M.M.R. da Fonseca

Effect of cultivation parameters on the production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) and poly(3-hydroxybutyrate-4-hydroxybutyrate-3-hydroxyvalerate) by Cupriavidus necator using waste glycerol.

Short-chain polyhydroxyalkanoate co-polymers (poly(3-hydroxybutyrate-co-4-hydroxybutyrate)) (P(3HB-co-4HB)) and terpolymers (poly(3-hydroxybutyrate-4-hydroxybutyrate-3-hydroxyvalerate)) (P(3HB-4HB-3HV)) were produced using high-cell density fed-batch cultures of Cupriavidus necator DSM 545. C-source for growth and 3HB synthesis was waste glycerol (GRP) from a biodiesel plant. Incorporation of 4HB monomers was promoted by γ-butyrolactone (GBL). Propionic acid (PA), a stimulator of 4HB accumulation, increased the 4HB molar ratio 2-fold, but also acted as 3HV precursor, yielding P(3HB-4HB-3HV). Dissolved oxygen (DOC) was a key parameter for % PHA accumulation and volumetric productivity (Prod(vol)). 4HB molar ratio increased in the presence of PA and with extended accumulation time. By manipulating DOC and cultivation time, P(3HB-4HB) with between 11.4 and 21.5 molar% of 4HB were attained. Similarly, P(3HB-4HB-3HV) was obtained with 4HB molar% between 24.8% and 43.6% and 3HV% from 5.6% to 9.8%. Mw varied between 5.5 × 10(5) and 1.37 × 10(6)Da. PHA production from GRP helps reducing production costs with concomitant GRP valorization.

Contribution of response surface design to the development of glycerolysis systems catalyzed by commercial immobilized lipases

Abstract Two commercial immobilized lipases (“Lipozyme® IM” and “Novozym® 435”) were tested as biocatalysts for the glycerolysis of olive residue oil in n -hexane aimed at the production of monoglycerides (MG) and diglycerides (DG). A central composite rotatable design (CCRD) was followed to model and optimize glycerolysis as a function of both the amount of biocatalyst ( L ) and of the molar ratio glycerol/triglycerides (Gly/TG). For both biocatalysts, the production of free fatty acids (FFA) was described by second order models. In terms of MG and DG production, as well as of TG conversion, the best fits were obtained with first-order models. The highest MG productions were in the range 43–45% (w/w, on the basis of total fat) for both biocatalysts tested at a (Gly/TG) ratio of one. In the case of “Novozym 435”, the lowest load used (12%, w/w) gave the best results, in contrast with “Lipozyme IM” with which a concentration of about 26% (w/w) was necessary to obtain the highest production. Under these conditions, the amount of FFA produced was about 2% and 10% (w/w), respectively, for “Novozym 435” and “Lipozyme IM” catalyzed systems. Considering both FFA production and lipase loading, “Novozym 435” was shown to be a better biocatalyst for the glycerolysis of olive residue oil in n -hexane, aimed at the production of MG, than “Lipozyme IM”.

Response surface modelling of the production of ω-3 polyunsaturated fatty acids-enriched fats by a commercial immobilized lipase

Abstract The aim of this study was to model the production of fats, enriched with ω-3 polyunsaturated fatty acids (ω-3 PUFA) for nutraceutical purposes, via the response surface methodology. These fats were obtained by transesterification of palm oil stearin (POS) with a concentrate (EPAX 2050TG) of triglycerides enriched with ω-3 PUFA and soybean oil, catalysed by a commercial immobilized Candida antarctica lipase (“Novozym 435”). The initial water activity ( a w ) of the biocatalyst, POS and EPAX 2050TG concentrations, time and temperature showed a significant effect on the transesterification reaction, as well as on the competing reactions of hydrolysis and lipid oxidation. Depending on the factors included, the transesterification reaction was described either by first- or second-order models. The production of free fatty acids, which is ascribed both to the hydrolytic reaction and the mechanism of lipase-catalysed transesterification, showed a second-order dependence on the initial a w of the biocatalyst.

