Journal of Industrial Microbiology & Biotechnology
This work aimed at the production of stabilized derivatives of Thermomyces lanuginosus lipase (TL... more This work aimed at the production of stabilized derivatives of Thermomyces lanuginosus lipase (TLL) by multipoint covalent immobilization of the enzyme on chitosan-based matrices. The resulting biocatalysts were tested for synthesis of biodiesel by ethanolysis of palm oil. Different hydrogels were prepared: chitosan alone and in polyelectrolyte complexes (PEC) with κ-carrageenan, gelatin, alginate, and polyvinyl alcohol (PVA). The obtained supports were chemically modified with 2,4,6-trinitrobenzene sulfonic acid (TNBS) to increase support hydrophobicity, followed by activation with different agents such as glycidol (GLY), epichlorohydrin (EPI), and glutaraldehyde (GLU). The chitosan-alginate hydrogel, chemically modified with TNBS, provided derivatives with higher apparent hydrolytic activity (HAapp) and thermal stability, being up to 45-fold more stable than soluble lipase. The maximum load of immobilized enzyme was 17.5 mg g−1 of gel for GLU, 7.76 mg g−1 of gel for GLY, and 7.65 mg g−1 of gel for EPI derivatives, the latter presenting the maximum apparent hydrolytic activity (364.8 IU g−1 of gel). The three derivatives catalyzed conversion of palm oil to biodiesel, but chitosan-alginate-TNBS activated via GLY and EPI led to higher recovered activities of the enzyme. Thus, this is a more attractive option for both hydrolysis and transesterification of vegetable oils using immobilized TLL, although industrial application of this biocatalyst still demands further improvements in its half-life to make the enzymatic process economically attractive.
Biodiesel Cyanobacteria Lipid feedstock Synechococcus sp. PCC7942 Enzymatic catalysis Cyanobacter... more Biodiesel Cyanobacteria Lipid feedstock Synechococcus sp. PCC7942 Enzymatic catalysis Cyanobacteria have several advantages as lipid feedstock for biodiesel production compared to microalgae. These benefits include the ability to increase lipid content via genetic manipulation, a high growth rate, nitrogenfixation and a simpler cell wall, which aids in lipid extraction. Moreover, these bacteria have the capability to convert solar energy into biomass that is two times greater than microalgae and ten times greater than corn and sugar cane. The aim of this study was to optimize the cultivation conditions of Synechococcus sp. PCC7942 and to evaluate the lipid feedstock to generate biodiesel via an enzymatic route. For this purpose, the effect of light intensity (50-150 μmol m −2 s −1 ) and the concentration of Na 2 CO 3 (0.5-1.5 g L −1 ) were studied according to a 2 2 full-factorial design. Under optimized conditions, the biomass productivity (Q P ) of 124.0 ± 3.2 mg L −1 day −1 , lipid content of 29.0 ± 2.1% and lipid productivity of 35.9 ± 0.5 mg L −1 day −1 were obtained. The fatty acid profile indicated differences when the medium was enriched with Na 2 CO 3 and cultured in high light intensity, resulting in an increase in unsaturated fatty acids, particularly linoleic acid, which accounts for 40% of the fatty acids in Synechococcus sp. PCC7942. The lipid feedstock was characterized and used as feedstock for biodiesel synthesis using Novozym® 435 as a catalyst and ethanol as an acyl acceptor. The biocatalyst was able to form ethyl esters from all of the fatty acids present in the lipid feedstock, attaining a conversion of 97.1%. Analysis of Synechococcus sp. PCC7942 lipids indicated the potential of this cyanobacterium as a biodiesel feedstock. The primary fatty acids of this cyanobacterium were C16-C18, which is suitable for the production of good-quality biodiesel.
