A membrane bioreactor for the biotransformation of α-pinene oxide to isonovalal by Pseudomonas fluorescens NCIMB 11671

In this work the biotransformation of α-pinene oxide to isonovalal using resting cells of Pseudomonas fluorescens NCIMB 11671 was evaluated in a membrane bioreactor for biotransformations (MBB). Since the membrane area required to obtain optimum productivities was calculated to be very large (1,000 m² m⁻³), and not possible to fit into the laboratory reactor used, we initially evaluated performance with lower membrane areas (71 m² m⁻³) in a batch system with both the substrate and product in the organic phase. This resulted in low productivities due to mass transfer limitations, so an optimum feeding rate of 0.1 g α-pinene oxide h⁻¹ g_cells ⁻¹ added directly to the reactor contents was determined in batch culture to minimise inhibition. The MBB was then operated continuously for the production of isonovalal, and a final concentration of 108 g l⁻¹ was obtained in the organic reservoir after nearly 400 h of operation (0.32 g-isonovalal l⁻¹ h⁻¹), and the reaction was found not to be mass transfer limited. Finally, the relative viability of the cells was measured using fluorescent probes, and their half-life was found to be almost 2 months, confirming the ability of the MBB to facilitate biotransformations with inhibitory substrates and products.     

Optimization of isonovalal production from α-pinene oxide using permeabilized cells of <Emphasis Type="Italic">Pseudomonas rhodesiae</Emphasis> CIP 107491

Optimization studies on the synthesis of isonovalal from alpha-pinene oxide by Pseudomonas rhodesiae CIP 107491 operated in a biphasic medium are presented. Three key parameters are identified. The first is the need for a permeabilization of cells by freezing them and then treating the thawed material with an organic solvent such as chloroform, toluene or diethyl ether. This operation allows both enzyme release into the aqueous phase outside the cells and an improvement in the transport properties of both substrate and product across the cell membrane, strongly increasing reaction rates. The second is that the enzyme alpha-pinene oxide lyase, which exhibits an irreversible inactivation by isonovalal (or a by-product), presents a constant turn-over, i.e., the total product synthesis is proportional to the biomass loading and is close to 108 mmol (16.4 g) isonovalal l(-1) g(-1) biomass. The third phenomenon is that the biphasic system used is not phase-transfer-limited, a feature attributed to the spontaneous formation of an oil-in-water emulsion. It is thus possible to carry out a very efficient process, allowing the recovery of 2.63 mol isonovalal l(-1) (400 g l(-1)) from 25 g biomass l(-1) in 2.5 h, corresponding to an average reaction rate as high as 0.70 mmol min(-1) g(-1) cells (160 g l(-1) h(-1)).     

Production of trans-2-methyl-5-isopropylhexa-2,5-dienoic acid by Pseudomonas rhodesiae CIP 107491

The feasibility of trans-2-methyl-5-isopropylhexa-2,5-dienoic acid (novalic acid) accumulation using the alpha-pinene degradation pathway of Pseudomonas rhodesiae CIP 107491 was studied. This appeared possible by using concentrated living bacterial cells produced under oxygen limitation with alpha-pinene as sole carbon source. The second step of the process, the bioconversion itself, had to be performed without oxygen limitation due to the need for cofactor regeneration. Results showed that a not yet reported cofactor-dependent enzymatic isomerization of isonovalal into novalal was likely to occur and that both aldehyde isomers could be oxidized to the corresponding acid. Precursors tested, alpha-pinene oxide and isonovalal had a strong permeabilization effect on bacterial cells. This effect, which increased from the oxide to the aldehyde, led to an inactivation of the respiratory chain and to acids synthesis stop. Present results allowed to obtain about 12 g/L acids (80% novalic acid) with an average yield close to 50% after 12h reaction in a biphasic system using alpha-pinene oxide as precursor .     

