Post-Consumer Poly(ethylene terephthalate) (PET) Depolymerization by Yarrowia lipolytica: A Comparison between Hydrolysis Using Cell-Free Enzymatic Extracts and Microbial Submerged Cultivation.

Several microorganisms have been reported as capable of acting on poly(ethylene terephthalate) (PET) to some extent, such as Yarrowia lipolytica, which is a yeast known to produce various hydrolases of industrial interest. The present work aims to evaluate PET depolymerization by Y. lipolytica using two different strategies. In the first one, biocatalysts were produced during solid-state fermentation (SSF-YL), extracted and subsequently used for the hydrolysis of PET and bis(2-hydroxyethyl terephthalate) (BHET), a key intermediate in PET hydrolysis. Biocatalysts were able to act on BHET, yielding terephthalic acid (TPA) (131.31 µmol L-1), and on PET, leading to a TPA concentration of 42.80 µmol L-1 after 168 h. In the second strategy, PET depolymerization was evaluated during submerged cultivations of Y. lipolytica using four different culture media, and the use of YT medium ((w/v) yeast extract 1%, tryptone 2%) yielded the highest TPA concentration after 96 h (65.40 µmol L-1). A final TPA concentration of 94.3 µmol L-1 was obtained on a scale-up in benchtop bioreactors using YT medium. The conversion obtained in bioreactors was 121% higher than in systems with SSF-YL. The results of the present work suggest a relevant role of Y. lipolytica cells in the depolymerization process.

Improved production of biocatalysts by Yarrowia lipolytica using natural sources of the biopolyesters cutin and suberin, and their application in hydrolysis of poly (ethylene terephthalate) (PET).

Since plastic pollution emerged as an urgent environmental problem, different biocatalysts have been tested for poly(ethylene terephthalate) (PET) hydrolysis. This work evaluated three different possible inducers for lipases and/or esterases, two natural sources of biopolymers (apple peels and commercial cork) and PET, as supplements in the solid-state fermentation of soybean bran by Yarrowia lipolytica. The obtained enzymatic extracts displaying different levels of lipase and esterase activities were then tested for PET depolymerization. Supplementation with 5 or 20 wt% of commercial cork led to an increase of 16% in lipase activity and to an increase of 131% in esterase activity, respectively. PET supplementation also led to an increase in the esterase activity of the enzymatic extracts (up to 69%). Enzymes produced in the screening step were able to act as biocatalysts in PET hydrolysis. Enzymatic extracts obtained in fermentation samples supplemented with 20 wt% PET and 20 wt% apple peels led to the highest terephthalic acid concentration (21.2 µmol L-1) in 7 days, whereas enzymes produced in commercial cork media were more efficient for bis(2-hydroxyethyl) terephthalate (BHET) hydrolysis, one of the key-PET hydrolysis intermediates. Results suggest a good potential of the biocatalysts produced by Y. lipolytica IMUFRJ 50,682 in a low-cost media for subsequent utilization in PET depolymerization reactions. This is one of the few reports on the use of a yeast for this application.

Choline chloride-based deep eutectic solvent as an inhibitor of metalloproteases (collagenase and elastase) in cosmetic formulation.

Green chemistry and engineering are potential alternatives for achieving higher sustainability and lower generation of hazardous compounds in chemical product design, production, and use. Deep Eutectic Solvents (DES) are characterized as green solvents and have become increasingly attractive due to their characteristic design solvents. In this work, two DES (choline chloride (ChCl)/glycerol and ChCl/Urea), aqueous solutions of the DES-forming components, and green tea extracts obtained with DES were used as anti-ageing active in cosmetic products using in vitro tests to inhibit extracellular matrix metalloproteases (such as collagenase and elastase). Finally, the stability of the formulations with DES as a cosmetic active was also evaluated. The results showed that DES based on ChCl/Urea and ChCl/glycerol exhibited remarkable inhibition values of collagenase (91.1 and 92.7%, respectively) and elastase (49.8 and 45.7%, respectively). However, pure urea displayed better inhibition values (66%) for elastase, possibly due to its direct contribution to intramolecular hydrogen bonds. ChCl/glycerol showed remarkable stability in the average cube diameter values, which may indicate no change in the conformation of the micellar structure of the cosmetic formulation. Moreover, the formulation containing this DES remained stable at room temperature. Given the remarkable results, DES can be applied in cosmetic products for anti-ageing purposes.

Recovery of ß-carotene from pumpkin using switchable natural deep eutectic solvents.

