Biotransformation of ethylene glycol to glycolic acid by Yarrowia lipolytica: A route for poly(ethylene terephthalate) (PET) upcycling.

Biological recycling of PET waste has been extensively investigated recently to tackle plastic waste pollution, and ethylene glycol (EG) is one of the main building blocks recovered from this process. Wild-type Yarrowia lipolytica IMUFRJ 50682 can be a biocatalyst to biodepolymerize PET. Herein, we report its ability to perform oxidative biotransformation of EG into glycolic acid (GA): a higher value-added chemical with varied industrial applications. We found that this yeast tolerates high EG concentrations (up to 2 M) based on maximum non-inhibitory concentration (MNIC) tests. Whole-cell biotransformation assays using resting yeast cells showed GA production uncoupled to cell growth metabolism, and 13 C nuclear magnetic resonance (NMR) analysis confirmed GA production. Moreover, higher agitation speed (450 vs. 350 rpm) resulted in a 1.12-fold GA production improvement (from 352 to 429.5 mM) during Y. lipolytica cultivation in bioreactors after 72 h. GA was constantly accumulated in the medium, suggesting that this yeast may also share an incomplete oxidation pathway (i.e., it is not metabolized to carbon dioxide) as seen in acetic acid bacterial group. Additional assays using higher chain-length diols (1,3-propanediol, 1,4-butanediol, and 1,6-hexanediol) revealed that C4 and C6 diols were more cytotoxic, suggesting that they underwent different pathways in the cells. We found that this yeast consumed extensively all these diols, however, 13 C NMR analysis from supernatant identified solely the presence of 4-hydroxybutanoic acid from 1,4-butanediol, along with GA from EG oxidation. Findings reported herein reveal a potential route for PET upcycling to a higher value-added product.

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.

Novel efficient enzymatic synthesis of the key-reaction intermediate of PET depolymerization, mono(2-hydroxyethyl terephthalate) - MHET.

Poly(ethylene terephthalate) (PET) is one of the main synthetic plastics produced worldwide. The extensive use of this polymer causes several problems due to its low degradability. In this scenario, biocatalysts dawn as an alternative to enhance PET recycling. The enzymatic hydrolysis of PET results in a mixture of terephthalic acid (TPA), ethylene glycol (EG), mono-(2-hydroxyethyl) terephthalate (MHET) and bis-(2-hydroxyethyl) terephthalate (BHET) as main products. This work developed a new methodology to quantify the hydrolytic activity of biocatalysts, using BHET as a model substrate. The protocol can be used in screening enzymes for PET depolymerization reactions, amongst other applications. The very good fitting (R2 = 0.993) between experimental data and the mathematical model confirmed the feasibility of the Michaelis-Menten equation to analyze the effect of BHET concentration (8-200 mmol L-1) on initial hydrolysis rate catalyzed by Humicola insolens cutinase (HiC). In addition to evaluating the effects of enzyme and substrate concentration on the enzymatic hydrolysis of BHET, a novel and straightforward method for MHET synthesis was developed using an enzyme load of 0.025 gprotein gBHET-1 and BHET concentration of 60 mmol L-1 at 40 °C. MHET was synthesized with high selectivity (97 %) and yield (82 %). The synthesized MHET properties were studied using differential scanning calorimetry (DSC), thermogravimetry (TGA), and proton nuclear magnetic resonance (1H NMR), observing the high purity of the final product (86.7 %). As MHET is not available commercially, this synthesis using substrate and enzyme from open suppliers adds new perspectives to monitoring PET hydrolysis reactions.

Experimental and mathematical modeling approaches for biocatalytic post-consumer poly(ethylene terephthalate) hydrolysis.