A membrane bioreactor for biotransformations of hydrophobic molecules

The Membrane Bioreactor for Biotransformations (MBB) is based on the aqueous/organic two-phase system, and uses a tubular silicone rubber membrane to separate the two liquid phases. This avoids the key problem associated with direct contact two-phase processes, specifically, product emulsification. The baker's yeast mediated reduction of geraniol to citronellol was used as a model biotransformation to demonstrate MBB operation. Values for the overall mass transfer coefficient were determined for geraniol, (2.0 x 10(-5) ms-1), and for citronellol, (2.1 x 10(-5) ms-1) diffusion across the silicone rubber membrane. Using these values, and the specific activity of the biocatalyst (5 nmols-1g biomass-1), a suitable membrane surface area: biomass ratio was determined as 2.4 x 10(-3) m2g biomass-1. The bioreactor was operated at this surface area: biomass ratio and achieved a product accumulation rate 90-95% that of a conventional direct contact two-phase system. The slight reduction in product accumulation rate was shown not to be due to mass transfer limitations with respect to reactant delivery or product extraction. Copyright 1998 John Wiley & Sons, Inc.     

Enhanced bioproduction of carvone in a two-liquid-phase partitioning bioreactor with a highly hydrophobic biocatalyst

The microbial biotransformation of (-)-trans-carveol to the flavor and fragrance compound (R)-(-)-carvone by Rhodococcus erythropolis DCL14 was carried out in a 3 L two phase partitioning bioreactor with an immiscible liquid second phase in an effort to improve upon the reactor performance achieved in a single aqueous phase system. The purpose of employing the liquid second phase is to minimize biotransformation rate inhibition due to the accumulation of the toxic substrate (cis-carveol) and product (carvone) in the aqueous phase. 1-Dodecene was chosen as the solvent for this application because it is biocompatible, non-biodegradable and has a superior affinity for the target product (carvone) relative to the other solvents tested. However, when 1-dodecene was used in the biotransformation, the extremely hydrophobic R. erythropolis DCL14 created an emulsion with the organic solvent with significant sequestering of the cells into the organic phase and negligible substrate conversion. To overcome these operational difficulties, silicone oil, which is considered a liquid polymer, was used with the aim of preventing emulsification and sequestration of cells in the non-aqueous phase. Although some emulsification of the water-silicone oil was again created by the cells, operability was improved and, in fed-batch mode, the system was able to convert approximately 2(1/2) times more carveol than a benchmark single aqueous phase system before substrate/product toxicity caused the biotransformation to stop. This study has demonstrated enhancement of a microbial biotransformation for the production of a high value nutraceutical compound via the use of a second partitioning phase, along with operational challenges arising from the use of a highly hydrophobic organism in such systems.     

Modeling the acid–base properties of glutathione in different ionic media, with particular reference to natural waters and biological fluids

The acid-base properties of γ-L-glutamyl-L-cysteinyl-glycine (glutathione, GSH) were determined by potentiometry (ISE-H(+), glass electrode) in pure NaI((aq)) and in NaCl((aq))/MgCl(2(aq)), and NaCl((aq))/CaCl(2(aq)) mixtures, at T = 298.15 K and different ionic strengths (up to I(c) ~ 5.0 mol L(-1)). In addition, the activity coefficients of glutathione were also determined by the distribution method at the same temperature in various ionic media (LiCl((aq)), NaCl((aq)), KCl((aq)), CsCl((aq)), MgCl(2(aq)), CaCl(2(aq)), NaI((aq))). The results obtained were also used to calculate the Specific ion Interaction Theory (SIT) and Pitzer coefficients for the dependence on medium and ionic strength of glutathione species, as well as the formation constants of weak Mg(j)H( i )(GSH)((i+2j-3)) and Ca(j)H(i)(GSH)((i+2j-3)) complexes. Direct calorimetric titrations were also carried out in pure NaCl((aq)) and in NaCl((aq))/CaCl(2(aq)) mixtures at different ionic strengths (0.25 ≤ I (c )/mol L(-1) ≤ 5.0) in order to determine the enthalpy changes for the protonation and complex formation equilibria in these media at T = 298.15 K. Results obtained are useful for the definition of glutathione speciation in any aqueous media containing the main cations of natural waters and biological fluids, such as Na(+), K(+), Mg(2+), and Ca(2+). Finally, this kind of systematic studies, where a series of ionic media (e.g., all alkali metal chlorides) is taken into account in the determination of various thermodynamic parameters, is useful for the definition of some trends in the thermodynamic behavior of glutathione in aqueous solution.     