The aim of the present research was to develop green and sustainable extraction procedure for ß-carotene recovery from pumpkin. A series of hydrophobic natural deep eutectic solvents (NADESs) based onfatty acids were prepared to establish high extraction efficiency of ß-carotene and to increase stability of extracted carotenoids from the pumpkin. To intensify extraction process, NADES composed of C8 and C10 fatty acids (3:1) was selected and coupled with ultrasound assisted extraction. Response surface methodology and artificial neural network model (ANN) model was adopted to analyze significance of extraction parameters demonstrating high prediction levels of the ß-carotene yield, experimentally confirming the maximum ß-carotene content of 151.41 µg/mL at the optimal process condition. Extracted carotenoids in the optimal NADES extract have shown high stability during the storing period of 180 days. A switchable-hydrophilicity eutectic solvent system has been introduced as a successful way to recover extracted carotenoids from the NADES solvent. It was capable of precipitating 90% of carotenoids present in the extract. The proposed procedure is simple, easily scalable and has minimal impact on operators and the environment.

Decolorization and detoxification of different azo dyes by Phanerochaete chrysosporium ME-446 under submerged fermentation.

Azo dyes are widely used in the textile industry due to their resistance to light, moisture, and oxidants. They are also an important class of environmental contaminant because of the amount of dye that reaches natural water resources and because they can be toxic, mutagenic, and carcinogenic. Different technologies are used for the decolorization of wastewater containing dyes; among them, the biological processes are the most promising environmentally. The aim of this study was to evaluate the potential of Phanerochaete chrysosporium strain ME-446 to safely decolorize three azo dyes: Direct Yellow 27 (DY27), Reactive Black 5 (RB5), and Reactive Red 120 (RR120). Decolorization efficiency was determined by ultraviolet-visible spectrophotometry and the phytotoxicity of the solutions before and after the fungal treatment was analyzed using Lactuca sativa seeds. P. chrysosporium ME-446 was highly efficient in decolorizing DY27, RB5, and RR120 at 50 mg L-1, decreasing their colors by 82%, 89%, and 94% within 10 days. Removal of dyes was achieved through adsorption on the fungal mycelium as well as biodegradation, inferred by the changes in the dyes' spectral peaks. The intensive decolorization of DY27 and RB5 corresponded to a decrease in phytotoxicity. However, phytotoxicity increased during the removal of color for the dye RR120. The ecotoxicity tests showed that the absence of color does not necessarily translate to an absence of toxicity.

Biological Approaches for Extraction of Bioactive Compounds From Agro-industrial By-products: A Review.

Bioactive compounds can provide health benefits beyond the nutritional value and are originally present or added to food matrices. However, because they are part of the food matrices, most bioactive compounds remain in agroindustrial by-products. Agro-industrial by-products are generated in large quantities throughout the food production chain and can-when not properly treated-affect the environment, the profit, and the proper and nutritional distribution of food to people. Thus, it is important to adopt processes that increase the use of these agroindustrial by-products, including biological approaches, which can enhance the extraction and obtention of bioactive compounds, which enables their application in food and pharmaceutical industries. Biological processes have several advantages compared to nonbiological processes, including the provision of extracts with high quality and bioactivity, as well as extracts that present low toxicity and environmental impact. Among biological approaches, extraction from enzymes and fermentation stand out as tools for obtaining bioactive compounds from various agro-industrial wastes. In this sense, this article provides an overview of the main bioactive components found in agroindustrial by-products and the biological strategies for their extraction. We also provide information to enhance the use of these bioactive compounds, especially for the food and pharmaceutical industries.

Enzyme-assisted extraction of carotenoids and phenolic compounds from sunflower wastes using green solvents.

The aim of this work is to develop an optimized enzymatic assisted extraction methodology to extract carotenoids and phenolic compounds from sunflower wastes (petals and florets) using natural hydrophobic green solvents. Several natural green hydrophobic solvents were used as well as natural hydrophobic eutectic solvents composed of d,l-menthol and different acids, with different hydrophobicity. The multi-enzyme complex Viscozyme® was used to disrupt the cell wall of petals and disc florets. The extracted carotenoids content into the hydrophobic phase was quantified using UV-Vis spectrophotometry and the carotenoids profile was studied using high-performance liquid and thin layer chromatography. The amount of total sugars in the aqueous phase was also analyzed using the dinitrosalicylic acid (DNS) method to infer about the enzymatic action in cell wall. Phenolic compounds also in the aqueous phase were analyzed by Folin Denis method. The eutectic solvent d,l-menthol:d,l-lactic acid (M:HLac) (1:2) was the best solvent for extraction of carotenoids from sunflower wastes, with 147 ppm of carotenoids extracted, in comparison to 115 ppm obtained with the standard solvent, n-hexane. In what concerns phenolic compounds, M:HLac was again better than the standard solvent. The use of the multi-enzyme complex Viscozyme® had different responses, depending on the solvent tested. For the green solvent M:HLac, the enzyme improved the carotenoids extraction, achieving 335 ppm of carotenoids in the extract. The role of enzyme, solvent, water and sunflower quantity in the carotenoid extraction was evaluated and optimized through a central composite rotatable design (CCRD), using the M:HLac as solvent. According to the analysis of CCRD, the most efficient extractions were carried out using more solvent and less raw material, whose best result reached 1449 mg carotenoids/100 g biomass ppm of carotenoids. This work emphasizes the possibility of developing more sustainable enzyme-assisted separation processes, through the substitution of toxic solvents with natural, environmentally friendly, solvents.

