Response surface modeling of glycerolysis catalyzed by Candida rugosa lipase immobilized in different polyurethane foams for the production of partial glycerides

Monoglycerides (MG) and diglycerides (DG) are the most widely used emulsifiers in food and pharmaceutical industries. In this study, MG and DG were produced by inter-esterification of refined olive residue oil with glycerol (glycerolysis), in n-hexane, catalyzed by Candida rugosa lipase immobilized in different biocompatible hydrophilic polyurethane foams, A and B. These foams, with aquaphilicities of 3.7 and 2.8, were prepared with a toluene diisocyanate (“Hypol FHP 2002™”) and a diphenylmethane diisocyanate (“Hypol FHP X4300^TM”) pre-polymer, respectively.Response surface methodology was used for modeling the reaction, as a function of the molar ratio glycerol/triglycerides (Gly/TG, 0.5–2.0) and the initial water activity (a_w) of the biocatalyst (A, 0.24–0.91; B, 0.37–0.91). Experiments were carried out following a central composite rotatable design. With lipase in foam A, production of MG and DG could be described by first order polynomials. With foam B, MG and DG production could be fitted to concave and flat surfaces, described by a second and a first orders polynomials, respectively.The best productions of MG and DG were achieved with the lipase in the less hydrophilic foam, B: at 24 h reaction time, 32% (w/w) MG and 18% (w/w) DG were obtained, when the initial a_w of the biocatalyst was 0.83, with a Gly/TG of 1.     

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 (P_EtOH), butyric acid (P_BA) and ester (P_EB), 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 Cmicro_EtOH in both foams and Cmicro_BA in “FHP 5000” foams, was obtained upon lipase immobilization. P_EB 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.     

Production of manganese peroxidase and laccase in laboratory-scale bioreactors by Phanerochaete chrysosporium

The production of ligninolytic enzymes by the fungus Phanerochaete chrysosporium BKM-F-1767 (ATCC 24725) in laboratory-scale bioreactors was studied. One bioreactor was filled with cubes of polyurethane foam and the other with cubes of nylon sponge, in order to determine the more suitable carrier to produce high ligninolytic enzyme activities by this fungus. Both cultivations were carried out in batch. Manganese-dependent peroxidase activities about 600 U l^у were achieved in the bioreactor filled with cubes of nylon sponge, while up to 500 U l^у were detected in that filled with cubes of polyurethane foam. Furthermore, quite high levels of laccase appeared in both cultures: maximum activities of 114 U l^у and 62 U l^у were obtained on nylon and polyurethane supports, respectively.     

Lipase production by immobilized Candida rugosa cells

Summary Immobilization of Candida rugosa cells on a solid support for extracellular lipase production has been explored. The use of Ca-alginate beads and of mixed matrix of polyurethane foam/Ca-alginate beads enabled us to operate a batch and a continuous four-phase fluidized bed bioreactor. Cells co-entrapped together with polyurethane into Ca-alginate did not show higher lipase production levels than the cells entrapped in Ca-alginate gels. The addition of gum arabic to the medium greatly enhanced lipase production without affecting the hydrodynamic operating conditions significantly. This fact demonstrates that the reactor system is limited in terms of organic substrate dispersion and direct contact with cells. Correspondence to: C. Solà     

Ferrous iron oxidation by foam immobilized Acidithiobacillus ferrooxidans: Experiments and modeling