The cyanobacterium Microcystis aeruginosa strain NPCD-1, isolated from sewage treatment plant and... more The cyanobacterium Microcystis aeruginosa strain NPCD-1, isolated from sewage treatment plant and characterized as a non-microcystin producer by mass spectrometry and molecular analysis, was found to be a source of lipid when cultivated in ASM-1 medium at 25 • C under constant white fluorescent illumination (109 mol photon m −2 s −1 ). In these conditions, biomass productivity of 46.92 ± 3.84 mg L −1 day −1 and lipid content of 28.10 ± 1.47% were obtained. Quantitative analysis of fatty acid methyl esters demonstrated high concentration of saturated fatty acids (50%), palmitic (24.34%) and lauric (13.21%) acids being the major components. The remaining 50% constituting unsaturated fatty acids showed higher concentrations of oleic (26.88%) and linoleic (12.53%) acids. The feasibility to produce biodiesel from this cyanobacterial lipid was demonstrated by running enzymatic transesterification reactions catalyzed by Novozym ® 435 and using palm oil as feedstock control. Batch experiments were carried out using tertbutanol and iso-octane as solvent. Results showed similarity on the main ethyl esters formed for both feedstocks. The highest ethyl ester concentration was related to palmitate and oleate esters followed by laurate and linoleate esters. However, both reaction rates and ester yields were dependent on the solvent tested. Total ethyl ester concentrations varied in the range of 44.24-67.84 wt%, corresponding to ester yields from 80 to 100%. Iso-octane provided better solubility and miscibility, with ester yield of 98.10% obtained at 48 h for reaction using the cyanobacterium lipid, while full conversion was achieved in 12 h for reaction carried out with palm oil. These results demonstrated that cyanobacterial lipids from M. aeruginosa NPCD-1 have interesting properties for biofuel production.
Lipase de Candida rugosa na forma livre foi usada na síntese de butirato de butila pela esterific... more Lipase de Candida rugosa na forma livre foi usada na síntese de butirato de butila pela esterificação direta de n-butanol com ácido butírico. Um planejamento fatorial completo 2 4 foi empregado para determinar a influência da razão molar do álcool para ácido (1: 0,5-2,5), concentração de agente dessecante (0-20%), concentração de enzima (40-80 mg) e temperatura de incubação (30-60 °C) no rendimento de esterificação. A concentração de agente dessecante foi o fator mais significativo na esterificação, sendo sua influência negativa. O progresso da esterificação foi favorecido para substratos contendo ácido em excesso, mesmo em baixa concentrações de enzima (40 mg), sendo a conversão de 50%. Na região avaliada, a formação do butirato de butila (26,67 g/L) foi maximizada para razão molar (1:2,5), ausência de agente dessecante, concentração de enzima (80 mg) e temperatura de incubação de 30 °C. Unitermos • Lípase • Candida rugosa • Esterificação • Planejamento de • experimentos
Recebido em 21/6/04; aceito em 26/1/05; publicado na web em 30/6/05 STUDY OF THE INFLUENCE OF SOL... more Recebido em 21/6/04; aceito em 26/1/05; publicado na web em 30/6/05 STUDY OF THE INFLUENCE OF SOLVENT, CARBOHYDRATE AND FATTY ACID IN THE ENZYMATIC SYNTHESIS OF SUGAR ESTERS BY LIPASES. The aim of this work was to gain knowledge of enzymatic processes for the synthesis fatty acid esters of sugar, with the objective to develop an enzymatic process for the preparation of non-toxic biodegradable surface-active agents derived entirely from renewable resources. A wide range of data were collected for reaction conditions involving different sugars (glucose, fructose and sucrose), fatty acids (oleic, palmitic, lauric), solvents (hexane, heptane and t-butanol) and different sources of lipases in both free and immobilized forms. As a solvent t-butanol provided the best conditions to create a catalytic liquid phase in which the reaction occurs. Sugars were preferentially esterified in the following order: fructose > glucose > sucrose, depending on the enzyme preparation. For fructose no influence was found concerning de acyl donor and similar rates were achieved for all tested fatty acids. Ester synthesis was maximized for substrates containing fructose, lauric or oleic acids, t-butanol and lipase from porcine pancreas immobilized on polysiloxane-polyvinyl alcohol particles. Under such conditions molar conversions were higher than 50%.