Inhibitory effects of substrate and product on the carvone biotransformation activity of <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis>

The aqueous substrate and product toxicity thresholds in the microbial biotransformation of (-)-trans-carveol to the fragrance/flavor compound (R)-(-)-carvone by Rhodococcus erythropolis were determined. Above aqueous phase concentrations of approx. 500 mg carveol/l and 200-600 mg carvone/l, the biotransformation activity of the biocatalyst was inhibited. This biotransformation was undertaken in a single aqueous phase 3 l [corrected] reactor in which a total of 5 ml carveol (mixture of isomers) was added before the biotransformation rate decreased significantly. The carvone volumetric productivity was 31 mg/lh. Although the growth of the organism post-exposure was not affected, dramatic morphological changes in response to the accumulation of the inhibitory substrate and product were observed.     

The effect of terpenoid extracts from 15 pine species on the feeding behavioural sequence of the late instars of the pine processionary caterpillar Thaumetopoea pityocampa

The feeding behaviour of the pine processionary (PPC) caterpillar Thaumetopoea pityocampa Den. and Schiff. (Lepidoptera, Thaumetopoeidae) in L3-L4 stages was explored by means of laboratory arena feeding trials and natural substrates. In the bioassays, volatile extracts of 15 pine species, 8 of which are naturally growing in Greece, were incorporated. An analytical model was developed based on the principle of multinomial logit regression with five outcomes on the basis of the behavioural feeding sequence of the caterpillars. The outcomes were the five steps in which the feeding behavioural sequence was decomposed. The model's suitability (MacFadden's rho(2)=0.229, P<10(-4)) was examined when including 10 terpenes that were judged significant through a stepwise canonical discriminant analysis. The proposed model was superior to a random one and the two models resulting from the addition and subtraction of 4 terpenes to the already 10 existing compounds. The most informative model was built on the terpenes caryophyllene oxide, terpinolene, myrcene, germacrene D, eudesmol, limonene, beta-pinene, beta-caryophyllene, alpha-pinene and manoyl oxide. The background terpenes were present in the model and of particular importance. No special behavioural role, either as promoter or inhibitor could be assigned to the individual volatile metabolites, since no constant pattern among behavioural steps was observed. For instance, beta-caryophyllene while acts as promoter of attraction and trial bite it is a suppressor of partial feeding and strongly inhibits complete needle consumption. The monoterpene limonene, on the other hand, seems to be a suppressor of partial and complete feeding. The overall methodological scheme and the analytical modelling tool could be proved a suitable research protocol in unfolding the ecological role of a complex mixture of secondary metabolites. Those who develop safe practical systems can use this.     

Direct measurements of intervessel pit membrane hydraulic resistance in two angiosperm tree species

The hydraulic resistance of pit membranes was measured directly in earlywood vessels of Fraxinus americana and Ulmus americana. The area-specific resistance of pit membranes (r(mem)) was higher than modeled or measured values obtained previously for hardwood species, with r(mem) of 5.24 × 10(3) MPa·s·m(-1) for Fraxinus and 2.56 × 10(3) MPa·s·m(-1) for Ulmus. The calculated resistance of pit canals was three orders of magnitude below total pit resistance indicating that pit membranes contributed the majority of resistance. Scanning electron microscopy indicated that pit membranes of Ulmus were thinner and more porous than those of Fraxinus, consistent with the difference in r(mem) between the species. Measurements of average vessel diameter and length and area of wall overlap with neighboring vessels were used to partition the vascular resistance between vessel lumen and pit membrane components. Pit membrane resistance accounted for 80% of the total resistance in Fraxinus and 87% in Ulmus in 2-yr-old branch sections. However, measurements of vessel dimensions in the trunk suggest that the division of resistance between pit membrane and lumen components would be closer to co-limiting in older regions of the tree. Thus, pit membrane resistance may be of greater relative importance in small branches than in older regions of mature trees.     