Ferrous iron bio‐oxidation by Acidithiobacillus ferrooxidans immobilized on polyurethane foam was investigated. Cells were immobilized on foams by placing them in a growth environment and fully bacterially activated polyurethane foams (BAPUFs) were prepared by serial subculturing in batches with partially bacterially activated foam (pBAPUFs). The dependence of foam density on cell immobilization process, the effect of pH and BAPUF loading on ferrous oxidation were studied to choose operating parameters for continuous operations. With an objective to have high cell densities both in foam and the liquid phase, pretreated foams of density 50 kg/m³ as cell support and ferrous oxidation at pH 1.5 to moderate the ferric precipitation were preferred. A novel basket‐type bioreactor for continuous ferrous iron oxidation, which features a multiple effect of stirred tank in combination with recirculation, was designed and operated. The results were compared with that of a free cell and a sheet‐type foam immobilized reactors. A fivefold increase in ferric iron productivity at 33.02 g/h/L of free volume in foam was achieved using basket‐type bioreactor when compared to a free cell continuous system. A mathematical model for ferrous iron oxidation by Acidithiobacillus ferrooxidans cells immobilized on polyurethane foam was developed with cell growth in foam accounted by an effectiveness factor. The basic parameters of simulation were estimated using the experimental data on free cell growth as well as from cell attachment to foam under nongrowing conditions. The model predicted the phase of both oxidation of ferrous in shake flasks by pBAPUFs as well as by fully activated BAPUFs for different cell loadings in foam. Model for stirred tank basket bioreactor predicted within 5% both transient and steady state of the experiments closely for the simulated dilution rates. Bio‐oxidation at high Fe²⁺ concentrations were simulated with experiments when substrate and product inhibition coefficients were factored into cell growth kinetics. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Photosynthesis,growth and hydrocarbon production ofBotryococcus braunii immobilized by entrapment and adsorption in polyurethane foams

Summary Direct entrapment of the hydrocarbonrich algaBotryococcus braunii was examined using eleven polyurethane prepolymers. A high toxicity was observed in several foams. With five of the tested prepolymers, nevertheless, a large part of the algal population can survive entrapment and substantial photosynthetic capacity, ca. 40–60% relative to free controls, was retained one day after immobilization. However, prolonged batches under standard conditions revealed a long-term toxicity; as a result the photosynthetic capacity and hydrocarbon production of the entrapped cultures were strongly reduced relative to free controls. Immobilization ofB. braunii was also achieved, with a loading yield of ca. 70%, via adsorption on FHP 4000 and FHP 5000 foams. Subsequent batch cultures under shaken and airlift conditions revealed a substantial release, ca. 30% of free cells, at the end of the cultures. However, the release from these adsorbed cultures was no higher than from directly entrappedB. braunii. Furthermore, no toxic effects were noted in the adsorbed cultures; the showed active growth, high photosynthetic capacity and produced quite large amounts of hydrocarbons, the chemical structure and the relative abundance of which were not altered by immobilization. Taking into account cell leakage, it appears that adsorbed cultures exhibit a similar, and sometimes even higher metabolic activity than free controls; thus, under air-lift conditions, high biomass and hydrocarbon productivities can be achieved.     

ON THE GUSTATORY EFFECTS OF GYMNEMIC ACID AND MIRACULIN IN DOG, PIG AND RABBIT

The effects of gymnemic acid, 1 mg/ml, and miraculin, 1 mg/mlor in a tablet (Miralin^TM) on the taste response to sucrose,citric acid, NaCl and quinine have been studied in dog, pigand rabbit. In the dog gymnemic acid suppressed the responseto sucrose for about 5 min in two out of three animals. It alsoaffected the response to NaCl. Miraculin caused a slight short-lastingincrease in the response to acid. No effects of the taste modifierswere observed in the pig. In the rabbit gymnemic acid did notsuppress the response to sucrose while miraculin enhanced theresponse to citric acid in one out of three animals for a fewminutes.     