Production of monoglycerides by glycerolysis of olive oil with immobilized lipases: effect of the water activity

The production of monoglycerides by glycerolysis of olive oil catalyzed by lipases from Candida rugosa, Chromobacterium viscosum and Rhizopus sp. immobilized in a hydrophylic polyurethane foam was investigated. The effect of the amount of aqueous phase used for foam polymerization on the competing reactions of glycerolysis and hydrolysis was studied. The highest monoglyceride production was achieved with the C. rugosa lipase which was thus selected for subsequent studies.The extent to which hydrolysis and glycerolysis occur and the influence of the initial aw of the system on both reactions were also investigated. In glycerolytic reaction systems, initial rates of fatty acid release were always higher than in hydrolytic systems. At aw values lower than 0.43, hydrolysis was completely repressed, although glycerolysis still occurred. This suggests that hydrolysis of the ester bond in the glyceride, promoted by glycerol, is the first reaction step.In glycerolysis, initial rates of FFA and DG production increased exponentially with the initial aw of the system.The lowest total conversion (in terms of % TG consumed) at 48 hours was obtained at intermediate aw values; higher conversions at extreme aw indicated an increase in hydrolytic and glycerolytic rates, at high and low aw respectively.The yield of MG increased with decreasing aw. The highest yield of MG (∼70%, w/w) was obtained at the lowest aw used (0.23). The initial aw of the reaction system is an important parameter in glycerolysis.

Solvent selection for the biotransformation of terpenes by Pseudomonas putida

Abstract A two-phase reaction system for the biotransformation of terpenes was implemented in order to protect the microorganism from the toxic effects of substrates, intermediates and products and to prevent the oxidation of the compounds by oxygen and/or water. Preliminary studies were carried out with a Pseudomonas putida wild strain. Growth of the wild strain was not affected by the presence of 0.5% (v/v) α-pinene. However, the presence of α-pinene oxide in concentrations above 0.2% (v/v) had a progressive inhibitory effect on growth (complete inhibition occurred at 0.5%). Since substrate and/or product toxicity can often be overcome by the use of an organic solvent reservoir, we searched for a biocompatible solvent. Solvents with a log P close to 4.5 were the most effective in minimising α-pinene oxide toxicity, especially 1-dodecanol, as judged from growth and oxygen uptake experiments. Although the growth rate was 25% lower in the presence of 2% (v/total volume) of organic phase, growth and oxygen consumption rates were not further affected by the introduction of 0.5% (v/total volume) of α-pinene oxide in the solvent phase. The substrate α-pinene itself (log P 4.9) was tested as a solvent reservoir for α-pinene oxide and found effective.

Glycerolysis of olive oil: batch operational stability of Candida rugosa lipase immobilized in hydrophilic polyurethane foams

The operational stability of the Candida rugosa lipase immobilized in a hydrophilic polyurethane foam was evaluated in consecutive batches for the glycerolysis of olive oil in n-hexane, aimed at the production of monoglycerides.Glycerol controlled the glycerolysis in the system under study, since it is both a substrate and a powerful water binder that reduces the water activity of the reaction medium and of the microenvironment. Two sets of experiments were carried out under different glycerol/triglyceride ratios. After 345 hours of consecutive 23 hours batches no lipase inactivation was observed.

