. X. Py2 showed, in general, a low tendency to form aggregates under the tested conditions; A. simplex and R. erythropolis aggregated mainly in the presence of low log P solvents; and Mycobacterium. sp. cells showed a high ability to aggregate. [ABSTRACT FROM AUTHOR]

Rhodococcus opacus PW4 cells were found to produce trans- and cis-carveol and/or carvone as result of limonene metabolism, depending on the type and concentration of the carbon source used for cell growth. In aqueous systems, cells grown on ethanol and toluene only produced trans-carveol, whilst cells grown on limonene and on toluene with a larger head-space available produced both trans-carveol and carvone. In biphasic systems, limonene was converted to trans- and cis-carveol as well as to carvone, regardless of the carbon source used, although carveol and carvone production rates were higher in toluene and limonene grown cells, respectively. A good and stable emulsion was obtained in a magnetically stirred two-phase reactor but both trans-carveol and carvone were produced at low rates: 0.08 and 0.02 nmol/min mg prot, respectively. No cis-carveol was formed. When (−)-carveol was added, carvone production increased 4.7 fold to 0.12 nmol/min mg prot. Using an aerated two-phase reactor, carvone production was enhanced even with cells grown on toluene. The highest trans- and cis-carveol and carvone production rates were attained with cells grown on limonene by continuously supplying limonene to the reactor through the air stream, carvone production reaching 0.58 nmol/min mg prot. The best trans-/cis-carveol ratio (2.26) was observed with cells grown on toluene when limonene was supplied in the gas phase. When 50 mM limonene was added initially, carvone was produced 27.9 and 141.4 times faster than trans-carveol with cells grown on toluene and limonene, respectively. [Copyright &y& Elsevier]

The effect of some solvents, present in different amounts, upon whole cells of Rhodococcus erythropolis DCL14 carrying out the biotransformation of (-)-carveol to (-)-carvone was studied. The solvents tested were ethyl butyrate, n -hexane, cyclohexane, iso -octane, n -dodecane, dimethyl sulfoxide, bis(2-ethylhexyl) phthalate and FC-70. The volumes of each solvent corresponded to organic:aqueous phase ratios of 0.0005, 0.0025, 0.005, 0.025 and 0.2. To assess any potential solvent protection towards substrate toxicity, assays were carried out at two initial carveol concentrations (15 and 50 mM). Carvone accumulation was followed by gas chromatography. Cell viability, several aspects of cell morphology and the ability to form clusters were monitored by fluorescence microscopy. Principal components analysis (PCA) was used as a tool to explain the differences in the observations of the multidimensional data set obtained from the multiple conditions. PCA using the different volumes of each solvent as variable suggests that the variability of the observations can be summarised in six components which represent 79.4% of the variance of the data. Conversely, using cell and solvent data to perform the PCA, 97.1% of the variance of the data can be summarised in three components, the first two capturing 91.0% of the information. These components seem to represent solvent toxicity and a protective effect of the solvent from carveol toxicity. [ABSTRACT FROM AUTHOR]

Whole cells of Rhodococcus erythropolis DCL14 present carveol dehydrogenase (CDH) activity, which allows them to stereoselectively carry out the oxidation of the (+)-cis and (−)-trans-carveol to (+)- and (−)-carvone, respectively . When a diastereomeric mixture of (−)-carveol was supplied for biotransformation, the (−)-trans-carveol was converted to (−)-carvone. When the cells grow on limonene or cyclohexanol the major activity is NAD-dependent. The relatively low water solubility of carveol and carvone was overcome through the implementation of an organic:aqueous system. The prolonged productivity of such a system depends on cell viability, since viable cells are naturally able to regenerate the co-factor. Fluorescence microscopy was used to off-line monitor cell viability during the time course of the biotransformation.n-Dodecane was the solvent that allowed the highest retention of both cell activity and viability. The most adequate phase ratio was 1:5, at which, for ODs higher than 0.57, a stable emulsion is formed. At an OD of 0.46 only half of the solvent was emulsified. Loss of viability increased with the OD, probably due to oxygen depletion. The maximum specific production rate was obtained at an initial carveol concentration of 125 mM. At this scale (60 ml flasks) the best aeration rate was 0.01 vvm, both with regard to viability and stability of the emulsion. Carvone was found to be toxic, causing cell death at concentrations above 50 mM. [Copyright &y& Elsevier]

Biocatalysis with whole cells in aqueous–organic systems is extremely attractive for conversion of substrates with low water solubility, specially if co-factor regeneration is required. However, in the latter case, cells should be able to stay viable, which makes both reactor configuration and operation conditions important.Three reactor configuration types were tested: mechanically stirred direct contact reactor, silicone tube membrane reactor and air-driven column reactor. All reactors were operated with pulse additions of carveol. The maximum trans-carveol conversion (92%) and specific production rate (1.69 mg carvone/h mg prot) were obtained with the mechanically stirred reactor at, respectively, ambient temperature, in 50 mM, pH 7.0 phosphate buffer, and at 28 °C in mineral medium. The highest productivity (0.164 mg carvone/h ml org) was attained with the air-driven column reactor after the cells had been adapted to the presence of solvent, substrate and product. [Copyright &y& Elsevier]

The enantiomeric ratio (E) is commonly used to evaluate enzyme-catalysed kinetic resolutions. Chen et al. (1982) proposed a model for the enantiomeric ratio, which relates the extent of substrate conversion and the enantiomeric excess. The model, however, does not apply to cases where the substrate initially contains unequal amounts of enantiomers. This paper describes the adaptation of Chen''s model to cases where the reagent consists of a mixture of diastereomers present in unequal initial amounts and gives product as a single diastereomer. Diastereomeric ‘resolutions’ of (+)-limonene-1,2-epoxide and of (−)-carveol were used to validate this model. [Copyright &y& Elsevier]

The fatty acid (FA) composition of the bacterial membrane of Cupriavidus necator DSM 545 was assessed during the time course of two-stage fed-batch cultivations for the production of short-chain polyhydroxyalkanoates (PHA). Changes in the relative proportion of straight, methyl and cyclopropyl saturated, unsaturated, hydroxy substituted and polyunsaturated FA were observed, depending on the C sources and cultivation conditions used to favor the synthesis of poly(3-hydroxybutyrate) (P(3HB)), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P(3HB-co-4HB)) or poly(3-hydroxybutyrate-4-hydroxybutyrate-3-hydroxyvalerate) (P(3HB-4HB-3HV)), under N limiting conditions. The relative percentage of each FA class was studied using glucose or waste glycerol (GRP), as main C source for P(3HB) production. The FA profile was also assessed when GRP was used together with i) γ-butyrolactone (GBL) (precursor of 4HB monomers) for P(3HB-4HB) synthesis and ii) GBL and propionic acid (PA) (3HV precursor) to yield P(3HB-4HB-3HV). The effect of GBL and PA utilization as PHA monomer precursors on the FA profile of the cell membrane was studied under two different dissolved oxygen concentrations (DOC)., (Copyright © 2013 Elsevier B.V. All rights reserved.)