Spruce Bark-Extracted Lignin and Tannin-Based Bioresin-Adhesives: Effect of Curing Temperatures on the Thermal Properties of the Resins.

In this study, formaldehyde-free bioresin adhesives were synthesised from lignin and tannin, which were obtained from softwood bark. The extraction was done via organosolv treatment and hot water extraction, respectively. A non-volatile, non-toxic aldehyde, glyoxal, was used as a substitute for formaldehyde in order to modify the chemical structure of both the lignin and tannin. The glyoxal modification reaction was confirmed by ATR-FTIR spectroscopy. Three different resin formulations were prepared using modified lignin along with the modified tannin. The thermal properties of the modified lignin, tannin, and the bioresins were assessed by DSC and TGA. When the bioresins were cured at a high temperature (200 °C) by compression moulding, they exhibited higher thermal stability as well as an enhanced degree of cross-linking compared to the low temperature-cured bioresins. The thermal properties of the resins were strongly affected by the compositions of the resins as well as the curing temperatures.

Organosolv Fractionation of Birch Sawdust: Establishing a Lignin-First Biorefinery.

The use of residual biomass for bioconversions makes it possible to decrease the output of fossil-based chemicals and pursue a greener economy. While the use of lignocellulosic material as sustainable feedstock has been tried at pilot scale, industrial production is not yet economically feasible, requiring further technology and feedstock optimization. The aim of this study was to examine the feasibility of replacing woodchips with residual sawdust in biorefinery applications. Woodchips can be used in value-added processes such as paper pulp production, whereas sawdust is currently used mainly for combustion. The main advantages of sawdust are its large supply and a particle size sufficiently small for the pretreatment process. Whereas, the main challenge is the higher complexity of the lignocellulosic biomass, as it can contain small amounts of bark and cambium. Here, we studied the fractionation of birch sawdust by organosolv pretreatment at two different temperatures and for two different durations. We evaluated the efficiency of fractionation into the three main fractions: lignin, cellulose, and hemicellulose. The cellulose content in pretreated biomass was as high as 69.2%, which was nearly double the amount in untreated biomass. The obtained lignin was of high purity, with a maximum 4.5% of contaminating sugars. Subsequent evaluation of the susceptibility of pretreated solids to enzymatic saccharification revealed glucose yields ranging from 75% to 90% after 48 h but reaching 100.0% under the best conditions. In summary, birch sawdust can be successfully utilized as a feedstock for organosolv fractionation and replace woodchips to simplify and lower the costs of biorefinery processes.

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.

Acid Assisted Organosolv Delignification of Beechwood and Pulp Conversion towards High Concentrated Cellulosic Ethanol via High Gravity Enzymatic Hydrolysis and Fermentation.

BACKGROUND: Future biorefineries will focus on converting low value waste streams to chemical products that are derived from petroleum or refined sugars. Feedstock pretreatment in a simple, cost effective, agnostic manner is a major challenge. METHODS: In this work, beechwood sawdust was delignified via an organosolv process, assisted by homogeneous inorganic acid catalysis. Mixtures of water and several organic solvents were evaluated for their performance. Specifically, ethanol (EtOH), acetone (AC), and methyl- isobutyl- ketone (MIBK) were tested with or without the use of homogeneous acid catalysis employing sulfuric, phosphoric, and oxalic acids under relatively mild temperature of 175 °C for one hour. RESULTS: Delignification degrees (DD) higher than 90% were achieved, where both AC and EtOH proved to be suitable solvents for this process. Both oxalic and especially phosphoric acid proved to be good alternative catalysts for replacing sulfuric acid. High gravity simultaneous saccharification and fermentation with an enzyme loading of 8.4 mg/gsolids at 20 wt.% initial solids content reached an ethanol yield of 8.0 w/v%. CONCLUSIONS: Efficient delignification combining common volatile solvents and mild acid catalysis allowed for the production of ethanol at high concentration in an efficient manner.

Lignin from Hardwood and Softwood Biomass as a Lubricating Additive to Ethylene Glycol.

Ethylene glycol (EG)-based lubricant was prepared with dissolved organosolv lignin from birch wood (BL) and softwood (SL) biomass. The effects of different lignin types on the rheological, thermal, and tribological properties of the lignin/EG lubricants were comprehensively investigated by various characterization techniques. Dissolving organosolv lignin in EG results in outstanding lubricating properties. Specifically, the wear volume of the disc by EG-44BL is only 8.9% of that lubricated by pure EG. The enhanced anti-wear property of the EG/lignin system could be attributed to the formation of a robust lubrication film and the strong adhesion of the lubricant on the contacting metal surface due to the presence of a dense hydrogen bonding (H-bonding) network. The lubricating performance of EG-BL outperforms EG-SL, which could be attributed to the denser H-bonding sites in BL and its broader molecular weight distribution. The disc wear loss of EG-44BL is only 45.7% of that lubricated by EG-44SL. Overall, H-bonding is the major contributor to the different tribological properties of BL and SL in EG-based lubricants.