Pseudomonas rhodesiae PF1: A New and Efficient Biocatalyst for Production of Isonovalal from α-pinene Oxide

A new bacterial strain, identified as Pseudomonas rhodesiae PF1 and deposited under the accession number CIP 107491, is presented. It is very active for the production of the acyclic compound Z-2-methyl-5-isopropyl-hexa-2,5-dien-1-al (isonovalal) from &#102 -pinene oxide. Enzyme synthesis is induced by culturing cells on &#102 -pinene; growing bacteria also have the ability to synthesize the epoxide derivative of &#102 -pinene. Isonovalal production was performed without aeration and with concentrated resting cells previously frozen at &#109 20°C, and subsequently thawed in a water-organic solvent, two-phase system. The organic layer was hexadecane, the volume ratio being 1:1. The best results achieved allowed recovery of c.a. 60 g/l organic solvent of isonovalal in 2.5 h operation, which is the most efficient process to date in the area of terpene biotransformations.     

Nitroxide radicals. Controlled release from and transport through biomimetic and hollow fibre membranes

Stable nitroxide radicals have found wide applications in chemistry and biology and they have some potential applications in medicine due to their antioxidant properties. Nitrocellulose filters impregnated with lipid-like substances are used as an imitation of biomembranes and could be used as a controlled drug release vehicle, while experiments with hollow fibres can be useful in the modelling of a drug delivery via blood vessels. This paper describes mechanisms of the nitroxide transport in four different model systems, i.e. a) exit of nitroxide into aqueous solution from porous nitrocellulose filters, impregnated with organic solvents, b) transport of nitroxides through the impregnated membrane from one into another aqueous solution, c) transport of nitroxides from bulk phase of organic solvents through the impregnated membrane into aqueous phase with ascorbic acid, and d) transport of nitroxides from liquid organic phase into aqueous solution through porous hollow fibres. The results are analysed in terms of mass transfer resistance of a membrane, organic and aqueous phase, based on nitroxide diffusion and distribution coefficients. Ascorbic acid reduced nitroxides in water and enhanced the rate of their transfer due to the decrease of transport resistance of unstirred aqueous layers. It is demonstrated that in the case of biomembranes the rate limiting step could be the transport through unstirred aqueous layers and membrane/water interface.     

Nitroxide radicals. Controlled release from and transport through biomimetic and hollow fibre membranes

Stable nitroxide radicals have found wide applications in chemistry and biology and they have some potential applications in medicine due to their antioxidant properties. Nitrocellulose filters impregnated with lipid-like substances are used as an imitation of biomembranes and could be used as a controlled drug release vehicle, while experiments with hollow fibres can be useful in the modelling of a drug delivery via blood vessels. This paper describes mechanisms of the nitroxide transport in four different model systems, i.e. a) exit of nitroxide into aqueous solution from porous nitrocellulose filters, impregnated with organic solvents, b) transport of nitroxides through the impregnated membrane from one into another aqueous solution, c) transport of nitroxides from bulk phase of organic solvents through the impregnated membrane into aqueous phase with ascorbic acid, and d) transport of nitroxides from liquid organic phase into aqueous solution through porous hollow fibres. The results are analysed in terms of mass transfer resistance of a membrane, organic and aqueous phase, based on nitroxide diffusion and distribution coefficients. Ascorbic acid reduced nitroxides in water and enhanced the rate of their transfer due to the decrease of transport resistance of unstirred aqueous layers. It is demonstrated that in the case of biomembranes the rate limiting step could be the transport through unstirred aqueous layers and membrane/water interface.     

