Strategy for the analysis of lignocellulosic biomass to select a viable transformation route in the Colombian context.

Lignocellulosic biomass is widely available in the world. However, a consensus has yet to be established to evaluate the biomass valorization alternatives. The chemical composition is the primary technical limitation in selecting a transformation route to obtain value-added products. In this paper, the bagasse from non-centrifuged sugar (NCS) production and Pinus patula (PP) wood chips were analyzed in terms of complete chemical composition to establish their potential for selecting the transformation routes. A strategy to select the best route based on the chemical composition was applied and a feedstock criteria model was proposed. Schemes were obtained and compared using a bioprocess selection strategy proposed in previous works. As a result, the preliminary biorefinery schemes were finally defined. The assessment of schemes derived from the outlined strategy included technical, economic, environmental, and social analyses. The environmental evaluation was complemented with a geolocation assessment, revealing a 0.75-ton CO2-eq/yr contribution to the carbon footprint for local distribution. The sustainability index for the PP biorefinery and the bagasse from NCS production was analyzed, resulting in indices of 44.8 and 60.9, respectively. These values were primarily derived from the economic and environmental analyses of both processes.

Ether Bond Cleavage of a Phenylcoumaran ß-5 Lignin Model Compound and Polymeric Lignin Catalysed by a LigE-type Etherase from Agrobacterium sp.

A LigE-type beta-etherase enzyme from lignin-degrading Agrobacterium sp. has been identified, which assists degradation of polymeric lignins. Testing against lignin dimer model compounds revealed that it does not catalyse the previously reported reaction of Sphingobium SYK-6 LigE, but instead shows activity for a ß-5 phenylcoumaran lignin dimer. The reaction products did not contain glutathione, indicating a catalytic role for reduced glutathione in this enzyme. Three reaction products were identified: the major product was a cis-stilbene arising from C-C fragmentation involving loss of formaldehyde; two minor products were an alkene arising from elimination of glutathione, and an oxidised ketone, proposed to arise from reaction of an intermediate with molecular oxygen. Testing of the recombinant enzyme against a soda lignin revealed the formation of new signals by two-dimensional NMR analysis, whose chemical shifts are consistent with the formation of a stilbene unit in polymeric lignin.

Comparative Analysis of Trifluoracetic Acid Pretreatment for Lignocellulosic Materials.

Lignocellulosic materials are usually processed toward C5 and C6 corresponding sugars. Trifluoroacetic acid (TFA) is a pretreatment method to solubilize hemicellulose to sugars such xylose without degrading cellulose. However, this pretreatment has not been compared to other processes. Thus, this paper focuses on the techno-economic comparison of the C5-C6 production of C5-C6 as raw materials platforms using non-centrifuged sugarcane bagasse (NCSB) and Pinus patula wood chips (PP). Hydrolysates using TFA 2.5 M as an acid were characterized through HPLC regarding arabinose, galactose glucose, xylose, and mannose sugars. Then, simulations of the processes according to the experimental results were done. The economic assessment was performed, and compared with some common pretreatments. The mass and energy balances of the simulations indicate that the process can be compared with other pretreatments. From the economic perspective, the main operating expenditures (OpEx) are related to raw materials and capital depreciation due to the cost of TFA corrosion issues. The processes showed a CapEx and OpEx of 0.99 MUSD and 6.59 M-USD/year for NCSB, and 0.97 MUSD and 4.37 MUSD/year for PP, considering a small-scale base (1 ton/h). TFA pretreatment is innovative and promising from a techno-economic perspective.

Modification of a Marine Pine Kraft Lignin Sample by Enzymatic Treatment with a Pycnoporus cinnabarinus Laccase.

Here, we report work on developing an enzymatic process to improve the functionalities of industrial lignin. A kraft lignin sample prepared from marine pine was treated with the high-redox-potential laccase from the basidiomycete fungus Pycnoporus cinnabarinus at three different concentrations and pH conditions, and with and without the chemical mediator 1-hydroxybenzotriazole (HBT). Laccase activity was tested in the presence and absence of kraft lignin. The optimum pH of PciLac was initially 4.0 in the presence and absence of lignin, but at incubation times over 6 h, higher activities were found at pH 4.5 in the presence of lignin. Structural changes in lignin were investigated by Fourier-transform infrared spectroscopy (FTIR) with differential scanning calorimetry (DSC), and solvent-extractable fractions were analyzed using high-performance size-exclusion chromatography (HPSEC) and gas chromatography-mass spectrometry (GC-MS). The FTIR spectral data were analyzed with two successive multivariate series using principal component analysis (PCA) and ANOVA statistical analysis to identify the best conditions for the largest range of chemical modifications. DSC combined with modulated DSC (MDSC) revealed that the greatest effect on glass transition temperature (Tg) was obtained at 130 U g cm-1 and pH 4.5, with the laccase alone or combined with HBT. HPSEC data suggested that the laccase treatments led to concomitant phenomena of oligomerization and depolymerization, and GC-MS revealed that the reactivity of the extractable phenolic monomers depended on the conditions tested. This study demonstrates that P. cinnabarinus laccase can be used to modify marine pine kraft lignin, and that the set of analytical methods implemented here provides a valuable tool for screening enzymatic treatment conditions.

