Enzymatic esterification of dihydroxystearic acid

Dihydroxystearic acid (DHSA) ester was synthesized enzymatically to overcome the problems associated with chemical processes. Immobilized enzyme, Lipozyme IM and Novozym 435, were employed as catalysts in the esterification reaction between DHSA and monohydric alcohol. Various factors that may affect the esterification reaction were studied, such as initial water content (a _w ), organic solvent, substrate concentration and the influence of alcohol chain length. It was found that the percent conversion was higher in organic solvents with log P (the logarithm of the partition coefficient of solvent in octanol/water system) from 2.0 to 4.0. The reaction was not affected by a _w from 0.09 to 0.96. Increasing the mole ratio of alcohol to acid above 2.0 did not increase the percent converions of ester. The ester was identified by Fourier transform infrared and ¹³C nuclear magnetic resonance spectroscopy.     

Combined Effect of Agitation/Aeration and Fed‐Batch Strategy on Ubiquin‐ one‐10 Production by Pseudomonas diminuta

The effects of aeration rate and agitation speed on ubiquinone‐10 (CoQ10) submerged fermentation in a stirred‐tank reactor using Pseudomonas diminuta NCIM 2865 were investigated. CoQ10 production, biomass formation, glycerol utilization, and volumetric mass transfer coefficient (k_La) were affected by both aeration and agitation. An agitation speed of 400 rpm and aeration rate of 0.5 vvm supported the maximum production (38.56 mg L^–1) of CoQ10 during batch fermentation. The fermentation run supporting maximum production had an k_La of 27.07 h^–1 with the highest specific productivity and CoQ10 yield of 0.064 mg g^–1h^–1 and 0.96 mg g^–1 glycerol, respectively. Fermentation kinetics performed under optimum aeration and agitation showed the growth‐associated constant (a = 5.067 mg g^–1) to be higher than the nongrowth‐associated constant (β = 0.0242 mg g^–1h^–1). These results were successfully utilized for the development of fed‐batch fermentation, which increased the CoQ10 production from 38.56 mg L^–1 to 42.85 mg L^–1.     

Regioselective acylation of flavonoids catalyzed by lipase in low toxicity media

Flavonoid fatty esters were prepared by acylation of flavonoids (rutin and naringin) by fatty acids (C8, C10, C12), catalyzed by immobilized lipase from Candida antarctica in various solvent systems. The reaction parameters affecting the conversion of the enzymatic process, such as the nature of the organic solvent and acyl donor used, the water activity (a_w) of the system, as well as the acyl donor concentration have been investigated. At optimum reaction conditions, the conversion of flavonoids was 50—60% in tert‐butanol at a_w less than 0.11. In all cases studied, only flavonoid monoester was identified, which indicates that this lipase‐catalyzed esterification is regioselective.     

Enzymatic alcoholysis of triolein to produce wax ester

An alcoholysis reaction between triolein and oleyl alcohol catalyzed by Lipozyme and Novozyme was carried out to produce oleyl oleate, a wax ester. The effects of various reaction parameters such as time, reaction temperature, amount of enzyme, molar ratio of substrates (oleyl alcohol/triolein), various organic solvents used and the initial water activity, a_w of the reaction system were studied. The best conditions tested to produce wax ester were respectively, incubation time, 5 h; temperature, 50 °C for Lipozyme and 60 °C for Novozyme; weight of enzyme, 0.30 g and molar ratio of oleyl alcohol to triolein, 6:1. The use of organic solvents greatly influenced the activity of lipase. Generally, the activity of lipase was high in nonpolar solvents with log P values greater than 2.50. Heptane and hexane were the best solvents tested. The enzymatic synthesis of oleyl oleate was best carried out at a_w 0.32. Analysis of the yield of the products of the reaction at optimized reaction condition using Lipozyme showed that 75.66% oleyl oleate was produced. © 2001 Society of Chemical Industry     

Enantioselective esterification reaction using immobilized Candida rugosa lipase on poly(N‐vinyl‐2‐pyrrolidone‐co‐styrene) hydrogel