The capacity for thermal protection of photosynthetic electron transport varies for different monoterpenes in Quercus ilex

Heat stress resistance of foliar photosynthetic apparatus was investigated in the Mediterranean monoterpene-emitting evergreen sclerophyll species Quercus ilex. Leaf feeding with fosmidomycin, which is a specific inhibitor of the chloroplastic isoprenoid synthesis pathway, essentially stopped monoterpene emission and resulted in the decrease of the optimum temperature of photosynthetic electron transport from approximately 38 degrees C to approximately 30 degrees C. The heat stress resistance was partly restored by fumigation with 4 to 5 nmol mol(-1) air concentrations of monoterpene alpha-pinene but not with fumigations with monoterpene alcohol alpha-terpineol. Analyses of monoterpene physicochemical characteristics demonstrated that alpha-pinene was primarily distributed to leaf gas and lipid phases, while alpha-terpineol was primarily distributed to leaf aqueous phase. Thus, for a common monoterpene uptake rate, alpha-terpineol is less efficient in stabilizing membrane liquid-crystalline structure and as an antioxidant in plant membranes. Furthermore, alpha-terpineol uptake rate (U) strongly decreased with increasing temperature, while the uptake rates of alpha-pinene increased with increasing temperature, providing a further explanation of the lower efficiency of thermal protection by alpha-terpineol. The temperature-dependent decrease of alpha-terpineol uptake was both due to decreases in stomatal conductance, g(w), and increased volatility of alpha-terpineol at higher temperature that decreased the monoterpene diffusion gradient between the ambient air (F(A)) and leaf (F(I); U = g(w)[F(A) - F(I)]). Model analyses suggested that alpha-pinene reacted within the leaf at higher temperatures, possibly within the lipid phase, thereby avoiding the decrease in diffusion gradient, F(A) - F(I). Thus, these data contribute to the hypothesis of the antioxidative protection of leaf membranes during heat stress by monoterpenes. These data further suggest that fumigation with the relatively low atmospheric concentrations of monoterpenes that are occasionally observed during warm windless days in the Mediterranean canopies may significantly improve the heat tolerance of nonemitting vegetation that grows intermixed with emitting species.     

Characterization of monoterpene biotransformation in two pseudomonads

Aims: To study the metabolic profile of Pseudomonas rhodesiae and Pseudomonas fluorescens in water–organic solvent systems using terpene substrates for both growth and biotransformation processes and to determine the aerobic or anaerobic status of these degradation pathways. Materials and Methods: Substrates from pinene (α‐pinene, α‐pinene oxide, β‐pinene, β‐pinene oxide, turpentine) and limonene (limonene, limonene‐1,2‐oxide, orange peel oil) families were tested. For the bioconversion, the terpene‐grown biomass was concentrated and used either as whole cells or as a crude enzymatic extract. Conclusion: Pseudomonas rhodesiae was the most suitable biocatalyst for the production of isonovalal from α‐pinene oxide and did not metabolize limonene. Pseudomonas fluorescens was a more versatile micro‐organism and metabolized limonene in two different ways. The first (anaerobic, cofactor‐independent, noninducible) allowed limonene elimination by synthesizing α‐terpineol. The second (aerobic, cofactor‐dependent) involved limonene‐1,2‐oxide as an intermediate for energy production through a β‐oxidation process. Significance and Impact of the Study: Enzymatic isomerization of β‐ to α‐pinene was described for the first time for both strains. Alpha‐terpineol production by P. fluorescens was very efficient and appeared as a promising alternative for the commercial production of this bioflavour.     

