Bernardo Dias Ribeiro

Production and Use of Lipases in Bioenergy: A Review from the Feedstocks to Biodiesel Production

Lipases represent one of the most reported groups of enzymes for the production of biofuels. They are used for the processing of glycerides and fatty acids for biodiesel (fatty acid alkyl esters) production. This paper presents the main topics of the enzyme-based production of biodiesel, from the feedstocks to the production of enzymes and their application in esterification and transesterification reactions. Growing technologies, such as the use of whole cells as catalysts, are addressed, and as concluding remarks, the advantages, concerns, and future prospects of enzymatic biodiesel are presented.

Toxicity of ionic liquids toward microorganisms interesting to the food industry

Considering the potential applications of ionic liquids (ILs) as solvents in biotechnological processes such as food production via microbial synthesis, the work presented here aimed to evaluate the toxicity of these new solvents to microorganisms of interest in the food industry. Following the international standard method of the CLSI (Clinical and Laboratory Standards Institute), the maximum non-toxic concentration (MNTC) was determined for nine ILs (containing an imidazolium, cholinium or phosphonium cation) toward nine microorganisms (the bacteria Bacillus subtilis, Lactobacillus delbrueckii subs. delbrueckii, Pseudomonas aeruginosa, the actinobacteria Streptomyces drozdowiczii, the yeasts Saccharomyces cerevisiae, Yarrowia lipolytica, Kluyveromyces marxianus, and the filamentous fungi Aspergillus brasiliensis and Rhizopus oryzae). Among the bacteria, B. subtilis and P. aeruginosa were more tolerant to hydrophilic imidazolium ILs with [C2mim] cations combined with [EtSO4], [EtSO3] and [Cl] anions. In the presence of hydrophilic choline and phosphonium based-ILs, the Gram-negative bacterium P. aeruginosa was more resistant than others. The same effect was observed for the [NTf2]-based ILs, in which only P. aeruginosa could grow. Regarding the fungi, A. brasiliensis and R. oryzae tolerated high concentrations of ILs. Among the yeasts, only Y. lipolytica was tolerant to all tested ILs. In general, ILs containing choline as the cationic moiety were more biocompatible since they allowed the growth of all the studied microorganisms.

Enzyme-enhanced extraction of phenolic compounds and proteins from flaxseed meal.

Flaxseed (Linum usitatissimum) meal, the main byproduct of the flaxseed oil extraction process, is composed mainly of proteins, mucilage, and phenolic compounds. The extraction methods of phenolics either commonly employed the use of mixed solvents (dioxane/ethanol, water/acetone, water/methanol, and water/ethanol) or are done with the aid of alkaline, acid, or enzymatic hydrolysis. This work aimed at the study of optimal conditions for a clean process, using renewable solvents and enzymes, for the extraction of phenolics and proteins from flaxseed meal. After a screening of the most promising commercial preparations based on different carbohydrases and proteases, a central composite rotatable design and a mixture design were applied, achieving as optimal results a solution containing 6.6 and 152 g kg−1  meal of phenolics and proteins, respectively. The statistical approach used in the present study for the enzyme-enhanced extraction of phenolics and proteins from the major flaxseed byproduct was effective. By means of the sequential experimental design methodology, the extraction of such compounds was increased 10-fold and 14-fold, when compared to a conventional nonenzymatic extraction.

An ethanol-based process to simultaneously extract and fractionate carotenoids from Mauritia flexuosa L. Pulp

