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     

Pre‐treatment and utilization of raw glycerol from sunflower oil biodiesel for growth and 1,3‐propanediol production by Clostridium butyricum

BACKGROUND: The objective of the present work is to report an efficient pre‐treatment process for sunflower oil biodiesel raw glycerol (SOB‐RG) and its fermentation to 1,3‐propanediol. RESULTS: The growth inhibition percentages of Clostridium butyricum DSM 5431 on grade A (pH 4.0) and grade B (pH 5.0) phosphoric acid‐treated SOB‐RG were similar to those of pure glycerol at 20 g glycerol L^?1; i.e., 18.5 ± 0.707% to 20.5 ± 0.7% inhibition. In grade A, growth inhibition was reduced from 85.25 ± 0.35% to 32 ± 1.4% (a 53.25% reduction) at 40 g glycerol L^?1 by washing grade A raw glycerol twice with n‐hexanol (grade A‐2). The kinetic parameters for product formation and substrate consumption in anaerobic batch cultures gave almost similar values at 20 g glycerol L^?1, while at 50 g glycerol L^?1 volumetric productivity (Q_p) and specific rate of 1,3‐propanediol formation (q_p) were improved from 1.13 to 1.85 g L^?1 h^?1 and 1.60 to 2.65 g g^?1 h^?1, respectively, by employing grade A‐2 raw glycerol, while the yields were similar (0.5–0.52 g g^?1). CONCLUSION: The results are important as the pre‐treatment of SOB‐RG is necessary to develop bioprocess technologies for conversion of SOB‐RG to 1,3‐propanediol. Copyright © 2008 Society of Chemical Industry     

Strain isolation and optimization of process parameters for bioconversion of glycerol to lactic acid

BACKGROUND: The crude glycerol from biodiesel production represents an abundant and inexpensive source which can be used as raw material for lactic acid production. The first aim of this investigation was to select a strain suitable for producing lactic acid from glycerol with a high concentration and productivity. The second aim was to obtain the optimum fermentation conditions, as a basis for large‐scale lactate production in the future. RESULTS: Eight bacterial strains, which could aerobically convert glycerol to lactic acid, were screened from soil samples. One of the strains, AC‐521, which synthesized lactic acid with a higher concentration, was identified based on its 16S rDNA sequences and physiological characteristics. These results indicated that this strain was a member of Escherichia coli. The optimal fermentation conditions for Escherichia coli AC‐521 were 42 °C, pH 6.5, 0.85 min^?1 (K_La). CONCLUSION: Escherichia coli AC‐521 suitable for producing lactic acid from glycerol with high concentration and productivity was identified. After 88 h of fed‐batch fermentation, both the lactic acid concentration and glycerol consumption reached maximum, giving 85.8 g L^?1 of lactic acid with a productivity of 0.97 g L^?1 h^?1 and a yield of 0.9 mol mol^?1 glycerol. Copyright © 2009 Society of Chemical Industry     

Inoculum strategies for Penicillium simplicissimum lipase production by solid‐state fermentation using a residue from the babassu oil industry

Two alternative inoculation strategies for lipase production by the fungus Penicillium simplicissimum were tested in solid‐state fermentation using a residue from the babassu oil industry (babassu cake). Conventional spore inoculation was compared with fungal pellets grown in liquid medium and with inocula consisting of fermented cake. Fungal pellets delayed lipase production whereas fermented cake accelerated enzyme synthesis, yielding a productivity of 0.45 U g^?1 h^?1, which is equivalent to the highest values obtained with conventional inocula. Therefore, a 2² factorial design was used to determine the best conditions for lipase production with fermented cake as inoculum strategy, varying the inoculum propagation time and inoculum concentration. Lipase activity and productivity reached 30 U g^?1 and 0.63 U g^?1 h^?1, respectively, with 10% inoculum and 36 h. Thus, fermented cake inocula increased production 1.5‐fold with 10 times fewer spores than in conventional inoculation, indicating that fermented solids are an interesting alternative for inoculum development in solid‐state fermentation, mainly for large‐scale processes. Copyright © 2007 Society of Chemical Industry     