Lipase from Candida rugosa was immobilized on poly(N‐vinyl‐2‐pyrrolidone‐co‐styrene) hydrogel (poly‐(VP‐co‐ST)) with ethylene dimethacrylate and α,α'‐azoisobutyronitrile, which act as crosslinker and initiator, respectively. Three different compositions of monomers were used, namely VP(%):ST(%), 10:90, 50:50, and 70:30 (wt(%)/wt(%)). The immobilized lipases were used in the enantioselective esterification of (R,S)‐2‐(4‐chlorophenoxy)‐propanoic acid with n‐tetradecanol. The optimum reaction condition of the enantioselective esterification for the native lipase and the poly(VP‐co‐ST) hydrogel immobilized lipases was determined with respect to temperature, solvents, and initial water activity (a_w). The optimum temperature obtained was 40°C, with the poly(VP‐co‐ST) hydrogel immobilized lipase VP(%)/ST(%):10:90 showing the highest enantiomeric excess. In the solvent effect studies, the best solvents for high enantioselectivity were chloroform and carbon tetrachloride. In the a_w studies, optimum α_w for NL, VP(%):ST(%), 10:90, and 50:50 was 0.328, while for VP(%):ST(%), 70:30, it was 0.55. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 92: 3381–3386, 2004     

In‐line mixing for production of citric acid by Candida lipolytica grown on n‐paraffins

This study reports on the effects of internal fermenter and external in‐line agitation and fed‐batch mode of operation on citric acid production from Candida lipolytica using n‐paraffin as the carbon source. An optimum range of fermenter agitation speeds in the range 800–1000 rpm corresponding to Reynolds numbers of 50433–62947 (based on initial batch conditions) seemed to give the best balance between substrate utilization for biomass growth and citric acid production. Proof of concept evidence is presented that indicates that an external in‐line agitator could be used in place of high speed internal agitation to increase citric acid production. However, more work is required to optimize the external agitator concept. Application of multiple fed‐batch feedings can be used to extend the batch fermentation and increase final citric acid concentrations and product yield. Experiments were conducted implementing a three‐cycle fed‐batch process which increased overall citric acid yields to 0.8–1.0 g citric acid g^?1 n‐paraffin, approximately 200% improvement from those found in the normal batch process. The three‐cycle fed‐batch mode of operation also increased the final citric acid concentration to 42 g dm^?3 from about 6 g dm^?3 for normal batch operation. Increased citric acid concentrations in three‐cycle fed‐batch mode was achieved at longer fermentation times. Copyright © 2004 Society of Chemical Industry     

Optimization of pullulan production from synthetic medium by Aureobasidium pullulans in a stirred tank reactor by response surface methodology

The production of pullulan from synthetic medium by Aureobasidium pullulans P56 in a stirred tank fermenter was investigated. The kinetics of polysaccharide, pullulan and biomass production was determined. Response surface methodology was used to investigate the effects of three factors (initial sugar concentration, aeration rate and agitation speed) on the concentration of pullulan in batch cultures of A pullulans. In the experiments, the range of values used for the three variables described were; 30–70 g dm^?3 initial sugar concentration, 200–600 rpm agitation speed and 1.0–3.0 vvm aeration rate. No previous work has used statistical analysis in determining the interactions among these variables in pullulan production. Results of the statistical analysis showed that the fit of the model was good in all cases. Aeration rate, agitation speed and sugar concentration had a strong linear effect on pullulan concentration. Moreover, pullulan concentration was significantly influenced by the negative quadratic effects of the given variables and by their positive or negative interactions with the exception that the interaction between agitation speed and aeration rate was insignificant (P > 0.05). Maximum pullulan concentration of 17.2 g dm^?3 was obtained at the optimum levels of process variables (initial sugar concentration 51.4 g dm^?3, aeration rate 2.36 vvm, agitation speed 345.3 rpm). These values were obtained by fitting of the experimental data to the model equation. Scanning electron microscope (SEM) photographs of polysaccharide particles containing different concentrations of pullulan were also taken to observe the morphological differences of the samples. Copyright © 2005 Society of Chemical Industry     