Modelling the microenvironment of a lipase immobilized in polyurethane foams

The effects of partition of substrates and product on the modelling of the microenvironment of an immobilized lipase were evaluated using Response Surface Methodology. The esterification of butyric acid with ethanol in n-hexane, catalyzed by Candida rugosa lipase immobilized in two biocompatible and relatively hydrophilic polyurethane foams (“Hypol FHP 2002™” and “Hypol FHP 5000™”) was used as the model system. For each set of initial conditions, the final concentration of substrates and ethyl butyrate in the microenvironment, at equilibrium, Cmicro, were estimated by mass balancing bulk and foams. The Cmicro values obtained were used to estimate the corresponding partition coefficients of ethanol (PEtOH), butyric acid (PBA) and ester (PEB), between the foams (microenvironment) and the bulk medium. Foams containing previously inactivated lipase, as well as lipase-free foams were used. For both substrates, Cmicro values were, in the majority of the experiments, higher than their macroenvironmental counterparts. The lowest Cmicro values were observed with the less hydrophilic foam (“FHP 5000”). A decrease of CmicroEtOH in both foams and CmicroBA in “FHP 5000” foams, was obtained upon lipase immobilization. PEB values were, in all cases, close to zero. This is beneficial in terms of the shift in reaction equilibrium, product recovery and alleviation of product inhibition effects.

Adsorption studies for the separation ofl-tryptophan froml-serine and indole in a bioconversion medium

Abstractl-tryptophan was produced froml-serine and indole by immobilized Escherichia coli cells in organic-aqueous systems. Selective adsorption was the method chosen to enable both product separation andl-serine reutilization. Amongst various adsorbents tested activated carbons and neutral polymeric resins (XAD-4 and XAD-7) showed good performance. The neutral resins could selectively concentrate thel-tryptophan from dilute aqueous solutions and adsorbed only 5% of the unconvertedl-serine. High separation factors (l-tryptophan/l-serine and indole/l-tryptophan) were obtained with these adsorbents. Despite a lower capacity, the XAD-7 resin had the advantage of desorbingl-tryptophan with basic or acidic solutions, while organic solvents were required to desorb, at the same concentration levels, this compound from XAD-4.In a packed bed column filled with XAD-4 resin or activated carbon, totall-tryptophan adsorption and recovery were achieved at linear velocities up to 5.0 cm/min and 3.2 cm/min respectively. Successive sorbent reutilization, following continuous sorption and elution steps, was carried out in packed bed columns with the neutral resins and activated carbon.Thel-form of tryptophan, after crystallization, was identified by HPTLC.

The Effect of Substrate Hydrophobicity on the Kinetic Behaviour of Immobilized Candida rugosa Lipase

Candida rugosa lipase (EC 3.1.1.3.) was immobilized in a hydrophilic polyurethane foam and used in the hydrolysis of olive oil, in H-hexane. The results obtained were compared with those from a previous study, in which the same lipase preparation was used in the esterification of ethanol with butyric acid.The initial rate of hydrolysis increased exponentially with increasing olive oil concentration. In contrast, for the esterification reaction, Michaelis-Menten kinetics with inhibition by both substrates, had been observed.The effect of medium viscosity, stirring conditions and size of immobilization particles could not explain the observed kinetics of the hydrolytic reaction. However, a direct relationship was observed between the log P values of the reaction medium and the initial rate of hydrolysis, i.e., activation of the immobilized Candida rugosa lipase appears to be promoted by a high hydrophobicity of the reaction medium.In the case of the esterification reaction, no similar correlation was found.

Diffusion in cell-free and cell immobilising κ-carrageenan gel beads with and without chemical reaction

Diffusion into and from kappa-carrageenan gel beads was studied, both in the absence and presence of bacterial cells, both with and without biochemical reaction. The solutes were indole, L-serine, and L-tryptophan. The reaction was that of indole and L-serine to give L-tryptophan. Established theory concerning diffusion of a single solute in cell-free gels was found to describe well the effect of the gel on diffusivity. Simultaneous diffusion of the three solutes resulted in lower diffusivities than those for individual solutes, suggesting the need to use multicomponent diffusion theory. The effect of cells on diffusion could only be accounted for by models assuming permeable cells. Diffusion with chemical reaction was reasonably well described by an effectiveness factor calculated using an effective diffusivity estimated from diffusion data without reaction. Copyright 1999 John Wiley & Sons, Inc.