A Putative Lignin Copper Oxidase from Trichoderma reesei.

The ability of Trichoderma reesei, a fungus widely used for the commercial production of hemicellulases and cellulases, to grow and modify technical soda lignin was investigated. By quantifying fungal genomic DNA, T. reesei showed growth and sporulation in solid and liquid cultures containing lignin alone. The analysis of released soluble lignin and residual insoluble lignin was indicative of enzymatic oxidative conversion of phenolic lignin side chains and the modification of lignin structure by cleaving the ß-O-4 linkages. The results also showed that polymerization reactions were taking place. A proteomic analysis conducted to investigate secreted proteins at days 3, 7, and 14 of growth revealed the presence of five auxiliary activity (AA) enzymes in the secretome: AA6, AA9, two AA3 enzymes), and the only copper radical oxidase encoded in the genome of T. reesei. This enzyme was heterologously produced and characterized, and its activity on lignin-derived molecules was investigated. Phylogenetic characterization demonstrated that this enzyme belonged to the AA5_1 family, which includes characterized glyoxal oxidases. However, the enzyme displayed overlapping physicochemical and catalytic properties across the AA5 family. The enzyme was remarkably stable at high pH and oxidized both, alcohols and aldehydes with preference to the alcohol group. It was also active on lignin-derived phenolic molecules as well as simple carbohydrates. HPSEC and LC-MS analyses on the reactions of the produced protein on lignin dimers (SS ßß, SS ßO4 and GG ß5) uncovered the polymerizing activity of this enzyme, which was accordingly named lignin copper oxidase (TrLOx). Polymers of up 10 units were formed by hydroxy group oxidation and radical formation. The activations of lignin molecules by TrLOx along with the co-secretion of this enzyme with reductases and FAD flavoproteins oxidoreductases during growth on lignin suggest a synergistic mechanism for lignin breakdown.

Fungal Treatment for the Valorization of Technical Soda Lignin.

Technical lignins produced as a by-product in biorefinery processes represent a potential source of renewable carbon. In consideration of the possibilities of the industrial transformation of this substrate into various valuable bio-based molecules, the biological deconstruction of a technical soda lignin by filamentous fungi was investigated. The ability of three basidiomycetes (Polyporus brumalis, Pycnoporus sanguineus and Leiotrametes menziesii) to modify this material, the resultant structural and chemical changes, and the secreted proteins during growth on this substrate were investigated. The three fungi could grow on the technical lignin alone, and the growth rate increased when the media were supplemented with glucose or maltose. The proteomic analysis of the culture supernatants after three days of growth revealed the secretion of numerous Carbohydrate-Active Enzymes (CAZymes). The secretomic profiles varied widely between the strains and the presence of technical lignin alone triggered the early secretion of many lignin-acting oxidoreductases. The secretomes were notably rich in glycoside hydrolases and H2O2-producing auxiliary activity enzymes with copper radical oxidases being induced on lignin for all strains. The lignin treatment by fungi modified both the soluble and insoluble lignin fractions. A significant decrease in the amount of soluble higher molar mass compounds was observed in the case of P. sanguineus. This strain was also responsible for the modification of the lower molar mass compounds of the lignin insoluble fraction and a 40% decrease in the thioacidolysis yield. The similarity in the activities of P. sanguineus and P. brumalis in modifying the functional groups of the technical lignin were observed, the results suggest that the lignin has undergone structural changes, or at least changes in its composition, and pave the route for the utilization of filamentous fungi to functionalize technical lignins and produce the enzymes of interest for biorefinery applications.

High-Throughput Analysis of Lignin by Agarose Gel Electrophoresis.

A high-throughput agarose gel electrophoresis (AGE) analytical method has been developed to separate lignin fractions according to their molecular weight (Mw), charge, and shape. Operating conditions to effect separation of species have been evaluated along with imaging parameters. Kraft, soda (Protobind), and Organosolv lignins showed distinct differences in migration. Bands were cut, extracted, and cross-analyzed by gel permeation chromatography (GPC), 1H NMR, and pyrolysis GC/MS to confirm their identity as lignin. The band intensity was correlated with lignin concentration by running serially diluted samples and imaging each lane to produce a precise calibration curve. The AGE technique was used to monitor and compare enzymatic, bacterial, chemical, and hydrothermal lignin digestions. Each method showed changes in lignin migration and band intensities over time. Low Mw species were seen in samples collected from the anode buffer tank. Though requiring further development, the AGE method can provide structural information about the lignin and is accessible to biological and chemistry laboratories.