Stability of Immobilized Candida sp. 99–125 Lipase for Biodiesel Production

The stability of the immobilized lipase from Candida sp. 99–125 during biodiesel production was investigated. The lipase was separately incubated in the presence of various reaction components such as soybean oil, oleic acid methyl ester, n‐hexane, water, methanol, and glycerol, or the lipase was stored at 60, 80, 100 and 120 °C. Thereafter the residual lipase activity was determined by methanolysis reaction. The results showed that the lipase was rather stable in the reaction media, except for methanol and glycerol. The stability study performed in a reciprocal shaker indicated that enzyme desorption from the immobilized lipase mainly contributed to the lipase inactivation in the water system. So the methanol and glycerol contents should be controlled more precisely to avoid lipase inactivation, and the immobilization method should be improved with regard to lipase desorption.     

Optimization of Enzymatic Synthesis of Tricaprylin in Ionic Liquids by Response Surface Methodology

Ten 1,3‐dialkylimidazolium‐based ionic liquids (ILs) have been investigated as media for the enzymatic synthesis of tricaprylin, in comparison with the conventional organic solvent hexane. The results suggested that the esterification activity of Novozym 435 was higher than Lypozyme RM IM in all the ILs assayed. Novozym 435 showed higher catalytic activity in ILs with anions Tf₂N^? and PF₆^? than in BF₄^? and hexane. FTIR analysis of the secondary structure of the lipase indicated that a smaller decrease of the α‐helix was observed in [C₄MIM] Tf₂N and [C₄MIM] PF₆ than [C₄MIM] BF₄ and hexane, indicating that the anions of ILs might be a key factor for the activity of lipase in ILs. Process parameters (amount of lipase, caprylic acid/glycerol molar ratio, temperature and their interactive effects) were optimized in 1‐butyl‐3‐methylimidazolium hexafluorophosphate ([C₄MIM]PF₆) using Novozym 435 by response surface methodology. When the reactions were performed with the lipase amount of 6.1 % substrate mass at a caprylic acid/glycerol molar ratio of 4.5:1 and 66.7 °C, a higher yield was reached up to 92.4 %.     

Fermentation and oxygen transfer characteristics in recombinant human growth hormone production by Pichia pastoris in sorbitol batch and methanol fed‐batch operation

BACKGROUND: The influence of methanol feed rate on recombinant human growth hormone (rhGH) production by Pichia pastoris hGH‐Mut⁺ in medium containing sorbitol was investigated at three different specific growth rates (µ), namely, 0.02 (MS‐0.02), 0.03 (MS‐0.03), and 0.04 (MS‐0.04). RESULTS: Increasing methanol feed rate above MS‐0.03 did not affect sorbitol consumption, showing that µ = 0.03 h^?1 is a threshold limiting value, above which sorbitol utilization became independent of methanol feed rate. Moreover, when sorbitol was consumed, no further cell growth was observed. Increase in methanol feed rate triggered cell synthesis and the highest cell concentration was obtained at MS‐0.04 as 48 g L^?1 (t = 18 h); whereas, the highest rhGH production, 270 mg L^?1, was obtained at MS‐0.03 as a consequence of lower extracellular protease production and higher AOX activity (41 U g^?1 CDW). Oxygen uptake rate increased with increasing µ, having the maximum value, 76.6 mmol m^?3 s^?1, at MS‐0.04. K_La had a tendency to increase with µ, having a maximum value of 0.15 s^?1 at MS‐0.04 (t = 15 h). CONCLUSION: By considering rhGH concentration and oxygen transfer characteristics, the bioprocess can be improved by a two‐stage feeding strategy, operating at MS‐0.04 at the beginning of fermentation, and thereafter shifting to MS‐0.03. Copyright © 2009 Society of Chemical Industry     

Sequential optimization of culture medium composition for extracellular lipase production by Bacillus sphaericus using statistical methods

