Development of Thermophilic Tailor-Made Enzyme Mixtures for the Bioconversion of Agricultural and Forest Residues

Even though the main components of all lignocellulosic feedstocks include cellulose, hemicellulose, as well as the protective lignin matrix, there are some differences in structure, such as in hardwoods and softwoods, which may influence the degradability of the materials. Under this view, various types of biomass might require a minimal set of enzymes that has to be tailor-made. Partially defined complex mixtures that are currently commercially used are not adapted to efficiently degrade different materials, so novel enzyme mixtures have to be customized. Development of these cocktails requires better knowledge about the specific activities involved, in order to optimize hydrolysis. The role of filamentous fungus Myceliophthora thermophila and its complete enzymatic repertoire for the bioconversion of complex carbohydrates has been widely proven. In this study, four core cellulases (MtCBH7, MtCBH6, MtEG5 and MtEG7), in the presence of other four accessory enzymes (mannanase, lytic polyssacharide monooxygenase MtGH61, xylanase, MtFae1a) and β-glucosidase MtBGL3, were tested as a 9-component cocktail against one model substrate (phosphoric acid swollen cellulose) and four hydrothermally pretreated natural substrates (wheat straw as an agricultural waste, birch and spruce biomass, as forest residues). Synergistic interactions among different enzymes were determined using a suitable design of experiments methodology. The results suggest that for the hydrolysis of the pure substrate (PASC), high proportions of MtEG7 are needed for efficient yields. MtCBH7 and MtEG7 are enzymes of major importance during the hydrolysis of pretreated wheat straw, while MtCBH7 plays a crucial role in case of spruce. Cellobiohydrolases MtCBH6 and MtCBH7 act in combination and are key enzymes for the hydrolysis of the hardwood (birch). Optimum combinations were predicted from suitable statistical models which were able to further increase hydrolysis yields, suggesting that tailor-made enzyme mixtures targeted towards a particular residual biomass can help maximize hydrolysis yields. The present work demonstrates the change from ‘one cocktail for all’ to ‘tailor-made cocktails’ that are needed for the efficient saccharification of targeted feed stocks prior to the production of biobased products through the biorefinery concept

Utilization of Volatile Fatty Acids from Microalgae for the Production of High Added Value Compounds

Volatile Fatty Acids (VFA) are small organic compounds that have attracted much attention lately, due to their use as a carbon source for microorganisms involved in the production of bioactive compounds, biodegradable materials and energy. Low cost production of VFA from different types of waste streams can occur via dark fermentation, offering a promising approach for the production of biofuels and biochemicals with simultaneous reduction of waste volume. VFA can be subsequently utilized in fermentation processes and efficiently transformed into bioactive compounds that can be used in the food and nutraceutical industry for the development of functional foods with scientifically sustained claims. Microalgae are oleaginous microorganisms that are able to grow in heterotrophic cultures supported by VFA as a carbon source and accumulate high amounts of valuable products, such as omega-3 fatty acids and exopolysaccharides. This article reviews the different types of waste streams in concert with their potential to produce VFA, the possible factors that affect the VFA production process and the utilization of the resulting VFA in microalgae fermentation processes. The biology of VFA utilization, the potential products and the downstream processes are discussed in detail.VOLATILE, a project funded by the European Union’s Horizon 2020 research and innovation program, under the grant agreement no. 720777. The Greek State Scholarships (Postdoc-Research Scholarships IKY). The Hellenic Foundation of Research and Innovation (ELIDEK) financial support (ELIDEK Scholarships for Ph.D. Students)

Isolation and modification of nano-scale cellulose from organosolv-treated birch through the synergistic activity of LPMO and endoglucanases

Nanocellulose isolation fromlignocellulose is a tedious and expensive processwith high energy and harsh chemical requirements, primarily due to the recalcitrance of the substrate, which otherwise would have been costeffective due to its abundance. Replacing the chemical steps with biocatalytic processes offers opportunities to solve this bottleneck to a certain extent due to the enzymes substrate specificity and mild reaction chemistry. In this work, we demonstrate the isolation of sulphate-free nanocellulose from organosolv pretreated birch biomass using different glycosyl-hydrolases, along with accessory oxidative enzymes including a lytic polysaccharide monooxygenase (LPMO). The suggested process produced colloidal nanocellulose suspensions (zeta-potential-19.4 mV) with particles of 7-20 nm diameter, high carboxylate content and improved thermostability (T-o= 301 degrees C, T-max= 337 degrees C). Nanocelluloseswere subjected to post-modification using LPMOs of different regioselectivity. The sample from chemical route was the least favorable for LPMO to enhance the carboxylate content, while that from the C1-specific LPMO treatment showed the highest increase in carboxylate content. (c) 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http:// creativecommons.org/licenses/by/4.0/)