Recebido em 27/3/07; aceito em 26/10/07; publicado na web em 13/8/08 MONOGLYCERIDES: PRODUCTION B... more Recebido em 27/3/07; aceito em 26/10/07; publicado na web em 13/8/08 MONOGLYCERIDES: PRODUCTION BY ENZYMATIC ROUTE AND APPLICATIONS: Monoglycerides (MAG) are non-ionic surfactants, widely used in the pharmaceutical, food and cosmetic industries. Although MAGs are manufactured on an industrial scale by chemical glycerolysis of oils and fats, new developments in lipase catalyzed synthesis have been studied as an alternative to the classical method seeking to use clean technology and green chemistry. In this work, different methods such as glycerolysis, selective hydrolysis of fats and oils, and esterification of fatty acids or transesterification of esters with glycerol are presented. The properties and applications of the monoglycerides are also included in this review.
Recebido em 12/12/03; aceito em 14/9/04; publicado na web em 4/2/05 APPLICATION OF LIPASES FOR WA... more Recebido em 12/12/03; aceito em 14/9/04; publicado na web em 4/2/05 APPLICATION OF LIPASES FOR WASTEWATER TREATMENT CONTAINING HIGH LEVELS OF LIPIDS. The process of anaerobic digestion has been greatly developed during the last decades for the treatment of wastewater from food industries as for example, wastewaters from sugar and starch manufacturing and from breweries. However, for wastewaters which contain high amounts of fats and proteins, such as those from slaughterhouses and dairies, anaerobic digestion was found to be troublesome, due to the potential of sludge flotation, the formation of scum layers of lipids at the surface of the reactor, which are not digested and the toxicity of the intermediate compounds (long chain fatty acids) generated during the anaerobic digestion of the wastewater. The addition of hydrolytic enzymes, such as lipases prior to the anaerobic digestion can minimize these problems. In this work, this strategy is reviewed and the technical issues that must be considered in determining its feasibility are discussed.
O presente trabalho teve por objetivo o estudo do efeito do pré-tratamento de efluentes de laticí... more O presente trabalho teve por objetivo o estudo do efeito do pré-tratamento de efluentes de laticínios por lipase pancreática na remoção de matéria orgânica e formação de metano por biodegradação anaeróbia. Em 24 horas de hidrólise, a maior formação de ácidos graxos, 54,36 mM, foi obtida em meio reacional contendo efluente bruto suplementado com 10 mM de íons cálcio a pH 8,0, ajustado com NaOH 1M e incubado com 0,5% m/v de enzima. No efluente hidrolisado submetido à biodigestão anaeróbia houve redução de cor e da demanda química de oxigênio de 80,9 e 77,5%, enquanto no efluente bruto as reduções equivalentes foram iguais a 45,0 e 13,9%. O volume de metano formado após 15 dias de biodigestão do efluente hidrolisado (437 mL) foi aproximadamente o dobro do obtido com o efluente bruto (209 mL).
The agricultural activity has been responsible for the production of the largest part of renewabl... more The agricultural activity has been responsible for the production of the largest part of renewable resources; therefore, great research effort has been dedicated to increase crop productivities in the already cultivated areas. One of the research lives good promise perspectives to be developed is referred to the use of fertilizers based on metals as zinc, manganese, copper, molybdenum, cobalt, among others. The objective of this work was to describe the effect of milling on copper, zinc and manganese oxides in liquid phase, for further application in crop fields. The oxides were acquired commercially and submitted to an industrial process for fertilizer formulation consisting of dispersion of these oxides in surfactants together with the element of interest for agriculture, followed by milling in the liquid phase, structuring, densification and finishing. After milling, the microstructure of the oxides was analyzed by scanning electron microscopy, to determine the morphology, and la...
A síntese orgânica catalisada por enzimas envolve um mecanismo complexo dependente do tipo de sub... more A síntese orgânica catalisada por enzimas envolve um mecanismo complexo dependente do tipo de substrato, enzima, solvente orgânico e teor de água no meio reacional. Neste trabalho foi estudado a influência de alguns desses parâmetros no rendimento da esterificação do butanol com ácido butírico, utilizando uma preparação enzimática comercial de lipase. A polaridade e natureza do solvente, bem como a razão molar entre o butanol e ácido butírico, foram considerados os fatores que mais influenciaram o desenvolvimento dessa síntese enzimática.