A sequential optimization strategy with the aid of statistical design of experiments was used to enhance the lipase (triacylglycerol acylhydrolases, EC 3.1.1.3) production by Bacillus sphaericus in submerged cultivation. A Plackett–Burman experimental design was used to evaluate the twelve medium components. Various vegetable oil inducers were tested for lipase production in the second step and the third step was to identify the optimal values of the significant medium components with sesame oil as the inducer using response surface methodology. A predictive model of the combined effects of the independent variables using response surface methodology and an artificial neural network was proposed. Unstructured kinetic models, a logistic model and a Luedeking–Piret model, were used to describe the cell mass and lipase production respectively. The significant variables affecting lipase production were found to be glucose, olive oil, peptone, NaCl and MnSO₄.H₂O. Sesame oil was found to be the best inducer for lipase production by Bacillus sphaericus. The maximum lipase activity of 4.45 U mL^?1, which was 1.5 times the maximum activity obtained in the Plackett–Burman experimental trials, was obtained at the optimum combination of medium constituents containing 12.695 g L^?1 glucose, 13.161 mL L^?1 sesame oil, 9.947 g L^?1 peptone, 3.25 g L^?1 NaCl, 0.5917 g L^?1 MnSO₄.H₂O and other insignificant components at the fixed level. The statistical design of experiments offers an efficient methodology to identify the significant variables and to optimize the factors with a minimum number of experiments for lipase production by Bacillus sphaericus. Copyright © 2007 Society of Chemical Industry     

Production of Lipase from Geotrichum candidum Using Corn Steep Liquor in Different Bioreactors

The production of an extracellular lipase using corn steep liquor (CSL) as the nitrogen source in the cultivation of Geotrichum candidum NRRLY‐552 was evaluated. The optimized conditions in shake flasks were CSL, 8.0 % w/v, soybean oil, 0.6 % w/v, pH 7.0, 30 °C, 250 rpm, and 48 h, resulting in a maximum lipase productivity of 0.438 U mL^?1 h^?1(U = the amount of enzyme required to liberate 1 μmol of fatty acid per minute). Scale‐up was evaluated with airlift and stirred tank reactors; the best conditions, respectively, were 1 vvm(volume of gas per volume of medium per minute) of aeration which resulted in 0.535 U mL^?1 h^?1 (32 h) and 1 vvm and 300 rpm resulting in 0.563 U mL^?1 h^?1 (16 h). To facilitate downstream processes, lipase production was also evaluated using CSL previously clarified with activated charcoal resulting in 0.275 U mL^?1 h^?1 (24 h) using 12 % (w/v) of clarified CSL in shake flasks. The obtained results showed that CSL leads to similar productivity compared to peptone using the same microorganism under similar conditions. In addition the cost of fermentation medium using CSL is much lower because it is a very inexpensive by‐product from corn processing.     

Lipase‐catalyzed synthesis of partial acylglycerols of acetoacetate

Immobilized Rhizomucor miehei lipase (Lipozyme RMIM) was employed in the synthesis of partial acylglycerols of acetoacetate (C₄H₆O₃, 3‐oxobutanoic acid). Both 1(3)‐sn‐monoacetoacetyl glycerol (MAcG) and 1,3‐sn‐diacetoacetyl glycerol (DAcG) resulting from esterification of glycerol with ethylacetoacetate were isolated and identified by ¹H and ¹³C NMR. An HPLC method for the separation and quantitation of these species was developed. The effects on product yield of coordinate variations in the amounts of enzyme, water, ethyl acetoacetate, and the ratio of immobilizing silica to glycerol were explored. This allowed creation of predictive equations relating these variables to product yield. Reaction conditions were thereby identified and validated under which maximum yields of MAcG and/or DAcG or total ester were predicted. The production of both MAcG and DAcG was markedly sensitive to water, with optimal yields obtained within a narrow range of added water contents. Substantial excesses of ethylacetoacetate ameliorated the inhibitory effect of water. MAcG was effectively produced with the lowest amount of RMIM investigated. It was necessary to use larger amounts of lipase to achieve high yields of DAcG, and even then actual yields were only 30% of theoretical maximum. In the absence of silica only MAcG was produced. Practical applications: Aceotacetyl esters of glycerol are potential prochiral building blocks for further chemical synthesis and exhibit the chemical reactivity of both the acetoacetyl moiety and of glycerol. They are thus of potential use in, for example, the production of biopolymers exhibiting any of a variety of features and functionalities. The use of enzymatic catalysis rather than chemical synthesis for their production offers advantages of reduced degradation and contamination due to the gentler reaction conditions characteristic of enzymatic catalysis, as well as a potential reduction in the costs to procure and dispose of catalyst.     