Characterization of the CAZy Repertoire from the Marine-Derived Fungus Stemphylium lucomagnoense in Relation to Saline Conditions.

Even if the ocean represents a large part of Earth's surface, only a few studies describe marine-derived fungi compared to their terrestrial homologues. In this ecosystem, marine-derived fungi have had to adapt to the salinity and to the plant biomass composition. This articles studies the growth of five marine isolates and the tuning of lignocellulolytic activities under different conditions, including the salinity. A de novo transcriptome sequencing and assembly were used in combination with a proteomic approach to characterize the Carbohydrate Active Enzymes (CAZy) repertoire of one of these strains. Following these approaches, Stemphylium lucomagnoense was selected for its adapted growth on xylan in saline conditions, its high xylanase activity, and its improved laccase activities in seagrass-containing cultures with salt. De novo transcriptome sequencing and assembly indicated the presence of 51 putative lignocellulolytic enzymes. Its secretome composition was studied in detail when the fungus was grown on either a terrestrial or a marine substrate, under saline and non-saline conditions. Proteomic analysis of the four S. lucomagnoense secretomes revealed a minimal suite of extracellular enzymes for plant biomass degradation and highlighted potential enzyme targets to be further studied for their adaptation to salts and for potential biotechnological applications.

Dual Antioxidant Properties and Organic Radical Stabilization in Cellulose Nanocomposite Films Functionalized by In Situ Polymerization of Coniferyl Alcohol.

A new biobased material based on an original strategy using lignin model compounds as natural grafting additive on a nanocellulose surface through in situ polymerization of coniferyl alcohol by the Fenton reaction at two pH values was investigated. The structural and morphological properties of the materials at the nanoscale were characterized by a combination of analytical methods, including Fourier transform infrared spectroscopy, liquid chromatography combined with mass spectrometry, nuclear molecular resonance spectroscopy, electron paramagnetic resonance spectroscopy, water sorption capacity by dynamic vapor sorption, and atomic force microscopy (topography and indentation modulus measurements). Finally, the usage properties, such as antioxidant properties, were evaluated in solution and the nanostructured casted films by radical 2,2'-diphenyl-1-picrylhydrazyl (DPPH•) scavenging tests. We demonstrate the structure-function relationships of these advanced CNC-lignin films and describe their dual functionalities and characteristics, namely, their antioxidant properties and the presence of persistent phenoxy radicals within the material.

Enhancing the Antioxidant Activity of Technical Lignins by Combining Solvent Fractionation and Ionic-Liquid Treatment.

A grass soda technical lignin (PB1000) underwent a process combining solvent fractionation and treatment with an ionic liquid (IL), and a comprehensive investigation of the structural modifications was performed by using high-performance size-exclusion chromatography, 31 P NMR spectroscopy, thioacidolysis, and GC-MS. Three fractions with distinct reactivity were recovered from successive ethyl acetate (EA), butanone, and methanol extractions. In parallel, a fraction deprived of EA extractives was obtained. The samples were treated with methyl imidazolium bromide ([HMIM]Br) by using either conventional heating or microwave irradiation. The treatment allowed us to solubilize 28 % of the EA-insoluble fraction and yielded additional free phenols in all the fractions, as a consequence of depolymerization and demethylation. The gain of the combined process in terms of antioxidant properties was demonstrated through 2,2-diphenyl-1-picrylhydrazyl (DPPH. ) radical-scavenging tests. Integrating further IL safety-related data and environmental considerations, this study paves the way for the sustainable production of phenolic oligomers competing with commercial antioxidants.

Sphingobacterium sp. T2 Manganese Superoxide Dismutase Catalyzes the Oxidative Demethylation of Polymeric Lignin via Generation of Hydroxyl Radical.

Sphingobacterium sp. T2 contains two extracellular manganese superoxide dismutase enzymes which exhibit unprecedented activity for lignin oxidation but via an unknown mechanism. Enzymatic treatment of lignin model compounds gave products whose structures were indicative of aryl-Cα oxidative cleavage and demethylation, as well as alkene dihydroxylation and alcohol oxidation. 18O labeling studies on the SpMnSOD-catalyzed oxidation of lignin model compound guiaiacylglycerol-ß-guaiacyl ether indicated that the an oxygen atom inserted by the enzyme is derived from superoxide or peroxide. Analysis of an alkali lignin treated by SpMnSOD1 by quantitative 31P NMR spectroscopy demonstrated 20-40% increases in phenolic and aliphatic OH content, consistent with lignin demethylation and some internal oxidative cleavage reactions. Assay for hydroxyl radical generation using a fluorometric hydroxyphenylfluorescein assay revealed the release of 4.1 molar equivalents of hydroxyl radical by SpMnSOD1. Four amino acid replacements in SpMnSOD1 were investigated, and A31H or Y27H site-directed mutant enzymes were found to show no lignin demethylation activity according to 31P NMR analysis. Structure determination of the A31H and Y27H mutant enzymes reveals the repositioning of an N-terminal protein loop, leading to widening of a solvent channel at the dimer interface, which would provide increased solvent access to the Mn center for hydroxyl radical generation.