Structural and functional studies of a Fusarium oxysporum cutinase with polyethylene terephthalate modification potential.

BACKGROUND: Cutinases are serine hydrolases that degrade cutin, a polyester of fatty acids that is the main component of plant cuticle. These biocatalysts have recently attracted increased biotechnological interest due to their potential to modify and degrade polyethylene terephthalate (PET), as well as other synthetic polymers. METHODS: A cutinase from the mesophilic fungus Fusarium oxysporum, named FoCut5a, was expressed either in the cytoplasm or periplasm of Escherichia coli BL21. Its X-ray structure was determined to 1.9Å resolution using molecular replacement. The activity of the recombinant enzyme was tested on a variety of synthetic esters and polyester analogues. RESULTS: The highest production of recombinant FoCut5a was achieved using periplasmic expression at 16°C. Its crystal structure is highly similar to previously determined Fusarium solani cutinase structure. However, a more detailed comparison of the surface properties and amino acid interactions revealed differences with potential impact on the biochemical properties of the two enzymes. FoCut5a showed maximum activity at 40°C and pH 8.0, while it was active on three p-nitrophenyl synthetic esters of aliphatic acids (C(2), C(4), C(12)), with the highest catalytic efficiency for the hydrolysis of the butyl ester. The recombinant cutinase was also found capable of hydrolyzing PET model substrates and synthetic polymers. CONCLUSIONS: The present work is the first reported expression and crystal structure determination of a functional cutinase from the mesophilic fungus F. oxysporum with potential application in surface modification of PET synthetic polymers. GENERAL SIGNIFICANCE: FoCut5a could be used as a biocatalyst in industrial applications for the environmentally-friendly treatment of synthetic polymers.

A thermostable GH26 endo-ß-mannanase from Myceliophthora thermophila capable of enhancing lignocellulose degradation.

The endomannanase gene em26a from the thermophilic fungus Myceliophthora thermophila, belonging to the glycoside hydrolase family 26, was functionally expressed in the methylotrophic yeast Pichia pastoris. The putative endomannanase, dubbed MtMan26A, was purified to homogeneity (60 kDa) and subsequently characterized. The optimum pH and temperature for the enzymatic activity of MtMan26A were 6.0 and 60 °C, respectively. MtMan26A showed high specific activity against konjac glucomannan and carob galactomannan, while it also exhibited high thermal stability with a half-life of 14.4 h at 60 °C. Thermostability is of great importance, especially in industrial processes where harsh conditions are employed. With the aim of better understanding its structure-function relationships, a homology model of MtMan26A was constructed, based on the crystallographic structure of a close homologue. Finally, the addition of MtMan26A as a supplement to the commercial enzyme mixture Celluclast® 1.5 L and Novozyme® 188 resulted in enhanced enzymatic hydrolysis of pretreated beechwood sawdust, improving the release of total reducing sugars and glucose by 13 and 12 %, respectively.

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, (13)C-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.

Bioconversion of Biomass-Derived Phenols Catalyzed by Myceliophthora thermophila Laccase.

Biomass-derived phenols have recently arisen as an attractive alternative for building blocks to be used in synthetic applications, due to their widespread availability as an abundant renewable resource. In the present paper, commercial laccase from the thermophilic fungus Myceliophthora thermophila was used to bioconvert phenol monomers, namely catechol, pyrogallol and gallic acid in water. The resulting products from catechol and gallic acid were polymers that were partially characterized in respect to their optical and thermal properties, and their average molecular weight was estimated via solution viscosity measurements and GPC. FT-IR and ¹H-NMR data suggest that phenol monomers are connected with ether or C-C bonds depending on the starting monomer, while the achieved molecular weight of polycatechol is found higher than the corresponding poly(gallic acid). On the other hand, under the same condition, pyrogallol was dimerized in a pure red crystalline compound and its structure was confirmed by ¹H-NMR as purpurogallin. The herein studied green synthesis of enzymatically synthesized phenol polymers or biological active compounds could be exploited as an alternative synthetic route targeting a variety of applications.

Biogas potential of organosolv pretreated wheat straw as mono and co-substrate: substrate synergy and microbial dynamics.