The environmental impact arising from poly(ethylene terephthalate) (PET) waste is notable worldwide. Enzymatic PET hydrolysis can provide chemicals that serve as intermediates for value-added product synthesis and savings in the resources. In the present work, some reaction parameters were evaluated on the hydrolysis of post-consumer PET (PC-PET) using a cutinase from Humicola insolens (HiC). The increase in PC-PET specific area leads to an 8.5-fold increase of the initial enzymatic hydrolysis rate (from 0.2 to 1.7 mmol L-1 h-1), showing that this parameter plays a crucial role in PET hydrolysis reaction. The effect of HiC concentration was investigated, and the enzymatic PC-PET hydrolysis kinetic parameters were estimated based on three different mathematical models describing heterogeneous biocatalysis. The model that best fits the experimental data (R2 = 0.981) indicated 1.68 mgprotein mL-1 as a maximum value of the enzyme concentration to optimize the reaction rate. The HiC thermal stability was evaluated, considering that it is a key parameter for its efficient use in PET degradation. The enzyme half-life was shown to be 110 h at 70 ºC and pH 7.0, which outperforms most of the known enzymes displaying PET hydrolysis activity. The results evidence that HiC is a very promising biocatalyst for efficient PET depolymerization.

A critical view on the technology readiness level (TRL) of microbial plastics biodegradation.

Accumulation of plastic wastes and their effects on the ecosystem have triggered an alarm regarding environmental damage, which explains the massive investigations over the past few years, aiming technological alternatives for their proper destination and valorization. In this context, biological degradation emerges as a green route for plastic processing and recycling in a circular economy approach. Some of the main polymers produced worldwide are poly(ethylene terephthalate) (PET), polyethylene (PE) and polypropylene (PP), which are among the most recalcitrant materials in the environment. In comparison to other polymers, PET biodegradation has advanced dramatically in recent years concerning microbial and enzymatic mechanisms, being positioned in a higher technology readiness level (TRL). Even more challenging, polyolefins (PE and PP) biodegradation is hindered by their high recalcitrance, which is mainly related to stable carbon-carbon bonds. Potential microbial biocatalysts for this process have been evaluated, but the related mechanisms are still not fully elucidated. This review aims to discuss the latest developments on key microbial biocatalysts for degradation of these polymers, addressing biodegradation monitoring, intellectual property, and TRL analysis of the bioprocessing strategies using biodegradation performance, process time and scale as parameters for the evaluation.

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.

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.

Process strategies to improve biocatalytic depolymerization of post-consumer PET packages in bioreactors, and investigation on consumables cost reduction.

Massive plastics production has raised concerns about low recycling rates and disposal of these materials in nature, causing environmental and economic impacts. Poly(ethylene terephthalate) (PET) is one of main polymers used for manufacture of plastic packaging (e.g. bottles, trays). Enzymatic recycling of PET has been a route of increasing study aiming at to recover its monomers (terephthalic acid and ethylene glycol), resulting in a circular production chain. In this study, investigation of pH control and fractionation of enzyme feeding were explored in post-consumed PET (PC-PET) hydrolysis reactions catalyzed by Humicola insolens cutinase (HiC) in stirred reactors. It was found that the unbuffered reaction provided of pH control by 0.5 M NaOH addition showed 2.39-fold improvement in the released monomers (to a total of 26.3 mM), comparatively to the Tris-HCl-buffered reaction. In addition, it was observed a possibility of reducing the enzyme loading used in the process by half, leading to an increase of 2.41-fold in the specific terephthalic acid concentration released per protein amount, whilst maintaining a high products concentration (97 mM). A simplified cost analysis of reaction consumables was performed, and the data reported here demonstrates that these alternative process strategies contribute to costs reduction on the enzymatic depolymerization reactions of PET.

Investigation of mitochondrial protein expression profiles of Yarrowia lipolytica in response to citric acid production.

Nitrogen-limiting condition is essential for citric acid production by Yarrowia lipolytica. Mitochondrial protein expression profiles of Y. lipolytica IMUFRJ 50,682 cells cultivated in biomass proliferation medium (YPG medium, yeast extract, peptone and glycerol) and citric acid production medium (CA medium) were analyzed to identify differences in expressed proteins in response to medium composition. The identification of 45 proteins in mitochondria of YPG medium cells and 48 proteins in mitochondria of CA medium cells were possible with proteomic analyses. Only 11 proteins were common to both conditions, showing a different expression pattern in relation to limiting and non-limiting nitrogen conditions. For both conditions, most proteins (52%-CA medium, 46%-YPG medium) were related to energy metabolism. CA medium cells expressed more carbohydrate metabolism proteins (six proteins) then YPG medium cells (three proteins) and the opposite was detected for translation proteins.