Δημιουργία μικροβιακής πλατφόρμας για την παραγωγή ενζύμων που αποικοδομούν τη λιγνοκυτταρίνη

Cellulose, the most abundant polysaccharide on Earth, is a remarkable pure organic polymericcomponent of plant material, consisting solely of 1,4-linked β-D-glucopyranose units held together in agiant straight chain molecule. In nature, a variety of microorganisms are known for producing a set ofenzymes, referred to as cellulases, that are capable of degrading this insoluble polymer to solublesugars, primarily cellobiose and glucose. The application and interest in cellulases has particularlyincreased in recent years with the utilization of the enzymes in the production of bioethanol from lignocelluloses. Myceliophthora thermophila (synonym Sporotrichum thermophile) is a thermophilicfilamentous fungus, isolated from soil in eastern Russia, classified as an ascomycete, and constitutesan exceptionally powerful cellulolytic organism; it synthesizes a complete set of enzymes necessary forthe breakdown of cellulose. The genome of this fungus has been recently sequenced and annotated,allowing systematic examination and identification of enzymes required for the decomposition oflignocellulosic biomass. In this thesis, the genes encoding five cellulases, including twoendoglucanases belonging to glycoside hydrolase families GH5 and GH7, two cellobiohydrolasesbelonging to the families GH6 and GH7 and one β-glycosidase belonging to the family GH3, werecloned and expressed in methylotrophic yeast P. pastoris, and their properties were investigated. Inaddition, the enzymes were produced in high cell density cultures, in the controlled environment offermenters. The protein’s overexpression in a host suitable for industrial production is important inorder to achieve low-cost and highly efficient production. The enzymes were purified to theirhomogeneity and were used for the development of tailor-made enzyme mixtures targeted towardsparticular feedstocks, including agricultural and forest residues, where they were tested for their abilityto maximize hydrolysis yields.Η κυτταρίνη αποτελεί το αφθονότερο οργανικό πολυμερές που συναντάται στη φύση, γεγονός που τηνκαθιστά κατάλληλη ως φτηνά αξιοποιήσιμη πηγή άνθρακα και πρώτη ύλη για ποικίλες βιοτεχνολογικέςεφαρμογές. Ως εκ τούτου, η απομόνωση νέων ενζύμων με δράση που στοχεύει στην αποικοδόμηση ήτην τροποποίηση των κυτταρινούχων υλικών κρίνεται απαραίτητη. Οι θερμόφιλοι οργανισμοίαποτελούν σημαντική πηγή κυτταρινολυτικών ενζύμων με πολλές βιοτεχνολογικές εφαρμογές, καθώςοι διεργασίες αυτές συνήθως απαιτούν συνθήκες υψηλής θερμοκρασίας. Ο Myceliophthora thermophilaείναι ένας θερμόφιλος αερόβιος μύκητας, ο οποίος αυξάνεται με μέγιστο ρυθμό σε θερμοκρασίες 45 –50 οC. Ο μύκητας παράγει πολλά θερμοσταθερά ένζυμα τα οποία έχουν απομονωθεί και χαρακτηριστείκαι χρησιμοποιούνται στη βιομηχανία σε βιοδιεργασίες που απαιτούν υψηλές θερμοκρασίες. Σκοπόςτης παρούσας εργασίας ήταν η απομόνωση και ετερόλογη έκφραση πέντε γονιδίων από το γονιδίωματου συγκεκριμένου μύκητα που κωδικοποιούν πρωτεΐνες που εμπλέκονται στην αποικοδόμηση τηςλιγνοκυτταρίνης, καθώς και η παραγωγή και ο καταλυτικός χαρακτηρισμός του ανασυνδυασμένωνενζύμων. Οι νουκλεοτιδικές αλληλουχίες δύο ενδογλουκανασών των οικογενειών GH5 και GH7, δυοκελλοβιοϋδρολασών των οικογενειών GH6 και GH7 και μιας β-γλυκοσιδάσης της οικογένειας GH3ανασύρθηκαν από τη βάση δεδομένων Genome Portal, ενισχύθηκαν, κλωνοποιήθηκαν στονκατάλληλο φορέα και χρησιμοποιήθηκαν για το μετασχηματισμό και την ετερόλογη έκφραση στοζυμομύκητα P. pastoris. Ακολούθησε καθαρισμός των παραγόμενων πρωτεϊνών, τα οποία εν συνεχείαχρησιμοποιήθηκαν για τη δημιουργία ενζυμικών μιγμάτων και δοκιμάστηκαν για την υδρολυτική τουςικανότητα έναντι φυσικών υποστρωμάτων αγροτικής και δασικής προέλευσης