The efficiency of recombinant Escherichia coli as biocatalyst for stereospecific epoxidation

Styrene is efficiently converted into (S)-styrene oxide by growing Escherichia coli expressing the styrene monooxygenase genes styAB of Pseudomonas sp. strain VLB120 in an organic/aqueous emulsion. Now, we investigated factors influencing the epoxidation activity of recombinant E. coli with the aim to improve the process in terms of product concentration and volumetric productivity. The catalytic activity of recombinant E. coli was not stable and decreased with reaction time. Kinetic analyses and the independence of the whole-cell activity on substrate and biocatalyst concentrations indicated that the maximal specific biocatalyst activity was not exploited under process conditions and that substrate mass transfer and enzyme inhibition did not limit bioconversion performance. Elevated styrene oxide concentrations, however, were shown to promote acetic acid formation, membrane permeabilization, and cell lysis, and to reduce growth rate and colony-forming activity. During biotransformations, when cell viability was additionally reduced by styAB overexpression, such effects coincided with decreasing specific epoxidation rates and metabolic activity. This clearly indicated that biocatalyst performance was reduced as a result of product toxicity. The results point to a product toxicity-induced biological energy shortage reducing the biocatalyst activity under process conditions. By reducing exposure time of the biocatalyst to the product and increasing biocatalyst concentrations, volumetric productivities were increased up to 1,800 micromol/min/liter aqueous phase (with an average of 8.4 g/L(aq) x h). This represents the highest productivity reported for oxygenase-based whole-cell biocatalysis involving toxic products.     

Degradation of alpha-pinene oxide and [2H7]-2,5,6-trimethyl-hept-(2E)-enoic acid by Pseudomonas fluorescens NCIMB 11761

When submerged cultured Pseudomonas fluorescens NCIMB 11761 was fed-batch supplemented with alpha-pinene oxide, a rapid formation of 2,6-dimethyl-5-methylene-hept-(2Z)-enal (I) (isonovalal) was observed. Biotransformation and isomerisation of (I) to the (2E)-isomer (II) (novalal) were enhanced by Lewatit OC 1064, a macroporous polystyrene adsorbent. Accelerated isomerisation in the presence of an amino donor (glycine) at pH 7.3 pointed to a merely chemical mechanism. A maximum yield of 48 g of aldehydesl(-1) was achieved, but quantitative analysis of the volatile fraction showed that the molar conversion of the pinene oxide substrate reached no more than 67%. To fill this gap of the mass balance, the acidic fraction was isolated. It contained several compounds which suggested a beta-oxidation-like catabolism starting from 2,6-dimethyl-5-methylene-hept-(2E)-enoic acid (III) (novalic acid). Using [2H7]-2,5,6-dimethyl-hept-(2E)-enoic acid as a conversion substrate and gas chromatography coupled to atomic emission detection and mass spectrometry a degradation pathway via labelled 3,4-dimethylpentenoic and methylpropanoic acids was evidenced. This pathway may play a predominant role in isoprenoid degradation by soil bacteria.     

One-electron reduction of S-nitrosothiols in aqueous medium

One-electron reduction of S-nitrosothiols (RSNO) has been studied using radiolytically produced reducing entity, the hydrated electron (e(aq)(-)), in aqueous medium. Both kinetics of the reaction and the mechanistic aspects of the decomposition of S-nitroso derivatives of glutathione, L-cysteine, N-acetyl-L-cysteine, N-acetyl-D,L-penicillamine, N-acetylcysteamine, L-cysteine methyl ester, and D,L-penicillamine have been investigated at neutral and acidic pH. The second-order rate constants of the reaction of e(aq)(-) with RSNOs were determined using a pulse radiolysis technique and were found to be diffusion controlled (10(10) dm(3) mol(-1) s(-1)) at neutral pH. The product analysis using HPLC, fluorimetry, and MS revealed the formation of thiol and nitric oxide as the major end products. It is therefore proposed that one-electron reduction of RSNO leads to the liberation of NO. There is no intermediacy of a thiyl radical as in the case of oxidation reactions of RSNOs. The radical anion of RSNO (RSN(*)O(-)) is proposed as a possible intermediate. The overall reaction could be written as RSNO + e(aq)(-) --H+--> RSH + (*)NO.     