Anaerobic digestion (AD) technology can potentially address the gap between energy demand and supply playing a crucial role in the production of sustainable energy from utilization of biogenic waste materials as feedstock. The biogas production from anaerobic digestion is primarily influenced by the chemical compositions and biodegradability of the feedstock. Organosolv-steam explosion offers a constructive approach as a promising pretreatment method for the fractionation of lignocellulosic biomasses delivering high cellulose content.This study showed how synergetic co-digestion serves to overcome the challenges of mono-digestion's low efficiency. Particularly, the study evaluated the digestibility of organosolv-steam pretreated wheat straw (WSOSOL) in mono as well as co-digesting substrate with cheese whey (CW) and brewery spent grains (BSG). The highest methane yield was attained with co-digestion of WSOSOL + CW (338 mL/gVS) representing an enhanced biogas output of 1-1.15 times greater than its mono digestion. An ammonium production was favored under co-digestion strategy accounting for 921 mg/L from WSOSOL + BSG. Metagenomic study was conducted to determine the predominant bacteria and archaea, as well as its variations in their populations and their functional contributions during the AD process. The Firmicutes have been identified as playing a significant role in the hydrolysis process and the initial stages of AD. An enrichment of the most prevalent archaea genera enriched were Methanobacterium, Methanothrix, and Methanosarsina. Reactors digesting simpler substrate CW followed the acetoclastic, while digesting more complex substrates like BSG and WSOSOL followed the hydrogenotrophic pathway for biomethane production. To regulate the process for an enhanced AD process to maximize CH4, a comprehensive understanding of microbial communities is beneficial.

Recalcitrant polysaccharide degradation by novel oxidative biocatalysts.

The classical hydrolytic mechanism for the degradation of plant polysaccharides by saprophytic microorganisms has been reconsidered after the recent landmark discovery of a new class of oxidases termed lytic polysaccharide monooxygenases (LPMOs). LPMOs are of increased biotechnological interest due to their implication in lignocellulosic biomass decomposition for the production of biofuels and high-value chemicals. They act on recalcitrant polysaccharides by a combination of hydrolytic and oxidative function, generating oxidized and non-oxidized chain ends. They are copper-dependent and require molecular oxygen and an external electron donor for their proper function. In this review, we present the recent findings concerning the mechanism of action of these oxidative enzymes and identify issues and questions to be addressed in the future.

Covalently bound humin-lignin hybrids as important novel substructures in organosolv spruce lignins.

Organosolv lignins (OSLs) are important byproducts of the cellulose-centred biorefinery that need to be converted in high value-added products for economic viability. Yet, OSLs occasionally display characteristics that are unexpected looking at the lignin motifs present. Applying advanced NMR, GPC, and thermal analyses, isolated spruce lignins were analysed to correlate organosolv process severity to the structural details for delineating potential valorisations. Very mild conditions were found to not fractionate the biomass, causing a mix of sugars, lignin-carbohydrate complexes (LCCs), and corresponding dehydration/degradation products and including pseudo-lignins. Employing only slightly harsher conditions promote fractionation, but also formation of sugar degradation structures that covalently incorporate into the oligomeric and polymeric lignin structures, causing the isolated organosolv lignins to contain lignin-humin hybrid (HLH) structures not yet evidenced as such in organosolv lignins. These structures effortlessly explain observed unexpected solubility issues and unusual thermal responses, and their presence might have to be acknowledged in downstream lignin valorisation.

Role and importance of solvents for the fractionation of lignocellulosic biomass.

Lignocellulosic biomass is one of the most important renewable materials to replace carbon-based fossil resources. Solvent-based fractionation is a promising route for fractionation of biomass into its major components. Processing is governed by the employed solvent-systems properties. This review sheds light on the factors governing both dissolution and potential reactivities of the chemical structures present in lignocellulose, highlighting how proper understanding of the underlying mechanisms and interactions between solute and solvent help to choose proper systems for specific fractionation needs. Structural and chemical differences between the carbohydrate-based structural polymers and lignin require very different solvents capabilities in terms of causing and eventually stabilizing conformational changes and consequent activation of bonds to be cleaved by other active components in the. A consideration of potential depolymerization events during dissolution and energetic aspects of the dissolution process considering the contribution of polymer functionalities allow for a mapping of solvent suitability for biomass fractionation.

Lytic polysaccharide monooxygenases as powerful tools in enzymatically assisted preparation of nano-scaled cellulose from lignocellulose: A review.

Nanocellulose, either in the form of fibers or crystals, constitutes a renewable, biobased, biocompatible material with advantageous mechanical properties that can be isolated from lignocellulosic biomass. Enzyme-assisted isolation of nanocellulose is an attractive, environmentally friendly approach that leads to products of higher quality compared to their chemically prepared counterparts. Lytic polysaccharide monooxygenases (LPMOs) are enzymes that oxidatively cleave the ß-1,4-glycosidic bond of polysaccharides upon activation of O2 or H2O2 and presence of an electron donor. Their use for treatment of cellulose fibers towards the preparation of nano-scaled cellulose is related to the ability of LPMOs to create nicking points on the fiber surface, thus facilitating fiber disruption and separation. The aim of this review is to describe the mode of action of LPMOs on cellulose fibers towards the isolation of nanostructures, thus highlighting their great potential for the production of nanocellulose as a novel value added product from lignocellulose.