Construction of wild-type Yarrowia lipolytica IMUFRJ 50682 auxotrophic mutants using dual CRISPR/Cas9 strategy for novel biotechnological approaches.

Yarrowia lipolytica IMUFRJ 50682 is a Brazilian wild-type strain with potential application in bioconversion processes which can be improved through synthetic biology. In this study, we focused on a combinatorial dual cleavage CRISPR/Cas9-mediated for construction of irreversible auxotrophic mutants IMUFRJ 50682, which genomic information is not available, thought paired sgRNAs targeting upstream and downstream sites of URA3 gene. The disruption efficiency ranged from 5 to 28 % for sgRNAs combinations closer to URA3's start and stop codon and the auxotrophic mutants lost about 970 bp containing all coding sequence, validating this method for genomic edition of wild-type strains. In addition, we introduced a fluorescent phenotype and achieved cloning rates varying from 80 to 100 %. The ura3Δ strains IMUFRJ 50682 were also engineered for ß-carotene synthesis as proof of concept. Carotenoid-producing strains exhibited a similar growth profile compared to the wild-type strain and were able to synthesized 30.54-50.06 mg/L (up to 4.8 mg/g DCW) of ß-carotene in YPD and YNB flask cultures, indicating a promisor future of the auxotrophic mutants IMUFRJ 50682 as a chassis for production of novel value-added chemicals.

Characterization and Application of Yarrowia lipolytica Lipase Obtained by Solid-State Fermentation in the Synthesis of Different Esters Used in the Food Industry.

Yarrowia lipolytica lipase obtained by solid-state fermentation was characterized and applied in the synthesis of esters with commercial value in the food industry. The effect of different conditions on the hydrolysis activity of this biocatalyst was evaluated in the presence of metal ions, solvents, detergents, several pH and temperature parameters, and different substrates. Storage stability was also studied. The solid biocatalyst produced in soybean meal was used in synthesis reactions aiming to produce short-, medium-, and long-chain esters. Results showed that the best fermentation condition to produce the biocatalyst was using soybean oil (3% w/w), moisture content (55% w/v), and inoculum of 2.1 mgdry biomass/gsoybean meal at 28 °C for 14 h. High substrate conversion for ethyl octanoate, cetyl stearate, and stearyl palmitate synthesis was achieved in the presence of non-polar solvents in less than 6 h using a substrate molar ratio of 1:1 at 38 °C with 10-15% (w/v) of biocatalyst. This work showed the high potential of Y. lipolytica lipase to be used in the synthesis of different esters. Also, that it can be considered an attractive and economical process alternative to obtain high-added value products.

Biocatalytic esterification of fatty acids using a low-cost fermented solid from solid-state fermentation with Yarrowia lipolytica.

This study aimed to evaluate the use of a lyophilized fermented solid (named solid enzymatic preparation, SEP), with lipase activity, as a low-cost biocatalyst for esterification reactions of fatty acids present in acid raw materials for biodiesel synthesis. The SEP was obtained by solid-state fermentation (SSF) of soybean bran using the strain of Yarrowia lipolytica IMUFRJ 50682 and contains the lipases secreted by this yeast. The esterification reaction of ethanol and the predominant fatty acids present in different acid oil sources for biodiesel production (oleic, linoleic, stearic and palmitic acids) was investigated. Oleic acid conversion of above 85% was obtained after 24 h, using 30 wt% of SEP and ethanol/oleic acid molar ratio of 1, at 30 °C, in a reaction medium with and without solvent (n-hexane). Similar results were achieved with stearic (79%), palmitic (82%) and linoleic (90%) acids. The reusability of SEP was investigated over ten successive batches by washing it with different solvents (ethanol, water or n-hexane) between the cycles of ethyl oleate synthesis. Washing with water allowed the SEP to be reused for six cycles maintaining over 80% of the conversion reached in the first cycle. These results show the potential of this biocatalyst to reduce the content of free fatty acids in acid oils for biodiesel synthesis with a potential to be applied in a broad plethora of raw materials.

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.

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.