Measurement of the overall volumetric oxygen transfer coefficient in alkane‐aqueous dispersions

BACKGROUND: A reliable procedure for the determination of the volumetric oxygen transfer coefficient (K_La) in hydrocarbon–aqueous dispersions is critical to the assessment of oxygen transfer in hydrocarbon‐based bioprocesses. The pressure step procedure (PSP) and gassing out procedure (GOP) for K_La determination were evaluated in n‐C_10–13 alkane–aqueous dispersions over agitation rates (600 to 1200 rpm) and alkane concentrations (up to 20%) typical of hydrocarbon‐based bioprocesses. RESULTS: K_La from the procedures diverged progressively as agitation increased, with K_La underprediction attenuated at high alkane. At 1200 rpm and 5% alkane, K_La from the GOP was underpredicted by 49%. Compared with K_La from the PSP, K_La from the PSP without nitrogen transport was the same, but K_La from the PSP without response lag (PSP‐lag) was underpredicted by approximately 50% at 1200 rpm. Moreover, K_La from the PSP‐lag was lower than K_La from the GOP, except at 1200 rpm and alkane concentration up to 5%. CONCLUSION: The PSP was superior to the GOP in alkane–aqueous dispersions, particularly at extreme agitation and low alkane concentrations. This was attributable to the probe response lag and the non‐uniformity of gas composition inherent in the GOP and independent of the rate of coincident nitrogen transport. Copyright © 2009 Society of Chemical Industry     

The effects of emulsified polydimethylsiloxane FG‐10 on the oxygen transfer coefficient (kLa) and lipase production by Staphylococcus warneri EX17

BACKGROUND: In this study the effects of the addition of emulsified polydimethylsiloxane (PMDS) FG‐10 on the oxygen transfer coefficient (k_La) of submerged cultures of Staphylococcus warneri EX17 and its lipase production is described. FG‐10 is an emulsified silicone capable of dissolving 50 times more oxygen than water. The combined effects of FG‐10 concentration and different conditions of agitation were optimized in bioreactors using statistical design tools, and the cultures were run using raw glycerol from biodiesel synthesis as the sole carbon source. RESULTS: The optimal conditions found to improve lipase production were FG‐10 concentration of 11.2% (v/v) and speed agitation of 527 rpm, respectively, producing around 861 U L^?1 of lipolytic activity, a maximal cell concentration of 8.4 g L^?1, and a k_La of 99 h^?1, values that are approximately 3 times higher than cultures without FG‐10. CONCLUSIONS: This is the first report in the literature on the use of this class of chemicals as oxygen carriers in microbial cultures and its effect on k_La and lipase production, demonstrating the potential use of FG‐10 in microbial cultures. Copyright © 2012 Society of Chemical Industry     

Aeration and mixing in vortex fermenters

The overall apparent volumetric gas—liquid mass transfer coefficient (k₉₅a) and the mixing time (t₉₅) were determined in a 240 dm³ vortex aerated fermenter over stirrer speed and air flow ranges of 300–800 rpm and 10–45 normal dm³ min^?1, respectively. The mass transfer data obtained in an aqueous salt solution (2.5 kg m^?3 NaCl in water) compared well with the measurements in a fermentation medium used in culture of certain microaerophilic bacteria. Over the ranges examined, the gas-liquid mass transfer coefficient depended only on air flow rate; the dependence was linear with flow. Mixing time declined with increasing agitation according to a power-law relationship. The mixing and mass transfer characteristics of the vortex aerated system were compared with that of a ‘standard’ stirred tank fermenter (27 dm³). The mixing time variations with respect to agitation rate were remarkably similar for the two types of fermenters examined.     