Mauritia vinifera (buriti) e uma palmeira nativa de diferentes regioes do Brasil, particularmente na regiao Amazonica. O oleo de buriti e rico em carotenoides, especialmente em β-caroteno. A demanda por fontes naturais de β-caroteno tem contribuido para aumentar a industrializacao do fruto de buriti pelas usinas de extracao de oleos vegetais. O processo mais adotado baseia-se em tecnologias convencionais envolvendo as etapas de despolpamento, secagem e prensagem da polpa para extracao do oleo, seguida da separacao dos carotenoides em uma fase liquida usando solventes orgânicos. Neste trabalho, foi avaliado o uso do etanol comercial para extracao e fracionamento simultâneo de carotenoides a partir da polpa de buriti. A materia-prima foi misturada com etanol, na proporcao 1:4 solvente/substrato, e mantida em banho termostatizado a 60oC, por 1 hora, sob agitacao constante de 30 rpm. A mistura foi filtrada sob vacuo e resfriada a 10oC resultando na formacao de duas fases. A composicao de carotenoides foi determinada por CLAE e indicou uma concentracao de β-caroteno 12 vezes maior na fase mais densa que na fase leve. O perfil de carotenoides foi similar ao obtido no oleo bruto de buriti, porem a concentracao de carotenoides totais foi 40% maior, indicando que o processo tecnologico avaliado e particularmente promissor para aplicacao industrial.

APPLICATION OF FOAM COLUMN AS GREEN TECHNOLOGY FOR CONCENTRATION OF SAPONINS FROM SISAL (Agave sisalana) AND JUÁ (Ziziphus joazeiro)

Abstract - Saponins, molecules classified as triterpenic or steroidal glycosides, are metabolites distributed in all the plant kingdom that can be used for the production of foods, cosmetics, and pharmaceuticals, as well as in soil bioremediation. Saponins are normally extracted from natural resources with water, ethanol and/or methanol, and then concentrated by liquid-liquid partitioning with n-butanol. An alternative concentration method is with a foam column, by which the saponins can be concentrated via preferential adsorption at a gas-liquid interface. Therefore, the objective of this work was the use of a foam column for the concentration of saponins from jua and sisal, evaluating parameters such as: initial working volume in the column, saponin concentration in the extracts from jua and sisal, air flow rate, pH, Raschig rings loading and operation time. When a gradient air flow rate and 25 g of Raschig rings were used, 82.6% of the jua saponins loaded onto the system were recovered in a 3.46-fold concentrated solution after 9 h of operation. Regarding sisal saponins, a concentration factor of 1.98 was observed with 90.5% of saponin recovery during 4.5 h of operation.

Biotechnology and green chemistry.