Organosolv fractionation of spruce bark using ethanol-water mixtures: Towards a novel bio-refinery concept.

The aim of this study was to assess the effect of hot water extraction followed by organosolv pretreatment on the enzymatic hydrolysability of spruce bark biomass. To this end, samples were pretreated at five different temperatures in the presence or not of acid catalyst. The cellulose content of pretreated biomass reached 49.6% w/w. During the enzymatic hydrolysis trials with 3% w/w dry solids, the final hydrolysis yield reached up to 70.1%, which corresponded to the release of 7.8 g/L of glucose. Whereas, the final hydrolysis yield obtained during the high-gravity enzymatic hydrolysis reached up to 43.5%. The concentration of released glucose was in range of 33.3 - 40.0 g/L with a hemicellulose sugars in a range of 5.5 - 6.6 g/L. These values are suitable for downstream bioconversion processes and represent a significant improvement over existing steam pretreatment methods.

Screening of Recombinant Lignocellulolytic Enzymes Through Rapid Plate Assays.

In the search for novel biomass-degrading enzymes through mining microbial genomes, it is necessary to apply functional tests during high-throughput screenings, which are capable of detecting enzymatic activities directly by way of plate assay. Using the most efficient expression systems of Escherichia coli and Pichia pastoris, the production of a high amount of His-tagged recombinant proteins could be thrived, allowing the one-step isolation by affinity chromatography. Here, we describe simple and efficient assay techniques for the detection of various biomass-degrading enzymatic activities on agar plates, such as cellulolytic, hemicellulolytic, and ligninolytic activities and their isolation using immobilized-metal affinity chromatography.

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

Nanocellulose isolation from lignocellulose is a tedious and expensive process with high energy and harsh chemical requirements, primarily due to the recalcitrance of the substrate, which otherwise would have been cost-effective 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 (ζ-potential -19.4 mV) with particles of 7-20 nm diameter, high carboxylate content and improved thermostability (To = 301 °C, Tmax = 337 °C). Nanocelluloses were 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.

A novel hybrid organosolv-steam explosion pretreatment and fractionation method delivers solids with superior thermophilic digestibility to methane.

Rising environmental concerns and the imminent depletion of fossil resources have sparked a strong interest towards the production of renewable energy such as biomethane. Inclusion of alternative feedstock's such as lignocellulosic biomass could further expand the production of biomethane. The present study evaluated the potential of a novel hybrid organosolv-steam explosion fractionation for delivering highly digestible pretreated solids from birch and spruce woodchips. The highest methane production yield was 176.5 mLCH4 gVS-1 for spruce and 327.2 mL CH4 gVS-1 for birch. High methane production rates of 1.0-6.3 mL min-1 (spruce) and 6.0-35.5 mL min-1 (birch) were obtained, leading to a rapid digestion, with 92% of total methane from spruce being generated in 80 h and 95% of that from birch in 120 h. These results demonstrate the elevated potential of the novel method to fractionate spruce and birch biomass and deliver cellulose-rich pretreated solids with superior digestibility.

Organosolv lignin hydrophobic micro- and nanoparticles as a low-carbon footprint biodegradable flotation collector in mineral flotation.

Flotation is a key step during mineral separation. Xanthates are the most commonly used collectors for recovering Cu, Ni, and Zn from sulphide ores. However, xanthates are fossil-based and toxic for the environment. The aim of this study was to evaluate the use of lignin nanoparticles and microparticles as sustainable and environmentally friendly collectors. Lignin particles demonstrated good selectivity toward Cu (chalcopyrite), with total recoveries exceeding 80% and grades of up to 8.6% w/w from a Cu-Ni ore in rougher flotation tests. When floating Zn-Pb-Cu ore, lignin nanoparticles could reduce the use of xanthates by 50%. Moreover, they outperformed xanthates alone, achieving total recoveries of up to 91%, 85%, and 98% for Cu, Pb, and Zn, respectively. These results prove the potential of lignin as a flotation collector.

Recent advances in organosolv fractionation: Towards biomass fractionation technology of the future.

Organosolv treatment is among the most promising strategies for valorising lignocellulosic biomass and could facilitate the transition towards enhanced utilization of renewable feedstocks. However, issues such as inefficient solvent recycle and fractionation has to be overcome. The present review aims to address these issues and discuss the role of the components present during organosolv treatment and their influence on the overall process. Thus, the review focuses not only on how the choice of solvent and catalyst affects lignocellulosic fractionation, but also on how the choice of treatment liquor influences the possibility for solvent recycling and product isolation. Several organic solvents have been investigated in combination with water and acid/base catalysts; however, the lack of a holistic approach often compromises the performance of the different operational units. Thus, an economically viable organosolv process should optimize biomass fractionation, product isolation, and solvent recycling.