Lipase‐catalyzed production of medium‐chain triacylglycerols from palm kernel oil distillate: Optimization using response surface methodology

Optimization of lipase‐catalyzed esterification for the production of medium‐chain triacylglycerols (MCT) from palm kernel oil distillate and glycerol was carried out in order to determine the factors that have significant effects on the reaction system and MCT yield. Novozyme 435 from Candida antarctica lipase was found to have the highest activity at 52.87 ± 0.03 U/g. This lipase also produced the highest MCT yield, which is 56.67%. The effect of different variables on MCT synthesis was studied with a two‐level five‐factor fractional factorial design. The various variables include (1) reaction temperature, (2) enzyme load, (3) molecular sieves concentration, (4) reaction time and (5) molar substrate ratio. Reaction temperature, reaction time and molar substrate ratio strongly affect MCT synthesis (p <0.05). However, enzyme load and molecular sieve concentration did not have a significant (p >0.05) influence on MCT yield. Therefore, the significant variables such as reaction temperature, reaction time and molar substrate ratio were further optimized through central composite rotatable design (CCRD). Comparisons between predicted and experimental values from the CCRD optimization procedures revealed good correlation, implying that the quadric response model satisfactorily expressed the percentage yield of MCT in the lipase‐catalyzed esterification. The optimum MCT yield is 73.3% by using 2 wt‐% enzyme dosage, a molecular sieves concentration of 1 wt‐%, a reaction temperature of 90 °C, a reaction time of 10 h and a molar substrate ratio of 4 : 1 (medium‐chain fatty acid/glycerol). Experiments to confirm the predicted results using the optimal parameters were conducted and an MCT yield of 70.21 ± 0.18% (n = 3) was obtained.     

Effect of acetic acid on lipid accumulation by glucose‐fed activated sludge cultures

BACKGROUND: The effect of acetic acid, a lignocellulose hydrolysis by‐product, on lipid accumulation by activated sludge cultures grown on glucose was investigated. This was done to assess the possible application of lignocellulose as low‐cost and renewable fermentation substrates for biofuel feedstock production. RESULTS: Biomass yield was reduced by around 54% at a 2 g L^?1 acetic acid dosage but was increased by around 18% at 10 g L^?1 acetic acid dosage relative to the control run. The final gravimetric lipid contents at 2 and 10 g L^?1 acetic acid levels were 12.5 ± 0.7% and 8.8 ± 3.2% w/w, respectively, which were lower than the control (17.8 ± 2.8% w/w). However, biodiesel yields from activated sludge grown with acetic acid (5.6 ± 0.6% w/w for 2 g L^?1 acetic acid and 4.2 ± 3.0% w/w for 10 g L^?1 acetic acid) were higher than in raw activated sludge (1–2% w/w). The fatty acid profiles of the accumulated lipids were similar with conventional plant oil biodiesel feedstocks. CONCLUSIONS: Acetic acid enhanced biomass production by activated sludge at high levels but reduced lipid production. Further studies are needed to enhance acetic acid utilization by activated sludge microorganisms for lipid biosynthesis. Copyright © 2011 Society of Chemical Industry     

Enhanced lipase production from Aeromonas sp. S1 using Sal deoiled seed cake as novel natural substrate for potential application in dairy wastewater treatment