Polybenzimidazole (PBI) molecular weight and Mark‐Houwink equation

The molecular weight, and intrinsic viscosity of polybenzimidazole (PBI) and its phosphonylated derivatives are reported. The relationship between intrinsic viscosity [η] and weight average molecular weight (M_w) for PBI has been established in H₂SO₄ and DMF‐LiCl. The Mark Houwink constants K_w of 5.2 × 10^?3 mL/g, α of 0.92 for H₂SO₄ solvent systems and, K_w of 3.2 × 10^?2 mL/g, α of 0.754 for DMF‐LiCl solvent system have been determined at M_w < 65,000. The intrinsic viscosity of PBI determined by the Huggins–Kraemer method was compared with a single point method, and found that both methods fit well for PBI in relatively low concentration solvent system, giving ～ 99% accuracy. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Study of the bio‐production of carotenoids by Sporidiobolus salmonicolor (CBS 2636) using pre‐treated agro‐industrial substrates

BACKGROUND: The increasing industrial demand for carotenoids has aroused interest in their bio‐production, and the need to reduce production costs has encouraged the use of low cost industrial substrates, such as agro‐industrial residues. Thus the objective of this research was the bio‐production of carotenoids by Sporidiobolus salmonicolor using agro‐industrial substrates (corn steep liquor and sugarcane molasses), pre‐treated with acids (sulphuric and phosphoric). RESULTS: Bio‐production was carried out in an orbital shaker using a 10% (v/v) inoculum, incubation at 25 °C, and agitation at 180 rpm for 120 h in a non‐illuminated environment. The carotenoids were recovered using liquid N₂ combined with dimethylsulphoxide for cell rupture, and an acetone/methanol mixture (7:3 v/v) for extraction. CONCLUSION: The complete second‐order design allowed for optimisation of the carotenoid concentration obtained from industrial substrates pre‐treated with acids (sulphuric and phosphoric), obtaining a total carotenoid content of 541.5 µg L^?1 using 10 g L^?1 sugarcane molasses, 5 g L^?1 corn steep liquor and 5 g L^?1 yeast hydrolysate at 25 °C, with agitation at 180 rpm and an initial pH of 4.0. Copyright © 2008 Society of Chemical Industry     

Synthesis of star‐shaped poly(ϵ‐caprolactone) by samarium‐based tetrafunctional initiator and its dilute‐solution properties

An in situ–generated tetrafunctional samarium enolate from the reduction of 1,1,1,1‐tetra(2‐bromoisobutyryloxymethyl)methane with divalent samarium complexes [Sm(PPh₂)₂ and SmI₂] in tetrahydrofuran has proven to initiate the ring‐opening polymerization of ?‐caprolactone (CL) giving star‐shaped aliphatic polyesters. The polymerization proceeded with quantitative conversions at room temperature in 2 h and exhibited good controllability of the molecular weight of polymer. The resulting four‐armed poly(?‐caprolactone) (PCL) was fractionated, and the dilute‐solution properties of the fractions were studied in tetrahydrofuran and toluene at 30°C. The Mark–Houwink relations for these solvents were [η] = 2.73 × 10^?2M_w^0.74 and [η] = 1.97 × 10^?2M_w^0.75, respectively. In addition, the unperturbed dimensions of the star‐shaped PCL systems were also evaluated, and a significant solvent effect was observed. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 102: 175–182, 2006     

Lipase/acyltransferase‐catalysed interesterification of fat blends containing n‐3 polyunsaturated fatty acids

The lipase/acyltransferase from Candida parapsilosis is an original biocatalyst that preferentially catalyses alcoholysis over hydrolysis in biphasic aqueous/organic media. In this study, the performance of the immobilised biocatalyst in the interesterification in solvent‐free media of fat blends rich in n‐3 polyunsaturated fatty acids (n‐3 PUFA) was investigated. The interesterification activity of this biocatalyst at a water activity (a_w) of 0.97 was similar to that of commercial immobilised lipases at a_w values lower than 0.5. Thus, the biocatalyst was further used at an a_w of 0.97. Response surface modelling of interesterification was carried out as a function of medium formulation, reaction temperature (55–75 °C) and time (30–120 min). Reaction media were blends of palm stearin (PS), palm kernel oil and triacylglycerols (TAG) rich in n‐3 PUFA (“EPAX 4510TG”; EPAX AS, Norway). The best results in terms of decrease in solid fat content were observed for longer reaction time (>80 min), lower temperature (55–65 °C), higher “EPAX 4510TG” content and lower PS concentration. Reactions at higher temperature led to final interesterified fat blends with lower free fatty acid contents. TAG with high equivalent carbon number (ECN) were consumed while acylglycerols of lower ECN were produced.     

