Valorization of bioethanol by-products to produce unspecific peroxygenase with Agrocybe aegerita: Technological and proteomic perspectives

Unspecific peroxygenase (UPO) presents a wide range of biotechnological applications. This study targets the use of by-products from bioethanol synthesis to produce UPO by Agrocybe aegerita. Solid-state and submerged fermentations (SSF and SmF) were evaluated, achieving the highest titers of UPO and laccase in SmF using vinasse as nutrients source. Optimized UPO production of 331 U/L was achieved in 50% (v:v) vinasse with an inoculum grown for 14 days. These conditions were scaled-up to a 4 L reactor, achieving a UPO activity of 265 U/L. Fungal proteome expression was analyzed before and after UPO activity appeared by shotgun mass spectrometry proteomics. Laccase, dye-decolorizing peroxidases (DyP), lectins and proteins involved in reactive oxygen species (ROS) production and control were detected (in addition to UPO). Interestingly, the metabolism of complex sugars and nitrogen sources had a different activity at the beginning and end of the submerged fermentationS.G., A.T. and G.E. thank their grants (BES-2017-081677, FJC2019-041664-I and RYC2018-024846-I, respectively) funded by MCIN/AEI/ 10.13039/501100011033, and by “ERDF A way of making Europe” and “ESF Investing in your future”. Authors would like to thank the use of USC Mass Spectrometry and Proteomics facilities and Bioetanol Galicia S.A. for the supply of the substrates used in the fermentations. The authors belong to the Galician Competitive Research Groups (GRC)_ ED431C-2021/37. The program is co-funded by FEDER (UE)S

Electrochemical oxidation of lignin for the simultaneous production of bioadhesive precursors and value-added chemicals

Electrochemical oxidation of lignin has been widely regarded as a clean and reliable alternative to obtain value-added products from lignin, such as vanillin or guaiacol. This work aims to go one step beyond the production of low molecular weight molecules and explore the possibility of using lignin residues from electrochemical treatments in the context of biorefinery. To this end, a two-way valorization of lignin by electrochemical oxidation is proposed, in order to obtain a liquid phase enriched in low molecular weight organic oligomers and a solid phase of modified lignin to be used as bioadhesive precursor. Hydroxylation of lignin by electrochemical oxidation using boron-doped diamond (BDD) anodes was observed according to the FTIR and MALDI-TOF results, concluding that an applied current density of 10 mA cm−2 leads to promising modifications for the formulation of bioadhesives. Furthermore, NIPU bioadhesives with electrochemically modified lignin were successfully prepared and tested for use in particleboard panels, showing satisfactory mechanical properties, and thus paving the way for more environmentally friendly lignin modification procedures for the wood industryJJC acknowledges financial support from Galician Government though a postdoctoral fellowship (ED481B-2021/015). SG-R and GE predoctoral and postdoctoral fellowships (BES-2017-081677 and RYC-2018-024846-I, respectively) were funded by MCIN/AEI/10.13039/501100011033 and by “ESF Investing in your future”. JJC, SG-R, TAL-C, GE and MTM belong to the Galician Competitive Research Group (GRC) ED431C-2021/37. LERMAB is financed by the French Agence Nationale de la Recherche (ANR) as part of the laboratory of excellence (LABEX) ARBRE. The authors would also like to thank the use of the analytical facilities of IR-Raman Spectroscopy Unit and Mass Spectrometry Unit from RIAIDT-USC.S

Fungal pretreatment: An alternative in second-generation ethanol from wheat straw

7 páginas, 3 figuras, 2 tablas -- PAGS nros. 7500-7506The potential of a fungal pretreatment combined with a mild alkali treatment to replace or complement current physico-chemical methods for ethanol production from wheat straw has been investigated. Changes in substrate composition, secretion of ligninolytic enzymes, enzymatic hydrolysis efficiency and ethanol yield after 7, 14 and 21 days of solid-state fermentation were evaluated. Most fungi degraded lignin with variable selectivity degrees, although only eight of them improved sugar recovery compared to untreated samples. Glucose yield after 21 days of pretreatment with Poria subvermispora and Irpex lacteus reached 69% and 66% of cellulose available in the wheat straw, respectively, with an ethanol yield of 62% in both cases. Conversions from glucose to ethanol reached around 90%, showing that no inhibitors were generated during this pretreatment. No close correlations were found between ligninolytic enzymes production and sugar yieldsThis work was supported mainly by the CENIT I+DEA project (funded by CDTI, Spain) and carried out in collaboration with Abengoa Bionergía Nuevas Tecnologías. Authors thank also the Galician government (I. Barreto program), DEMO-2 and Lignodeco EU projects for additional supports, and Novozymes for providing commercial enzymes. D.S. thanks a FPU fellowship from the MICINNPeer reviewe