Journal of Industrial Microbiology & Biotechnology
This work aimed at the production of stabilized derivatives of Thermomyces lanuginosus lipase (TL... more This work aimed at the production of stabilized derivatives of Thermomyces lanuginosus lipase (TLL) by multipoint covalent immobilization of the enzyme on chitosan-based matrices. The resulting biocatalysts were tested for synthesis of biodiesel by ethanolysis of palm oil. Different hydrogels were prepared: chitosan alone and in polyelectrolyte complexes (PEC) with κ-carrageenan, gelatin, alginate, and polyvinyl alcohol (PVA). The obtained supports were chemically modified with 2,4,6-trinitrobenzene sulfonic acid (TNBS) to increase support hydrophobicity, followed by activation with different agents such as glycidol (GLY), epichlorohydrin (EPI), and glutaraldehyde (GLU). The chitosan-alginate hydrogel, chemically modified with TNBS, provided derivatives with higher apparent hydrolytic activity (HAapp) and thermal stability, being up to 45-fold more stable than soluble lipase. The maximum load of immobilized enzyme was 17.5 mg g−1 of gel for GLU, 7.76 mg g−1 of gel for GLY, and 7.65 mg g−1 of gel for EPI derivatives, the latter presenting the maximum apparent hydrolytic activity (364.8 IU g−1 of gel). The three derivatives catalyzed conversion of palm oil to biodiesel, but chitosan-alginate-TNBS activated via GLY and EPI led to higher recovered activities of the enzyme. Thus, this is a more attractive option for both hydrolysis and transesterification of vegetable oils using immobilized TLL, although industrial application of this biocatalyst still demands further improvements in its half-life to make the enzymatic process economically attractive.
Biodiesel Cyanobacteria Lipid feedstock Synechococcus sp. PCC7942 Enzymatic catalysis Cyanobacter... more Biodiesel Cyanobacteria Lipid feedstock Synechococcus sp. PCC7942 Enzymatic catalysis Cyanobacteria have several advantages as lipid feedstock for biodiesel production compared to microalgae. These benefits include the ability to increase lipid content via genetic manipulation, a high growth rate, nitrogenfixation and a simpler cell wall, which aids in lipid extraction. Moreover, these bacteria have the capability to convert solar energy into biomass that is two times greater than microalgae and ten times greater than corn and sugar cane. The aim of this study was to optimize the cultivation conditions of Synechococcus sp. PCC7942 and to evaluate the lipid feedstock to generate biodiesel via an enzymatic route. For this purpose, the effect of light intensity (50-150 μmol m −2 s −1 ) and the concentration of Na 2 CO 3 (0.5-1.5 g L −1 ) were studied according to a 2 2 full-factorial design. Under optimized conditions, the biomass productivity (Q P ) of 124.0 ± 3.2 mg L −1 day −1 , lipid content of 29.0 ± 2.1% and lipid productivity of 35.9 ± 0.5 mg L −1 day −1 were obtained. The fatty acid profile indicated differences when the medium was enriched with Na 2 CO 3 and cultured in high light intensity, resulting in an increase in unsaturated fatty acids, particularly linoleic acid, which accounts for 40% of the fatty acids in Synechococcus sp. PCC7942. The lipid feedstock was characterized and used as feedstock for biodiesel synthesis using Novozym® 435 as a catalyst and ethanol as an acyl acceptor. The biocatalyst was able to form ethyl esters from all of the fatty acids present in the lipid feedstock, attaining a conversion of 97.1%. Analysis of Synechococcus sp. PCC7942 lipids indicated the potential of this cyanobacterium as a biodiesel feedstock. The primary fatty acids of this cyanobacterium were C16-C18, which is suitable for the production of good-quality biodiesel.