Improvement of lipase activity by synergistic immobilization on polyurethane and its application for large-scale synthesizing vitamin A palmitate

Abstract

We have developed an improved and effective method to immobilize lipase on hydrophobic polyurethane foam (PUF) with different modifications. PUF was treated with hydrochloric acid to increase the active sites and then the active carboxyl groups and amino groups were exposed. Enzyme activity of lipase immobilized on PUF-HCL (8000?U/g) was 50% higher than that of lipase immobilized on PUF (5300?U/g). There is an increase in the activity of the immobilized lipase on AA/PEI-modified support (115,000?U/g), a 2.17-fold increase compared to lipase immobilized on the native support was observed. The activity of immobilized lipases was dependent on the PEI molecular weight, with best results from enzyme immobilized on PUF-HCL-AA/PEI (MW 70,000?Da, 12,800?U/g)), which was 2.41 times higher compared to that of the same enzyme immobilized on PUF. These results suggest that the activity of immobilized lipase is influenced by the support surface properties, and a moderate support surface micro-environment is crucial for improving enzyme activity. Finally, the immobilized lipase was used for the production of vitamin A palmitate. The immobilized lipase can be reused for up to 18 times with a conversion rate above 90% for 12?h in a 3?L bioreactor.

• Research highlights

• An efficient immobilization protocol on polyurethane foam was developed

• Polyethyleneimine and acetic acid were used to regulate the micro-environment concurrently

• The activity of lipase immobilized on PUF-HCL-AA/PEI was improved by 2.41 times

• Immobilized lipase exhibited excellent operational stability for vitamin A palmitate synthesis

     

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.List of Symbols a_w thermodynamic activity of water - DG diglyceride (s) - FAME fatty acid methyl ester (s) - FFA free fatty acid (s) - FID flame ionization detector Gly glycerol - MG monoglyceride (s) - TG triglyceride (s) - TLC thin layer chromatography The authors are grateful to Profs. P. Adlercreutz, Technical University of Lund, Sweden, and J.M.S. Cabral, Instituto Superior Técnico, Lisbon, Portugal, for inspiring discussions and advice, to Prof. Helena Pereira, Instituto Superior de Agronomia (ISA), Lisbon, Portugal, for the use of GC equipment and to Mrs. Marlene Dionísio, ISA, for invaluable help with some of the experimental work.     

Biosynthesis and degradation of glycerides in external mycelium of Glomus mosseae

The activities of enzymes involved in the glyceride metabolism of Glomus mosseae external mycelium are reported. Total mycelial homogenates were incubated with radiolabeled triolein and palmitic acid for various times under different conditions. The results obtained demonstrate the capacity of G. mosseae external mycelium to synthesize and hydrolyze its own acylglycerides. Neutral lipid biosynthesis progressively increased along with root colonization. Incorporation of [¹⁴C]-palmitate was mainly into triacylglycerols and as a minor fraction into diacylglycerols. The activity of palmitoyl-CoA ligase in external mycelium also increased in parallel with mycorrhiza development. The hydrolysis of triacylglycerols was very low at the beginning of colonization and then increased. However, lipase activity was lower than that of acyl-CoA ligase even at late stages of colonization. Thus, triacylglycerol biosynthesis apparently prevails over degradation during G. mosseae mycelium development in the period examined.     

Structural basis of stereoselectivity in Candida rugosa lipase-catalyzed hydrolysis of secondary alcohols

Lipases are widely used catalysts for highly enantioselective resolution of chiral secondary alcohols. While stereopreference is determined predominantly by the substrate structure, stereoselectivity (enantioselectivity and diastereoselectivity) depends on the atomic details of interactions between substrate and lipase. Experimentally obtained stereoselectivity and activity in the hydrolysis of butanoic acid esters of two secondary alcohols with two neighboring stereocenters by Candida rugosa lipase have been investigated by computer-aided molecular modeling of tetrahedral substrate intermediates in complex with the lipase. Breakdown of these intermediates is considered to be the rate-limiting step. Steric interactions of stereoisomers with the side chain of catalytic histidine led to different orientations of the imidazole. The distance d(H_N)-O_alc) between H_N) of the imidazole side chain of catalytic histidine and the alcohol oxygen of the substrate was identified to correlate with the experimentally determined reactivity order of the four stereoisomers. Modeled distances d(H_N)-O_alc) were short (=1.8 Å) for RR stereoisomers, which were also found to be hydrolyzed most rapidly experimentally; distances d(H_N)-O_alc) were about 2 Å for SS and SR stereoisomers, which were converted at similar rates but at a lower rate than RR stereoisomers; finally, distances d(H_N)-O_alc) for SR stereoisomers were greater than 4 Å, in accordance with very slow conversion of SR stereoisomers.     