Growth Parameters and Survivability of Saccharomyces boulardii for Probiotic Alcoholic Beverages Development.

The aim of this research was to optimize the growth parameters (pH, ethanol tolerance, initial cell concentration and temperature) for Saccharomyces boulardii and its tolerance to in vitro gastrointestinal conditions for probiotic alcoholic beverage development. Placket-Burman screening was used to select only statistically significant variables, and the polynomial mathematical model for yeast growth was obtained by central composite rotatable design. Confirmation experiments to determine the kinetic parameters for yeast growth were carried out by controlling the temperature and pH. Soon after, the survivability of yeast was tested under in vitro conditions mimicking the human upper gastrointestinal transit. S. boulardii had suitable resistance to alcohol and gastrointestinal conditions for probiotic alcoholic beverage development.

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.

Factors influencing crude oil biodegradation by Yarrowia lipolytica

Yarrowia lipolytica is unique strictly aerobic yeast with the ability to efficiently degrade hydrophobic substrates such as n-alkenes, fatty acids, glycerol and oils. In the present work, a 2(4) full factorial design was used to investigate the influence of the independent variables of temperature, agitation, initial cell concentration and initial petroleum concentration on crude oil biodegradation. The results showed that all variables studied had significant effects on the biodegradation process. Temperature, agitation speed and initial cell concentration had positive effects, and initial petroleum concentration had a negative effect. Among the crude oil removal conditions studied, the best temperature and agitation conditions were 28ºC and 250 rpm, respectively.

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.

Factorial design to optimize biosurfactant production by Yarrowia lipolytica.

In order to improve biosurfactant production by Yarrowia lipolytica IMUFRJ 50682, a factorial design was carried out. A 2(4) full factorial design was used to investigate the effects of nitrogen sources (urea, ammonium sulfate, yeast extract, and peptone) on maximum variation of surface tension (Delta ST) and emulsification index (EI). The best results (67.7% of EI and 20.9 mN m(-1) of Delta ST) were obtained in a medium composed of 10 g 1(-1) of ammonium sulfate and 0.5 g 1(-1) of yeast extract. Then, the effects of carbon sources (glycerol, hexadecane, olive oil, and glucose) were evaluated. The most favorable medium for biosurfactant production was composed of both glucose (4% w/v) and glycerol (2% w/v), which provided an EI of 81.3% and a Delta ST of 19.5 mN m(-1). The experimental design optimization enhanced Delta EI by 110.7% and Delta ST by 108.1% in relation to the standard process.

Characterization of commercial amylases for the removal of filter cake on petroleum wells.

Drilling fluid has many functions, such as carry cuttings from the hole permitting their separation at the surface, cool and clean the bit, reduce friction between the drill pipe and wellbore, maintain the stability of the wellbore, and prevent the inflow of fluids from the wellbore and form a thin, low-permeable filter cake. Filter cake removal is an important step concerning both production and injection in wells, mainly concerning horizontal completion. The drilling fluids are typically comprised of starch, the most important component of the filter cake. A common approach to remove this filter cake is the use of acid solutions. However, these are non-specific reactants. A possible alternative is the use of enzymatic preparations, like amylases, that are able to hydrolyze starch. Wells usually operate in drastic conditions for enzymatic preparations, such as high temperature, high salt concentration, and high pressure. Thus, the main objective of this work was to characterize four enzymatic preparations for filter cake removal under open hole conditions. The results showed that high salt concentrations (204,000 ppm NaCl) in completion fluid decreased amylolytic activity. All enzymatic preparations were able to catalyze starch hydrolysis at all temperatures tested (30, 65, 80, and 95 degrees C). An increase of amylolytic activity was observed with the increase of pressure (100, 500 and 1,000 psi) for one commercial amylase.