The cyanobacterium Microcystis aeruginosa strain NPCD-1, isolated from sewage treatment plant and... more The cyanobacterium Microcystis aeruginosa strain NPCD-1, isolated from sewage treatment plant and characterized as a non-microcystin producer by mass spectrometry and molecular analysis, was found to be a source of lipid when cultivated in ASM-1 medium at 25 • C under constant white fluorescent illumination (109 mol photon m −2 s −1 ). In these conditions, biomass productivity of 46.92 ± 3.84 mg L −1 day −1 and lipid content of 28.10 ± 1.47% were obtained. Quantitative analysis of fatty acid methyl esters demonstrated high concentration of saturated fatty acids (50%), palmitic (24.34%) and lauric (13.21%) acids being the major components. The remaining 50% constituting unsaturated fatty acids showed higher concentrations of oleic (26.88%) and linoleic (12.53%) acids. The feasibility to produce biodiesel from this cyanobacterial lipid was demonstrated by running enzymatic transesterification reactions catalyzed by Novozym ® 435 and using palm oil as feedstock control. Batch experiments were carried out using tertbutanol and iso-octane as solvent. Results showed similarity on the main ethyl esters formed for both feedstocks. The highest ethyl ester concentration was related to palmitate and oleate esters followed by laurate and linoleate esters. However, both reaction rates and ester yields were dependent on the solvent tested. Total ethyl ester concentrations varied in the range of 44.24-67.84 wt%, corresponding to ester yields from 80 to 100%. Iso-octane provided better solubility and miscibility, with ester yield of 98.10% obtained at 48 h for reaction using the cyanobacterium lipid, while full conversion was achieved in 12 h for reaction carried out with palm oil. These results demonstrated that cyanobacterial lipids from M. aeruginosa NPCD-1 have interesting properties for biofuel production.
Lipase de Candida rugosa na forma livre foi usada na síntese de butirato de butila pela esterific... more Lipase de Candida rugosa na forma livre foi usada na síntese de butirato de butila pela esterificação direta de n-butanol com ácido butírico. Um planejamento fatorial completo 2 4 foi empregado para determinar a influência da razão molar do álcool para ácido (1: 0,5-2,5), concentração de agente dessecante (0-20%), concentração de enzima (40-80 mg) e temperatura de incubação (30-60 °C) no rendimento de esterificação. A concentração de agente dessecante foi o fator mais significativo na esterificação, sendo sua influência negativa. O progresso da esterificação foi favorecido para substratos contendo ácido em excesso, mesmo em baixa concentrações de enzima (40 mg), sendo a conversão de 50%. Na região avaliada, a formação do butirato de butila (26,67 g/L) foi maximizada para razão molar (1:2,5), ausência de agente dessecante, concentração de enzima (80 mg) e temperatura de incubação de 30 °C. Unitermos • Lípase • Candida rugosa • Esterificação • Planejamento de • experimentos
Recebido em 21/6/04; aceito em 26/1/05; publicado na web em 30/6/05 STUDY OF THE INFLUENCE OF SOL... more Recebido em 21/6/04; aceito em 26/1/05; publicado na web em 30/6/05 STUDY OF THE INFLUENCE OF SOLVENT, CARBOHYDRATE AND FATTY ACID IN THE ENZYMATIC SYNTHESIS OF SUGAR ESTERS BY LIPASES. The aim of this work was to gain knowledge of enzymatic processes for the synthesis fatty acid esters of sugar, with the objective to develop an enzymatic process for the preparation of non-toxic biodegradable surface-active agents derived entirely from renewable resources. A wide range of data were collected for reaction conditions involving different sugars (glucose, fructose and sucrose), fatty acids (oleic, palmitic, lauric), solvents (hexane, heptane and t-butanol) and different sources of lipases in both free and immobilized forms. As a solvent t-butanol provided the best conditions to create a catalytic liquid phase in which the reaction occurs. Sugars were preferentially esterified in the following order: fructose > glucose > sucrose, depending on the enzyme preparation. For fructose no influence was found concerning de acyl donor and similar rates were achieved for all tested fatty acids. Ester synthesis was maximized for substrates containing fructose, lauric or oleic acids, t-butanol and lipase from porcine pancreas immobilized on polysiloxane-polyvinyl alcohol particles. Under such conditions molar conversions were higher than 50%.