Effect of Different Pretreatment Methods on Birch Outer Bark: New Biorefinery Routes

A comparative study among different pretreatment methods used for the fractionation of the birch outer bark components, including steam explosion, hydrothermal and organosolv treatments based on the use of ethanol/water media, is reported. The residual solid fractions have been characterized by ATR-FTIR, 13C-solid-state NMR and morphological alterations after pretreatment were detected by scanning electron microscopy. The general chemical composition of the untreated and treated bark including determination of extractives, suberin, lignin and monosaccharides was also studied. Composition of the residual solid fraction and relative proportions of different components, as a function of the processing conditions, could be established. Organosolv treatment produces a suberin-rich solid fraction, while during hydrothermal and steam explosion treatment cleavage of polysaccharide bonds occurs. This work will provide a deeper fundamental knowledge of the bark chemical composition, thus increasing the utilization efficiency of birch outer bark and may create possibilities to up-scale the fractionation processes

Novel Routes in Transformation of Lignocellulosic Biomass to Furan Platform Chemicals: From Pretreatment to Enzyme Catalysis

The constant depletion of fossil fuels along with the increasing need for novel materials, necessitate the development of alternative routes for polymer synthesis. Lignocellulosic biomass, the most abundant carbon source on the planet, can serve as a renewable starting material for the design of environmentally-friendly processes for the synthesis of polyesters, polyamides and other polymers with significant value. The present review provides an overview of the main processes that have been reported throughout the literature for the production of bio-based monomers from lignocellulose, focusing on physicochemical procedures and biocatalysis. An extensive description of all different stages for the production of furans is presented, starting from physicochemical pretreatment of biomass and biocatalytic decomposition to monomeric sugars, coupled with isomerization by enzymes prior to chemical dehydration by acid Lewis catalysts. A summary of all biotransformations of furans carried out by enzymes is also described, focusing on galactose, glyoxal and aryl-alcohol oxidases, monooxygenases and transaminases for the production of oxidized derivatives and amines. The increased interest in these products in polymer chemistry can lead to a redirection of biomass valorization from second generation biofuels to chemical synthesis, by creating novel pathways to produce bio-based polymers

Formation of Lignin Nanoparticles by Combining Organosolv Pretreatment of Birch Biomass and Homogenization Processes

Valorization of lignocellulosic biomass into a biorefinery scheme requires the use of all biomass components; in this, the lignin fraction is often underutilized. Conversion of lignin to nanoparticles is an attractive solution. Here, we investigated the effect of different lignin isolation processes and a post-treatment homogenization step on particle formation. Lignin was isolated from birch chips by using two organosolv processes, traditional organosolv (OS) and hybrid organosolv-steam explosion (HOS-SE) at various ethanol contents. For post-treatment, lignin was homogenized at 500 bar using different ethanol:water ratios. Isolation of lignin with OS resulted in unshaped lignin particles, whereas after HOS-SE, lignin micro-particles were formed directly. Addition of an acidic catalyst during HOS-SE had a negative impact on the particle formation, and the optimal ethanol content was 50–60% v/v. Homogenization had a positive effect as it transformed initially unshaped lignin into spherical nanoparticles and reduced the size of the micro-particles isolated by HOS-SE. Ethanol content during homogenization affected the size of the particles, with the optimal results obtained at 75% v/v. We demonstrate that organosolv lignin can be used as an excellent starting material for nanoparticle preparation, with a simple method without the need for extensive chemical modification. It was also demonstrated that tuning of the operational parameters results in nanoparticles of smaller size and with better size homogeneity