BACKGROUND: Sal (Shorea robusta) deoiled seed cake extract (SDOCE) was assessed for its suitability as a cheap natural substrate for lipase production under submerged fermentation. The bacterial isolate Aeromonas sp. S1 isolated from dairy industry was used for lipase production. Both the isolate and its lipase were shown to be potential tools for treatment of dairy wastewater containing higher organic load. RESULTS: On substituting tributyrin with SDOCE, lipase production was enhanced 24‐fold (195 U mL^?1) compared with the initial 8.13 U mL^?1 lipase activity. Maximum lipase production was obtained at pH 8.0 and incubation temperature 30 °C. The lipase had pH and temperature optima of 10.0 and 55 °C, respectively. The isolate and its crude enzyme preparation were checked separately for applicability in dairy wastewater treatment. The isolate was able to reduce chemical oxygen demand (COD) by 93%, oil and grease (O&G) by 75%, and total suspended solids (TSS) by 47% after 96 h of treatment. Enzymatic preparation gave 86% reduction of COD after 12 h and 75 and 45% reduction of O&G and TSS, respectively, after 96 h of treatment. CONCLUSION: Overall, the study shows the usefulness of Sal seed deoiled cake, a cheap agro‐industrial by‐product for the production of lipase. The isolate and its lipase can also be used effectively for the treatment of dairy wastewater. Copyright © 2011 Society of Chemical Industry     

Production of α‐amylase under solid‐state fermentation utilizing coffee waste

Coffee industry substrates such as coffee pulp, coffee cherry husk, silver skin, spent coffee and mixtures of these coffee wastes (MC) were evaluated for their efficacy as sole carbon source for the synthesis of α‐amylase in solid‐state fermentation (SSF) using a fungal strain of Neurospora crassa CFR 308. For SSF with coffee pulp and with MC, α‐amylase activity of 3908 U g^?1 ds (units per gram of dry substrate) and 3870 U g^?1 ds, respectively, was observed. Parameters such as moisture (60%), pH (4.6), temperature (28 °C), particle size (1.0 mm), inoculum size (10⁷ spores g^?1 ds), and fermentation time (5 days) were optimized for enzyme synthesis, wherein 4981 and 4324 U g^?1 U g^?1 ds of α‐amylase activity was obtained in SSF with coffee pulp and MC, respectively. The enzyme production was further improved when the substrates were subjected to pre‐treatment by steaming. Accordingly, maximum α‐amylase activity of 7084 U g^?1 ds and 6342 U g^?1 ds was obtained with steam‐pretreated coffee pulp and MC, respectively, demonstrating them to be excellent sole carbon sources for synthesis of α‐amylase production. Copyright © 2009 Society of Chemical Industry     

Fed‐batch fermentation of olive mill wastewaters for lipase production

In the Mediterranean basin countries, huge amounts of olive mill wastewaters (OMW) are produced by the olive oil industry. It constitutes a serious environmental problem, nevertheless its composition turns OMW into a potential growth medium to lipolytic microorganisms. The aim of this work was to study lipase production as well as OMW degradation in fed‐batch cultures of Candida cylindracea CBS 7869, Candida rugosa CBS 2275 and Yarrowia lipolytica W29 (ATCC 20460). Besides the improvement of lipase production, the fed‐batch approach enhanced the effluent degradation, since it led to good COD and lipids reduction, both higher than 50%. C. rugosa achieved the highest value of lipase productivity (130 U L^?1 h^?1), in parallel with highest lipids reduction (77%). This study demonstrates that OMW are becoming a competitive and valuable growth medium in fermentation processes with lipolytic microorganisms. The fed‐batch strategy used proved to be an efficient approach to enhance lipase production from OMW and to reduce significantly the final organic load of the medium. Copyright © 2012 Society of Chemical Industry     

Production of lipolytic enzymes in batch cultures of Ophiostoma piceae

After an extensive fungal screening, an Ophiostoma piceae strain was selected for its ability to produce high concentrations of lipase and esterase to remove pitch components of wood pulp. Optimal conditions for growth and enzyme production were established first in shaken flasks. A medium containing rapeseed oil and mycological peptone resulted in a lipase activity of 736 U dm^?3 and an esterase activity of 1569 U dm^?3. When fermentation runs were implemented using the same medium under controlled conditions in 2‐dm³ fermenters, the lipase and esterase activities were increased to 1005 and 4006 U dm^?3 respectively. Further scale‐up was carried out in two stages to 20 dm³ and 72 dm³ (pilot‐scale) stirred tank reactors. The results proved that the cultures could be scaled‐up successfully from shaken flasks to pilot‐scale with increases of 47% and 146% in lipase and esterase activities respectively. © 2001 Society of Chemical Industry     