Recebido em 27/3/07; aceito em 26/10/07; publicado na web em 13/8/08 MONOGLYCERIDES: PRODUCTION B... more Recebido em 27/3/07; aceito em 26/10/07; publicado na web em 13/8/08 MONOGLYCERIDES: PRODUCTION BY ENZYMATIC ROUTE AND APPLICATIONS: Monoglycerides (MAG) are non-ionic surfactants, widely used in the pharmaceutical, food and cosmetic industries. Although MAGs are manufactured on an industrial scale by chemical glycerolysis of oils and fats, new developments in lipase catalyzed synthesis have been studied as an alternative to the classical method seeking to use clean technology and green chemistry. In this work, different methods such as glycerolysis, selective hydrolysis of fats and oils, and esterification of fatty acids or transesterification of esters with glycerol are presented. The properties and applications of the monoglycerides are also included in this review.
Recebido em 12/12/03; aceito em 14/9/04; publicado na web em 4/2/05 APPLICATION OF LIPASES FOR WA... more Recebido em 12/12/03; aceito em 14/9/04; publicado na web em 4/2/05 APPLICATION OF LIPASES FOR WASTEWATER TREATMENT CONTAINING HIGH LEVELS OF LIPIDS. The process of anaerobic digestion has been greatly developed during the last decades for the treatment of wastewater from food industries as for example, wastewaters from sugar and starch manufacturing and from breweries. However, for wastewaters which contain high amounts of fats and proteins, such as those from slaughterhouses and dairies, anaerobic digestion was found to be troublesome, due to the potential of sludge flotation, the formation of scum layers of lipids at the surface of the reactor, which are not digested and the toxicity of the intermediate compounds (long chain fatty acids) generated during the anaerobic digestion of the wastewater. The addition of hydrolytic enzymes, such as lipases prior to the anaerobic digestion can minimize these problems. In this work, this strategy is reviewed and the technical issues that must be considered in determining its feasibility are discussed.
O presente trabalho teve por objetivo o estudo do efeito do pré-tratamento de efluentes de laticí... more O presente trabalho teve por objetivo o estudo do efeito do pré-tratamento de efluentes de laticínios por lipase pancreática na remoção de matéria orgânica e formação de metano por biodegradação anaeróbia. Em 24 horas de hidrólise, a maior formação de ácidos graxos, 54,36 mM, foi obtida em meio reacional contendo efluente bruto suplementado com 10 mM de íons cálcio a pH 8,0, ajustado com NaOH 1M e incubado com 0,5% m/v de enzima. No efluente hidrolisado submetido à biodigestão anaeróbia houve redução de cor e da demanda química de oxigênio de 80,9 e 77,5%, enquanto no efluente bruto as reduções equivalentes foram iguais a 45,0 e 13,9%. O volume de metano formado após 15 dias de biodigestão do efluente hidrolisado (437 mL) foi aproximadamente o dobro do obtido com o efluente bruto (209 mL).
The agricultural activity has been responsible for the production of the largest part of renewabl... more The agricultural activity has been responsible for the production of the largest part of renewable resources; therefore, great research effort has been dedicated to increase crop productivities in the already cultivated areas. One of the research lives good promise perspectives to be developed is referred to the use of fertilizers based on metals as zinc, manganese, copper, molybdenum, cobalt, among others. The objective of this work was to describe the effect of milling on copper, zinc and manganese oxides in liquid phase, for further application in crop fields. The oxides were acquired commercially and submitted to an industrial process for fertilizer formulation consisting of dispersion of these oxides in surfactants together with the element of interest for agriculture, followed by milling in the liquid phase, structuring, densification and finishing. After milling, the microstructure of the oxides was analyzed by scanning electron microscopy, to determine the morphology, and la...
A síntese orgânica catalisada por enzimas envolve um mecanismo complexo dependente do tipo de sub... more A síntese orgânica catalisada por enzimas envolve um mecanismo complexo dependente do tipo de substrato, enzima, solvente orgânico e teor de água no meio reacional. Neste trabalho foi estudado a influência de alguns desses parâmetros no rendimento da esterificação do butanol com ácido butírico, utilizando uma preparação enzimática comercial de lipase. A polaridade e natureza do solvente, bem como a razão molar entre o butanol e ácido butírico, foram considerados os fatores que mais influenciaram o desenvolvimento dessa síntese enzimática.
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