A mesocosm study of physical-biological interactions in artificial sea ice: effects of brine channel surface evolution and brine movement on algal biomass

The impact of changing physico-chemical boundary conditions in sea ice on biological processes was investigated during a 20-day-long simulated freeze-melt cycle in an 180-m³ mesocosm filled with artificial seawater and addition of a mixed Arctic sea-ice community. Ice formation started at T_air of -15°C with a growth rate of 0.7-1.2 mm h^-1 for 10 days. The last 10 days (T_air of=-5°C), ice thickness remained around 20 cm. Ice temperature gradients inside the ice were linear and determined brine salinities. Brine was collected by means of centrifugation and its volume ranged from 5 to 30% of total ice volume. Surface areas of interconnected brine channels were determined with two similar techniques and maximum values ranged between 1.5 and 4.8 m² kg^-1ice. Measurements determined with a modified method varied considerably and differed by a maximal factor of 2.0-6.5. Brine channel surfaces increased during the experiment as a result of the warming of the ice. The inoculated algal community was dominated by flagellates <10 µm. The low diatom biomass increased in the ice after the air temperature rise with rates comparable to field data (µ=0.2-0.3 day^-1). Comparison with brine salinities points towards the hypothesis of vertical brine stability being a controlling factor for ice algal growth. We infer from brine channel surface measurements that persistence of brine channel surfaces during spring might be an important prerequisite for the commencement of net diatom biomass accumulation. Advantages and limitations of mesoscale mesocosms as alternatives in ice biological work are discussed.     

<Emphasis Type="Italic">Candida rugosa</Emphasis> lipase-catalyzed polyurethane degradation in aqueous medium

Candida rugosa lipase (EC 3.1.1.3) was used to degrade commercially-available solid poly(ester)urethane (Impranil) in an aqueous medium under different temperature, pH, enzyme and substrate concentrations. A mathematical model was developed and applied to represent the degradation kinetics of the solid polyurethane. Reaction optima were found to be pH 7 and 35°C. Diethylene glycol, a degradation byproduct, generation rate was measured to be 0.12 mg/l min and the activation energy was calculated as 9.121 kcal/gmol K. This information will be useful in developing bioreactors for practical applications to manage polyurethane wastes using lipase.     

Mesozooplankton community structure and grazing impact in the Polar Frontal Zone of the south Indian Ocean during austral autumn 2002

Mesozooplankton community structure and grazing impact were investigated at 13 stations in the Polar Frontal Zone during the second Marion Offshore Variability Ecosystem Study (MOVES II), conducted during April 2002. Total integrated chl- a biomass ranged between 11.17 and 28.34 mg chl- a m^-2 and was always dominated by nano- and picophytoplankton (<20 µm). Throughout the study, small copepods, mainly Oithona similis and Ctenocalanus vanus, numerically dominated the mesozooplankton community, composing up to 85% (range 30-85%) of the total abundance. Grazing activity of the four most abundant copepods ( O. similis, C. vanus, Calanus simillimus and Clausocalanus spp.), constituting up to 93% of total mesozooplankton abundance, was investigated using the gut fluorescence technique. Results of gut fluorescence analyses indicated that Calanus simillimus and Clausocalanus spp. exhibited diel variability in gut pigments with maximum values recorded at night. In contrast, O. similis and Ctenocalanus vanus did not demonstrate diel variation in gut pigment contents. Ingestion rates of the four copepods ranged from 23.23 to 1462,02 ng(pigm.)ind^-1day^-1. The combined grazing impact of the four copepods ranged between 1 and 36% of the phytoplankton standing stock per day, with the highest daily impact occurring at stations occupied in the vicinity of the Antarctic Polar Front (~35.86% at station 23). Among the copepods, O. similis and Ctenocalanus vanus represented the most important consumers of phytoplankton biomass, collectively responsible for up to 89% (range 15-89%) of the total daily grazing impact. Carbon specific ingestion rates of the copepods varied between 42 and 320% body carbon per day.     