Detection of cellular responses to toxicants by dielectrophoresis

The dielectrophoretic (DEP) crossover method has been applied to the detection of cell responses to toxicants. Time and dose responses of the human cultured leukemia (HL-60) line were measured for paraquat, styrene oxide (SO), N-nitroso-N-methylurea (NMU) and puromycin. These toxicants were chosen because of their different predominant mechanisms of action, namely membrane free radical attack, simultaneous membrane and nucleic acid attack, nucleic acid alkylation, and protein synthesis inhibition, respectively. For all treatments, the specific membrane capacitance (C(mem)) of the cells decreased while the specific membrane conductance (G(mem)) increased in dose- and time-dependent manners. The DEP responses correlated sensitively with alterations in cell surface morphology, especially folds, microvilli, and blebs, observed by scanning electron microscopy. The DEP method was more sensitive to agents that had a direct action on the membrane than to agents for which membrane alterations were secondary. The responses to paraquat and SO, which directly damaged the cell membrane, could be detected 15 min after exposure, while those for puromycin and NMU, which acted on intracellular targets, could be detected after 30 min. The detection times and dose sensitivity results showed that the DEP method is much faster and more sensitive than conventional cell and higher organism viability testing techniques. The feasibility of producing small instruments for toxicity detection and screening based on cellular dielectric responses is discussed.     

Optimization of oxygen mass transfer in a multiphase bioreactor with perfluorodecalin as a second liquid phase

Oxygenation is an important parameter involved in the design and operation of mixing-sparging bioreactors and it can be analyzed by means of the oxygen mass transfer coefficient (k(L)a). The operational conditions of a stirred, submerged aerated 2-L bioreactor have been optimized by studying the influence of a second liquid phase with higher oxygen affinity (perfluorodecalin or olive oil) in the k(L)a. Using k(L)a measurements, the influence of the following parameters on the oxygen transfer rate was evaluated: the volume of working medium, the type of impellers and their position, the organic phase concentration, the aqueous phase composition, and the concentration of inactive biomass. This study shows that the best experimental conditions were achieved with a perfluorodecalin volume fraction of 0.20, mixing using two Rushton turbines with six vertical blades and in the presence of YPD medium as the aqueous phase, with a k(L)a value of 64.6 h(-1). The addition of 20% of perfluorodecalin in these conditions provided a k(L)a enhancement of 25% when pure water was the aqueous phase and a 230% enhancement when YPD medium was used in comparison to their respective controls (no perfluorodecalin). Furthermore it is shown that the presence of olive oil as a second liquid phase is not beneficial to the oxygen transfer rate enhancement, leading to a decrease in the k(L)a values for all the concentrations studied. It was also observed that the magnitude of the enhancement of the k(L)a values by perfluorodecalin depends on the biomass concentration present.     

Phase transfer and biocatalyst behaviour during biotransformation of beta-ionone in a two-phase liquid system by immobilised Aspergillus niger

The biotransformation of beta-ionone by Aspergillus niger IFO 8541 entrapped in Ca-alginate beads was investigated in a two-phase liquid system, due to the low aqueous solubility of the precursor. Modelling of phase transfer processes of the substrate demonstrated that the solute was transferred from the organic droplets to the gas, giving a loss by stripping, and then from the gas to the aqueous solution where a chemical degradation occurred. The biological reaction took place after direct precursor transfer from the organic layer to the biocatalyst by surface adsorption. Studies on the biological process demonstrated the critical effect of the biomass content in the medium at the time at which beta-ionone was added. Optimum conditions involved fed-batch feedings of both precursor and carbon source (sucrose) after the biomass concentration reached a value close to 6.8g/l. The biotransformation process then took place at a constant rate of 0.046mmol/lh with a reaction yield, defined with respect to beta-ionone metabolised by the fungus, close to unity. Best results achieved in this study allowed to obtain 3.5g/l biological compounds after 400h reaction.