White biotechnology can be regarded as applied biocatalysis, with enzymes and microorganisms, aiming at industrial production of bulk and fine chemicals to food and animal feed additives. In its turn biocatalysis has many attractive features in the context of greenchemistry: mild reaction conditions (physiological pH and temperature); environmentally compatible catalysts and solvent (often water) combined with high activities; and chemo-, regio-, and stereoselectivities in multifunctional molecules. This affords processes which are shorter, generate less waste, and are, therefore, both environmentally and economically more attractive than conventional routes. This special issue includes aspects involving the use of white biotechnology (enzymes, microorganisms, and plant tissues) within the green chemistry concept, concerning the use of alternative solvents (supercritical fluids, pressurized gases, ionic liquids, and micellar systems) and energies (microwaves and ultrasound); sustainable approaches for production of fine and bulk chemicals (aromas, polymers, pharmaceuticals, and enzymes); use of renewable resources or agroindustrial residues; biocatalysts recycling; and waste minimization. This special issue contains six papers, where three are related to green synthesis of nanoparticles and one paper covers biological pretreatment for enzymatic hydrolysis and bioethanol production. Two papers regard the use of alternative reaction systems. In the first paper entitled “Green synthesis of silver nanoparticles using Pinus eldarica bark extract,” S. Iravani and B. Zolfaghari present the optimization of the biosynthesis production of silver nanoparticles through the evaluation of Pinus eldarica bark extract quantity, substrate concentration, temperature, and pH on the formation of such material. The preparation of nanostructured silver particles using P. eldarica bark extract provides an environmentally friendly option, as compared to currently available chemical and/or physical methods. The second paper, “Green and rapid synthesis of anticancerous silver nanoparticles by Saccharomyces boulardii and insight into mechanism of nanoparticle synthesis,” by A. Kaler et al. describes an ecofriendly method for the synthesis of silver nanoparticles (AgNPs) by cell free extract (CFE) of Saccharomyces boulardii. In addition to the optimization of relevant synthesis parameters as culture age, cell mass concentration, temperature, and reaction time, the paper presents particles characterization by UV-Visible spectroscopy, EDX (energy dispersive X-Rays) analysis, transmission electron microscopy, and zeta potential, as well as the elucidation of proteins/peptides role in nanoparticles formation and stability and their anticancer activity. The method therein described a method that does not require tedious downstream processing and it may be scaled up to develop a viable technology for the Ag-nanoparticle synthesis. In the third paper, “Biosynthesis, antimicrobial and cytotoxic effect of silver nanoparticles using a novel Nocardiopsis sp. MBRC-1,” P. Manivasagan et al. present another green approach for biosynthesis of nanoparticles using the culture supernatant of Nocardiopsis sp. MBRC-1 to achieve the reduction of silver ions from a silver nitrate solution. The obtained nanoparticles were characterized by UV-visible, TEM, FE-SEM, EDX, FTIR, and XRD spectroscopy. The prepared silver nanoparticles exhibited strong antimicrobial activity against bacteria and fungi. Cytotoxicity of biosynthesized AgNPs against in vitro human cervical cancer cell line (HeLa) showed a dose-response activity. In the fourth paper, “Biological pretreatment of rubberwood with Ceriporiopsis subvermispora for Enzymatic hydrolysis and bioethanol production,” F. Nazarpour et al. investigate a novel feedstock for enzymatic hydrolysis and bioethanol production using biological pretreatment: rubberwood (Hevea brasiliensis). To improve ethanol production, rubberwood was pretreated with white rot fungus Ceriporiopsis subvermispora to increase fermentation efficiency. The fungal pretreatment provides a cost-effective method for reducing the recalcitrance of rubberwood with high selectivity of lignin degradation rate and minimal cellulose loss for enzymatic hydrolysis and bioethanol production. In the fifth paper, the research of G. D. Yadav and S. Devendran entitled “Microwave assisted enzymatic kinetic resolution of (±)-1-phenyl-2-propyn-1-ol in non-aqueous media,” proposes a kinetic resolution of 1-phenyl-2-propyn-1-ol, an important chiral synthon, through esterification with acyl acetate. The authors investigate synergism between microwave irradiation and enzyme catalysis. The lipase (Novozym 435) catalyzed kinetic resolution under microwave irradiation. The maximum conversion of 48.78% was obtained in 2 h using 10 mg enzyme loading with equimolar concentration of alcohol and ester at 60°C under microwave irradiation. From the progress curve analysis, it was found that reaction followed the ping-pong bi-bi mechanism with dead end inhibition of alcohol. Beside the previous papers herein described, the preparation of chiral secondary alcohols using lipase catalyzed kinetic resolution is mild and clean as compared to chemical process. In the seventh paper entitled “Demonstration of redox potential of Metschnikowia koreensis for stereoinversion of secondary alcohols/1,2-diols,” by V. S. Meena et al. reports the Metschnikowia koreensis-catalyzed one-pot deracemization of secondary alcohols/1,2-diols and their derivatives with in vivo cofactor regeneration. This ecofriendly method afforded the product in high yield (88%) and excellent optical purity (>98% ee), minimizing the requirement of multistep reaction and expensive cofactor. Bernardo Dias Ribeiro Isabel Marrucho Luciana Goncalves Maria Alice Z. Coelho

Obtenção de extratos de guaraná ricos em cafeína por processo enzimático e adsorção de taninos