Fine-Tuned Enzymatic Hydrolysis of Organosolv Pretreated Forest Materials for the Efficient Production of Cellobiose

Non-digestible oligosaccharides (NDOs) are likely prebiotic candidates that have been related to the prevention of intestinal infections and other disorders for both humans and animals. Lignocellulosic biomass is the largest carbon source in the biosphere, therefore cello-oligosacharides (COS), especially cellobiose, are potentially the most widely available choice of NDOs. Production of COS and cellobiose with enzymes offers numerous benefits over acid-catalyzed processes, as it is milder, environmentally friendly and produces fewer by-products. Cellobiohydrolases (CBHs) and a class of endoglucanases (EGs), namely processive EGs, are key enzymes for the production of COS, as they have higher preference toward glycosidic bonds near the end of cellulose chains and are able to release soluble products. In this work, we describe the heterologous expression and characterization of two CBHs from the filamentous fungus Thermothelomyces thermophila, as well as their synergism with proccessive EGs for cellobiose release from organosolv pretreated spruce and birch. The properties, inhibition kinetics and substrate specific activities for each enzyme are described in detail. The results show that a combination of EGs belonging to Glycosyl hydrolase families 5, 6, and 9, with a CBHI and CBHII in appropriate proportions, can enhance the production of COS from forest materials, underpinning the potential of these biocatalysts in the production of NDOs

Cloning, expression and characterization of an ethanol tolerant GH3 β-glucosidase from Myceliophthora thermophile

The β-glucosidase gene bgl3a from Myceliophthora thermophila, member of the fungal glycosyl hydrolase (GH) family 3, was cloned and expressed in Pichia pastoris. The mature β-glucosidase gene, which results after the excision of one intron and the secreting signal peptide, was placed under the control of the strong alcohol oxidase promoter (AOX1) in the plasmid pPICZαC. The recombinant enzyme (90 kDa) was purified and characterized in order to evaluate its biotechnological potential. Recombinant P. pastoris efficiently secreted β-glucosidase into the medium and produced high level of enzymatic activity (41 U/ml) after 192 h of growth, under methanol induction. MtBgl3a was able to hydrolyze low molecular weight substrates and polysaccharides containing β-glucosidic residues. The Km was found to be 0.39 mM on p-β-NPG and 2.64 mM on cellobiose. Optimal pH and temperature for the p-β-NPG hydrolysis were 5.0 and 70 °C. The β-glucosidase exhibits a half life of 143 min at 60 °C. Kinetic parameters of inhibition were determined for D-glucose, D-xylose and D-gluconic acid, indicating tolerance of the enzyme for these sugars and oxidized products. The recombinant enzyme was stimulated by short chain alcohols and has been shown to efficiently synthesize methyl-D-glucoside in the presence of methanol due to its transglycosylation activity. The stability of MtBgl3a in ethanol was prominent, and it retained most of its original activity after we exposed it to 50% ethanol for 6 h. The high catalytic performance, good thermal stability and tolerance to elevated concentrations of ethanol, D-xylose and D-glucose qualify this enzyme for use in the hydrolysis of lignocellulosic biomass for biofuel production, as part of an efficient complete multi-enzyme cocktail.Validerad; 2013; Bibliografisk uppgift: Article numbere 46; 20130221 (ysko

Tailoring Celluclast® Cocktail’s Performance towards the Production of Prebiotic Cello-Oligosaccharides from Waste Forest Biomass

The main objective of this study focused on the sustainable production of cellobiose and other cellulose-derived oligosaccharides from non-edible sources, more specifically, from forest residues. For this purpose, a fine-tuning of the performance of the commercially available enzyme mixture Celluclast® was conducted towards the optimization of cellobiose production. By enzyme reaction engineering (pH, multi-stage hydrolysis with buffer exchange, addition of β-glucosidase inhibitor), a cellobiose-rich product with a high cellobiose to glucose ratio (37.4) was achieved by utilizing organosolv-pretreated birch biomass. In this way, controlled enzymatic hydrolysis combined with efficient downstream processing, including product recovery and purification through ultrafiltration and nanofiltration, can potentially support the sustainable production of food-grade oligosaccharides from forest biomass. The potential of the hydrolysis product to support the growth of two Lactobacilli probiotic strains as a sole carbon source was also demonstrated