Co-immobilization of amyloglucosidase and pullulanase for enhanced starch hydrolysis

Summary Amyloglucosidase and pullulanase were co-immobilized using a hydrophilic polyurethane foam (Hypol^® 2002). The combined amyloglucosidase and pullulanase activity of the immobilized enzyme was 32.2% ± 1.7% relative to the non-immobilized enzyme. The co-immobilized enzymes were capable of using a variety of glycogen and starch substrates. Co-immobilization of amyloglucosidase and pullulanase increased the glucose yield 1.6-fold over immobilized amyloglucosidase alone. No decrease in activity was observed after 4 months storage for the co-immobilized enzymes. The results suggest that co-immobilization of amyloglucosidase and pullulanase in polyurethane foams is a potentially useful approach for commercial starch hydrolysis. Offprint requests to: K. B. Storey     

In situ oxygen microelectrode measurements of bottom-ice algal production in McMurdo Sound, Antarctica

In-situ estimates of fast-ice algal productivity at Cape Evans, McMurdo Sound, in 1999 were lower than at the same site in previous years. Under-ice irradiance was between 0 and 8 µmol photons m^-2 s^-1; the ice was between 1.9 and 2.0 m thick and the algal biomass averaged 150 mg chl a m^-2, although values as high as 378 mg chl a m^-2 were recorded. Production on 11 and 12 November was between 0.053 and 1.474 mg C m^-2 h^-1. When the data from 11 November were fitted to a hyperbolic tangent function, a multilinear regression gave estimates for P_max of 0.571 nmol O₂ cm^-2 s^-1, an ! of 0.167 nmol O₂ cm^-2 s^-1 µmol^-1 photons m^-2 s^-1 and an E_k of 3.419 µmol photons m^-2 s^-1. A P_max of 2.674 nmol O₂ cm^-2 s^-1, an ! of 0.275 nmol O₂ cm^-2 s^-1 µmol^-1 photons m^-2 s^-1, r of 0.305 nmol O₂ cm^-2 s^-1 and an E_k of 9.724 µmol^-1 photons m^-2 s^-1 were estimated from the 12 November data. The sea-ice algal community was principally comprised of Nitzschia stellata, Entomoneis kjellmanii and Berkeleya adeliensis. Other taxa present included N. lecointei, Fragilariopsis spp., Navicula glaciei, Pleurosigma spp. and Amphora spp. Variations in the method for estimating the thickness of the diffusive boundary layer were not found to significantly affect the measurements of oxygen flux. However, the inability to accurately measure fine-scale variations in biomass is thought to contribute to the scatter of the P versus E data.     