Bundling the removal of emerging contaminants with the production of ligninolytic enzymes from residual streams

Enzymes offer interesting features as biological catalysts for industry: high specificity, activity under mild conditions, accessibility, and environmental friendliness. Being able to produce enzymes in large quantities and having them available in a stable and reusable form reduces the production costs of any enzyme-based process. Agricultural residues have recently demonstrated their potential as substrates to produce ligninolytic enzymes by different white rot fungi. In this study, the biotechnological production of a manganese peroxidase (MnP) by Irpex lacteus was conducted through solid-state fermentation (SSF) with wheat straw as substrate and submerged fermentation (SmF) employing wheat straw extract (WSE). The obtained enzyme cocktail also showed manganese-independent activity (MiP), related to the presence of a short MnP and a dye-decolorizing peroxidase (DyP) which was confirmed by shotgun proteomic analyses. In view of the enhanced production of ligninolytic enzymes in SmF, different parameters such as WSE concentration and nitrogen source were evaluated. The highest enzyme titers were obtained with a medium formulated with glucose and peptone (339 U/L MnP and 15 U/L MiP). The scale-up to a 30 L reactor achieved similar activities, demonstrating the feasibility of enzyme production from the residual substrate at different production scales. Degradation of five emerging pollutants was performed to demonstrate the high oxidative capacity of the enzyme. Complete removal of hormones and bisphenol A was achieved in less than 1 h, whereas almost 30% degradation of carbamazepine was achieved in 24 h, which is a significant improvement compared to previous enzymatic treatments of this compoundOpen Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This research was funded by the Spanish Ministry of Science and Innovation: MODENA Project (CTQ2016-79461-R) and the WooBAdh project (PCI2018-092866, ERA-CoBioTech program)S

Polymerization of coniferyl alcohol by Mn3+-mediated (enzymatic) oxidation : Effects of H2O2 concentration, aqueous organic solvents, and pH

The objective of this study was to evaluate the ability of one versatile peroxidase and the biocatalytically generated complex Mn(III)-malonate to polymerize coniferyl alcohol (CA) to obtain dehydrogenation polymers (DHPs) and to characterize how closely the structures of the formed DHPs resemble native lignin. Hydrogen peroxide was used as oxidant and Mn2+ as mediator. Based on the yields of the polymerized product, it was concluded that the enzymatic reaction should be performed in aqueous solution without organic solvents at 4.5pH6.0 and with 0.75H(2)O(2):CA ratio1. The results obtained from the Mn3+-malonate-mediated polymerization showed that the yield was almost 100%. Reaction conditions had, however, effect on the structures of the formed DHPs, as detected by size exclusion chromatography and pyrolysis-GC/MS. It can be concluded that from the structural point of view, the optimal pH for DHP formation using the presently studied system was 3 or 4.5. Low H2O2/CA ratio was beneficial to avoid oxidative side reactions. However, the high frequency of - linkages in all cases points to dimer formation between monomeric CA rather than endwise polymerization. (c) 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:81-90, 2018Peer reviewe

Functionalisation of organosolv lignin by enzymatic demethylation for bioadhesive formulation

Bio-based adhesives offer a sustainable alternative to formaldehyde-based adhesives, which is the most common product applied in wood-based panel manufacturing. Despite the undeniable interest in lignin as a bulk chemical for the synthesis of bio-based adhesives, the main problem is its low reactivity. Therefore, it is crucial to study techniques to increase their reactivity, such as demethylation. One of the parameters to monitor the success of the demethylation is the methanol concentration measured in the reaction medium and complementing these results with the use of non-quantitative analyses such as FT-IR, MALDI-ToF and SEC. In order to maximize the action of the enzyme, a preliminary screening was performed with laccases with different redox potentials, which were added to organosolv lignin in the form of purified enzymes and cocktails. Trametes versicolor laccase was selected as the best candidate for the demethylation of organosolv lignin, with a methanol released concentration of 107 mg/L. Based on these results, parameters affecting both enzyme action and enzyme-substrate interaction were evaluated. Optimal working conditions were strongly defined by the use of surfactants that enhanced lignin solubility (Tween-80) and mediators that favored enzyme catalysis to participate directly in the enzyme catalytic cycle (HBT). However, it was also observed that factors such as pH, temperature and aeration have a direct impact on the enzymatic demethylation of lignin. Considering the operational variables that allowed better demethylation values, the amount of methanol released increased to 363 mg/L, which means an improvement of 239 %. These results were also supported by the increase in molecular weight observed in the SEC analysis and the reduction of the intensity of the FT-IR bands related to the methyl/methoxyl groupsS