Summary Guarana-flavoured beverages are very popular in Brazil and have shown an excellent sales potential on foreign markets. According to Brazilian law, each 100 mL of guarana-flavoured beverages must contain between 0.02 g and 0.2 g of guarana seed or its equivalent. These levels are normally obtained by adding a concentrated hydroalcoholic extract or sugar syrup containing guarana extract, directly to the beverage. However, the use of more concentrated extracts is limited by the presence of tannins, which imparts astringency and a dark colour to the final product. In this work the development of an enzymatic process to obtain non-alcoholic guarana extracts with low tannin concentrations and high caffeine contents was studied using an experimental design and adsorption processes. By way of a fractional factorial design the quantities of 0.25% (v/v) pectinase and 0.1% (v/v) glucoamylase were determined, which were maintained in the central composite design, obtaining as the optimal conditions: 0.23% (v/v) cellulase, 0.86% (v/v) hemicellulase, 1% (v/v) alpha-amylase, 5.5 h extraction time, 200 rpm and 50 °C, producing a caffeine/tannin ratio of 1.65. Using a magnesium oxide adsorption process at 10% (w/v), a caffeine/tannin ratio of 7.3 was obtained.

Culture Miniaturization of Lipase Production by Yarrowia lipolytica

Background: The proposal to perform scale-down of culture systems (2 to 4 mL) could be more efficient for screen multiple formulations and operational conditions. For such, is important to evaluate if the kinetic parameters of a bioprocess are comparative to conventional lab-scale reactors. In the present study, the effects of different miniaturized systems were evaluated on growth and lipase production of Yarrowia lipolytica IMUFRJ 50682. Methods: Cultivations were conducted in a mini-scale version of Erlenmeyer flask of 10 mL (10EF); 24 deep-well microplate of 11 ml (24MTP/11 mL) and 24 deep-well microplate of 25 ml (24MTP/25 mL). Results: Similar specific growth rate (μ) was observed between miniaturized cultivations (0.27 h). Different lipase productivities values were obtained, the highest was achieved in 10EF (181 U.L -1 .h -1 ). Volumetric oxygen transfer coefficient (kLa) and maximum lipase production were improved with culture miniaturization in comparison with conventional shake-flasks. Bioreactor (1.5 L) cultivation showed similar growth kinetic, pH profile and kLa values, but an increase in the maximum productivity was observed. These finding showed the advantages of Y. lipolytica cultivations scale-down. Conclusion: These findings show the advantages of Y. lipolytica scale-down cultivations, and to perform scale-up for benchtop bioreactor directly from these miniaturized cultivations, without the need of gradual scale-up, representing a reduction in costs and working time. Taking into consideration the rare works about miniaturized cultivation with Y. lipolytica, this work opens the way to a better understanding of bioprocess and alternatives for process conduction. A R T I C L E H I S T O R Y Received: March 26, 2018 Revised: July 10, 2018 Accepted: July 16, 2018 DOI: 10.2174/2212711905666180730101010

CHAPTER 1:Principles of Green Chemistry and White Biotechnology

White Biotechnology can be regarded as Applied Biocatalysis, with enzymes and microorganisms, aiming at industrial production from bulk and fine chemicals to food and animal feed additives. In your turn, Biocatalysis has many attractive features in the context of Green Chemistry: mild reaction conditions (physiological pH and temperature), environmentally compatible catalysts and solvent (often water) combined with high activities and chemo-, regio- and stereoselectivities in multifunctional molecules. This affords processes which are shorter, generate less waste and are, therefore, both environmentally and economically more attractive than conventional routes. The objective of this chapter is to comprise a brief introduction of the classification of biotechnology areas, including white biotechnology, as well as present enzyme classification and markets, and green chemistry principles, which are the basis of this book.

CHAPTER 15:Trends and Perspectives in Green Chemistry and White Biotechnology

Innovation is one of challenges of development new technologies. In this chapter, some trends and perspectives will be covered showing that other non-thermal energy (ultrasound) can be used with biotechnology, besides neoteric solvents (fluorous and tunable solvents, aphrons, glymes, liquid polymers, alkyl carbonates) and new applications such as biodesalination and nanotechnology.