Comparison of lipase production by Geotrichum candidum in stirring and airlift fermenters

The production of lipase by Geotrichum candidum in both, stirred tank and airlift bioreactors were compared. G candidum an imperfect filamentous fungus, grows well in liquid medium, and produces a lipase with specific affinity for long‐chain fatty acids with cis‐9 double bonds but, lipase production is generally not efficient because the optimum medium composition and fermentation conditions are not known. Response surface methodology was used to optimize the agitation speed (100–500 rpm) and aeration (0.2–1.8 vvm) for production of lipase by G candidum in a bench‐scale stirred fermenter. A Central Composite Rotatable Design (CCRD) was used to optimize lipase activity and productivity. Lipase production in an airlift fermenter was also studied with aeration ranging from 1 to 3 vvm. A previously optimized culture medium containing 3.58% of peptone, 0.64% of soy oil and an initial pH of 7.0, was used in the experiments, incubating at 30°C. In the stirred reactor the optimum conditions of agitation and aeration for lipase production and productivity were 300 rpm and 1 vvm, leading to an activity of 20 U cm^?3 in 54 h of fermentation and 0.3900 (U cm^?3 h^?1) of productivity. The best aeration condition in the airlift fermenter was 2.5 vvm, which yielded similar lipase activity after 30 h of fermentation, resulting in a productivity of 0.6423 (U cm^?3 h^?1). In the absence of mechanical agitation similar lipase yields were achieved but in less time, resulting in productivity, about 60% greater than in a stirred fermenter; the lower energy demand for the same lipase yield offers economic advantages. Copyright © 2004 Society of Chemical Industry     

Electrochemical measurements for studying the polymerization process and critical point behaviors of hydrogels

We propose a new technique based on electrochemical measurements for studying the critical point behaviors of the sol–gel transition of acrylamide–N,N′‐methylene bisacrylamide hydrogels. In this technique, no chemical activator is used for accelerating the polymerization reaction. However, a potential difference is applied by means of silver and calomel electrodes placed in the reaction mixture. The silver electrode begins to be ionized and loses its electrons. The free radicals, ^?O₃S? O^?, H^?, and ^?OH, form on the silver electrode via persulfate dissociation. The polymerization is initiated by means of these free radicals. The current measured during the gelation processes passes through a maximum (a Gaussian‐like behavior) and varies linearly with the reaction time during linear polymerization. All the parameters (the monomer, initiator, and crosslinker concentrations, the applied voltage, and the stirring rate of the reacting mixture) affecting the current have been studied in detail. We show that the maxima appearing in the current–time plots correspond to the gelation thresholds, the so‐called sol–gel transition points. We also analytically prove that the current monitors the weight‐average degree of polymerization (DP_w) and the gel fraction (G) below and above the threshold, respectively. The scaling behaviors of DP_w and G have been tested near the gelation thresholds, and we have observed that the critical exponents γ and β, defined for DP_w and G, agree with the predictions by mean‐field theory. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Optimization of the production of chitosan from beet molasses by response surface methodology

Chitosan was produced by Rhizopus oryzae 00.4367 in shake flask culture and a stirred tank fermenter. Synthetic medium, treated and untreated beet molasses were used as cultivation media in shake flask cultures. In the stirred tank fermenter, the cultivation media were synthetic medium and untreated beet molasses. Shake flask culture containing untreated molasses with a sugar concentration of 40 g dm^?3 produced the maximum chitosan yield (961 mg dm^?3). Chitosan concentration reached its maximum value at the late exponential growth phase of R oryzae. In all experiments almost 8–10% of biomass and 32–38% of alkali‐insoluble material was extracted as chitosan. A central composite design was employed to determine the optimum values of process variables (aeration rate, agitation speed and initial sugar concentration) leading to maximum chitosan concentration in the stirred tank fermenter. In all cases, the fit of the model was found to be good. Aeration rate, agitation speed and initial sugar concentration had a strong linear effect on chitosan concentration. Moreover, the concentration of chitosan was significantly influenced by the negative quadratic effects of the given variables and by their positive or negative interactions. A maximum chitosan concentration of 1109.32 mg dm^?3 was obtained in untreated molasses medium containing an initial sugar concentration of 45.37 g dm^?3 with an aeration rate and agitation speed of 2.10 vvm and 338.93 rpm, respectively. Copyright © 2004 Society of Chemical Industry     