Water availability and carbon isotope discrimination in conifers

The stable C isotope composition ('¹³C) of leaf and wood tissue has been used as an index of water availability at both the species and landscape level. However, the generality of this relationship across species has received little attention. We compiled literature data for a range of conifers and examined relationships among landscape and environmental variables (altitude, precipitation, evaporation) and '¹³C. A significant component of the variation in '¹³C was related to altitude (discrimination decreased with altitude in stemwood, 2.53‰ km^-1 altitude, r²=0.49, and in foliage, 1.91‰ km^-1, r²=0.42), as has been noted previously. The decrease in discrimination with altitude was such that the gradient in CO₂ partial pressure into the leaf (P_a-P_i) and altitude were generally unrelated. The ratio of precipitation to evaporation (P/E) explained significant variation in P_a-P_i of stemwood (r²=0.45) and foliage (r²=0.27), but only at low (<0.8) P/E. At greater P/E there was little or no relationship, and other influences on '¹³C probably dominated the effect of water availability. We also examined the relationship between plant drought stress (O) and '¹³C within annual rings of stemwood from Pinus radiata and Pinus pinaster in south-western Australia. Differential thinning and fertiliser application produced large differences in the availability of water, nutrients and light to individual trees. At a density of 750 stems ha^-1, O and '¹³C were less (more negative) than at 250 stems ha^-1 indicating greater drought stress and less efficient water use, contrary to what was expected in light of the general relationship between discrimination and P/E. The greater '¹³C of trees from heavily thinned plots may well be related to an increased interception of radiation by individual trees and greater concentrations of nutrients in foliage - attributes that increase rates of photosynthesis, reduce P_i and increase '¹³C. '¹³C was thus modified to a greater extent by interception of radiation and by nutrient concentrations than by water availability and the '¹³C-O relationship varied between thinning treatments. Within treatments, the relationship between '¹³C and O was strong (0.38<r²<0.58). We conclude that '¹³C may well be a useful indicator of water availability or drought stress, but only in seasonally dry climates (P/E<1) and where variation in other environmental factors can be accounted for.     

Regulation of xylem sap flow in an evergreen, a semi-deciduous, and a deciduous Meliaceae species from the Amazon

The significance of phenological characteristics, stomatal conductance of the leaves, and stem water storage fluctuations for the regulation of xylem sap flow in an evergreen (Carapa guianensis Aubl.), in a semi-deciduous (Swietenia macrophylla King), and in a deciduous (Cedrela odorata L.) Meliaceae species was studied in a 7-year-old plantation near Manaus, Brazil. The study responds to the increasing demand for knowledge on the water relations of highly exploited timber trees of the Amazon. Xylem sap flow measurements indicated that the daily sap flow of Carapa (3.8 l day^-1 tree^-1 to 16.4 l day^-1 tree^-1) exceeded the daily sap flow of Swietenia (2.4 l day^-1 tree^-1 to 7.0 l day^-1 tree^-1) and Cedrela (1.6 l day^-1 tree^-1 to 11.6 l day^-1 tree^-1) during the entire year, although the highest flux densities were measured in Cedrela. The decrease in xylem sap flow observed in periods with low soil water potentials and high atmospheric vapor saturation deficits was more pronounced in the deciduous (Cedrela) and semi-deciduous species (Swietenia) than the evergreen species (Carapa). Carapa, which has the highest daily sap flow, had the highest biomass and sapwood portion. The high flux densities measured in Cedrela most likely result from the large earlywood vessels in this species. The seasonal variation of xylem sap flow of the three species was correlated with the stomatal conductance of the leaves measured by infiltration experiments. Stem water storage fluctuations in Carapa and Swietenia were predominantly due to transpiration; in Cedrela it was predominantly due to evaporative water loss on the stem surface during dry periods.     

Ferrous sulphate oxidation using Thiobacillus ferrooxidans cells immobilised in polyurethane foam support particles

Summary Oxidation of ferrous iron by Thiobacillus ferrooxidans cells passively immobilised in polyurethane foam particles, using both repeated batches and continuous operation, was studied in a laboratory-scale reactor. Repeated batches yielded complete oxidation at higher rates than single batches, providing resident inocula for subsequent batches. In continuous operation maximum ferric iron productivities were achieved at dilution rates well above theoretical washout values. At a dilution rate of 0.31 h^–1 [approximately three times the maximum specific growth rate (_max)], a productivity of 1.56 kg m^–3 h^–1, based on total ferric iron, or 1.0 kg m^–3 h^–1 based on dissolved ferric iron, was achieved. In addition, cells immobilised in the foam particles retained their oxidative ability for periods of up to 6 weeks when stored in the open air and could be reused immediately.