Optimization of lipase production by Penicillium simplicissimum in soybean meal

BACKGROUND: Lipase production by Penicillium simplicissimum using soybean meal as substrate has been investigated. A factorial design technique was used to evaluate the effects of incubation temperature, initial moisture of the meal and substrate supplementation with low cost supplements, on lipase production. Soybean oil and wastewater from a slaughterhouse, which is rich on oil and fat, corn steep liquor and yeast hydrolysate, were tested as supplementary carbon and nitrogen sources. RESULTS Cultivation conditions were optimized for the production of lipase by factorial design and response surface methodology. Results show that the microorganism produces very low protease activity (0.21 U gds^?1 dry substrate), which helps to maximize lipase production. Soybean meal without supplements appears to be the best medium of those tested for lipase production by P. simplicissimum. CONCLUSION: This work showed that temperature and moisture are the factors that most strongly influence lipase production by P. simplicissimum using soybean meal as substrate. The growth conditions that optimize lipase production are 27.5 °C using substrate with 550 g kg^?1 of initial moisture. In optimum conditions lipase activity of 30 U gds^?1 dry substrate was obtained. Copyright © 2007 Society of Chemical Industry     

The use of olive mill wastewater by wild type Yarrowia lipolytica strains: medium supplementation and surfactant presence effect

BACKGROUND: The aim of this work was to study the ability of two different wild type strains of the yeast Yarrowia lipolytica to grow on olive mill wastewater (OMW) and their potential to produce high‐value products such as lipases. Factors that affect cellular growth and OMW degradation were studied, such as nitrogen supplementation, cells concentration and surfactant addition. RESULTS: Both strains, W29 and IMUFRJ 50682, were able to grow in OMW with 19 g L^?1 of COD and approximately 800 mg L^?1 of total phenols concentration. The strain W29 presented the highest potential for extracellular lipase production in OMW medium. Lipase productivity was improved by the medium supplementation with ammonium sulphate up to 6 g L^?1, leading to 80% of COD degradation and 70% of total phenols reduction. The surfactant Tween 80 enhanced cell growth and COD degradation, but had a negative effect on lipase activity. CONCLUSIONS Y. lipolytica has a great potential for OMW valorisation by its use as culture medium for biomass and enzymes production. The operating conditions that favoured lipase production differ from the conditions that improve COD reduction. Copyright © 2008 Society of Chemical Industry     

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     

One‐Pot Lipase Entrapment Within Silica Particles to Prepare a Stable and Reusable Biocatalyst for Transesterification

In order to enhance the reusability, Rhizomucor miehei lipase was entrapped in a single step within silica particles having an oleic acid core (RML@SiO₂). Characterization of RML@SiO₂ by scanning and transmission electron microscopy and Fourier transform infrared studies supported the lipase immobilization within silica particles. The immobilized enzyme was employed for transesterification of cottonseed oil with methanol and ethanol. Under the optimum reaction conditions of a methanol‐to‐oil molar ratio of 12:1 or ethanol‐to‐oil molar ratio of 15:1, stirring speed of 250 revolutions/min (flask radius = 3 cm), reaction temperature of 40 °C, and biocatalyst concentration of 5 wt% (with respect to oil), more than 98 % alkyl ester yield was achieved in 16 and 24 h of reaction duration in case of methanolysis and ethanolysis, respectively. The immobilized enzyme did not require any buffer solution or organic solvent for optimum activity; hence, the produced biodiesel and glycerol were free from metal ion or organic molecule contamination. The activation energies for the immobilized enzyme‐catalyzed ethanolysis and methanolysis were found to be 34.9 ± 1.6 and 19.7 ± 1.8 kJ mol^?1, respectively. The immobilized enzyme was recovered from the reaction mixture and reused in 12 successive runs without significant loss of activity. Additionally, RML@SiO₂ demonstrated better reusability as well as stability in comparison to the native enzyme as the former did not lose the activity even upon storage at room temperature (25–30 °C) over an 8‐month period.