Continuous lipase‐catalyzed synthesis of hexyl laurate in a packed‐bed reactor: optimization of the reaction conditions in a solvent‐free system

BACKGROUND: Hexyl laurate has been applied widely in cosmetic industries and is synthesized by chemical methods with problems of cost, environmental pollution, and by‐products. In this study, Lipozyme^® IM77 (from Rhizomucor miehei) was used to catalyze the direct‐esterification of hexanol and lauric acid in a solvent‐free system by utilizing a continuous packed‐bed reactor, wherein the aforementioned difficulties could be overcome. Response surface methodology (RSM) and three‐level‐three‐factor Box‐Behnken design were employed to evaluate the effects of synthesis parameters, such as reaction temperature (45–65 °C), mixture flow rate (0.25–0.75 mL min^?1) and concentration of lauric acid (100–300 mmol L^?1) on the production rate (µmol min^?1) of hexyl laurate by direct esterification. RESULTS: The production rate was affected significantly by the mixture flow rate and lauric acid concentration. On the basis of ridge‐max analysis, the optimum synthesis conditions for hexyl laurate were as follows: 81.58 ± 1.76 µmol min^?1 at 55 °C, 0.5 mL min^?1 flow rate and 0.3 mol L^?1 lauric acid. CONCLUSION: The lipase‐catalyzed synthesis of hexyl laurate by Lipozyme^® IM‐77 in a continuous packed‐bed bioreactor and solvent‐free system was successfully developed; optimization of the reaction parameters was obtained by Box–Behnken design and RSM. Copyright © 2008 Society of Chemical Industry     

In situ reactive extraction of lactic acid from fermentation media

Extractive lactic acid fermentation was investigated in the presence of sunflower oil and Alamine‐336 (with oleyl alcohol as the diluent solvent). Lactic acid was produced in various media at 37 °C using Lactobacillus delbrueckii (NRRL‐B 445). First, the effects of oleyl alcohol (33.3%, v/v), immobilisation, and immobilisation in the presence of sunflower oil (5, 10, 15%, v/v) on lactic acid production were investigated. It was found that oleyl alcohol did not affect production while addition of sunflower oil increased lactic acid production from 10.22 to 16.46 gdm^?3. On the other hand, a toxic effect was observed for oleyl alcohol solutions containing 15–50% (v/v) Alamine‐336. A maximum total lactic acid concentration of 25.59 gdm^?3 was obtained when an oleyl alcohol solution containing 15% (v/v) Alamine together with immobilised cells with 15% (v/v) sunflower oil was used. This value was about 2.5 times that obtained from fermentation without organic solutions. © 2001 Society of Chemical Industry     

Improving k La determination in fungal fermentation,taking into account electrode response time

An alternative way for determining the oxygen mass transfer coefficient, k_La, based upon the traditional dynamic method, is proposed. The oxygen material balance equation in the liquid phase is integrated after insertion of the oxygen probe response time (first order type), and k_La values are determined by employing Marquardt's algorithm, considering as a weighting factor the model's sensitivity with respect to k_La. Bench‐scale fermentations of Aspergillus awamori, performed under different agitation (300–700 rpm) and aeration conditions (0.2–0.6 vvm), were utilized for calculating k_La values (0.0283–0.0874 s⁻¹), employing three methods: two so‐called traditional, the gas balancing and the dynamic methods, and the one proposed here. The latter method is shown to be as reliable as the aforementioned methods but is easier to apply when the oxygen level in the reactor is above the critical value. © 2000 Society of Chemical Industry