Imidazolium-Based Ionic Liquids as Efficient Reagents for the C-O Bond Cleavage of Lignin.

The demethylation of lignin in ionic liquids (ILs) was investigated by using pure lignin model monomers and dimers together with dioxane-isolated lignins from poplar, miscanthus, and maize. Different methylimidazolium ILs were compared and the samples were treated with two different heating processes: microwave irradiation and conventional heating in a sealed tube. The conversion yield and influence of the treatment on the lignin structure were assessed by 31 P NMR spectroscopy, size-exclusion chromatography, and thioacidolysis. The acidic methylimidazolium IL [HMIM]Br was shown to be an effective combination of solvent and reagent for the demethylation and depolymerization of lignin. The relatively mild reaction conditions, the clean work-up, and the ability to reuse the IL makes the described procedure an attractive and new green method for the conversion of lignin to produce phenol-rich lignin oligomers.

Use of food and packaging model matrices to investigate the antioxidant properties of biorefinery grass lignins.

The antioxidant properties of grass lignins recovered from an alkaline industrial process and from different ethanol organosolv pretreatment processes were compared using two types of tests: (i) classical radical 2,2'-diphenyl-1-picrylhydrazyl (DPPH(•)) scavenging tests in dioxane/water or ethanol and (ii) tests involving multiphasic systems (lipid dispersion in water or cellulose film suspended in ethanol). These multiphasic systems were representative of food and packaging matrices in view of high-value applications. All lignins, in solution or in the film, effectively scavenged radicals. Moreover, they were competitive with a food commercial rosemary extract to protect linoleic acid against oxidation. Whereas the DPPH(•) test in dioxane was not discriminant, differences appeared between lignins when the test was performed in ethanol or with the multiphasic systems. Moreover, radical scavenging activity was preserved in the film even after its immersion in ethanol. Structural analysis of lignins revealed that low-molar-mass phenolics, namely p-hydroxycinnamic acids and lignin depolymerization products, governed lignin antioxidant properties in the multiphasic systems.

Sugar cane stillage: a potential source of natural antioxidants.

Biorefinery of sugar cane is the first economic activity of Reunion Island. Some sugar cane manufactured products (juice, syrup, molasses) have antioxidant activities and are sources of both phenolic compounds and Maillard Reaction Products (MRP). The study aimed to highlight the global antioxidant activity of sugar cane stillage and understand its identity. Chromatographic fractionation on Sephadex LH-20 resin allowed the recovery of a MRP-rich fraction, responsible for 58 to 66% of the global antioxidant activity according to the nature of the sugar cane stillage (DPPH test), and a phenolic compounds-rich fraction for 37 to 59% of the activity. A good correlation was recorded between the antioxidant activity of the sugar cane stillage and its content in total reducing compounds amount (Folin-Ciocalteu assay), among them 2.8 to 3.9 g/L of phenolic compounds (in 5-caffeoylquinic acid equivalent). Preliminary experiments by HPLC-DAD-MS allowed to identify several free phenolic acids and gave clues to identify esters of quinic acids.

Hydrophobic properties conferred to Kraft pulp by a laccase-catalysed treatment with lauryl gallate.

Hydrophobic properties were conferred to a high-lignin-content Kraft pulp by a laccase-catalysed treatment in the presence of lauryl gallate (LG). The treatment resulted in a two-fold increase in contact angle and conferred water absorption resistance to the pulp. Kappa number was increased, indicating that some phenolic compounds were incorporated in the pulp. A control treatment with LG alone did not affect water absorption, demonstrating that laccase was essential to attain these new properties. The loss of hydrophobicity after an acetone Soxhlet extraction highlighted that adsorbed acetone-soluble compounds played a key role in the properties. GC-FID and HPSEC-UV analysis of the acetone extract indicated the formation of dodecanol and different phenolic oligomers. SEM images showed the treatment-induced changes in the fibre network. Additional experiments with various reaction times and reactant concentrations highlighted the role of LG oxidation products in the introduction of absorption resistance.

Isolation of structurally distinct lignin-carbohydrate fractions from maize stem by sequential alkaline extractions and endoglucanase treatment.

Sequential fractionation of extractive-free maize stems was carried out using two mild alkaline extractions (0.5 and 2 M NaOH, 20°C, 24h) before and after endoglucanase treatment. This procedure provided two lignin-carbohydrate fractions (LC1 and LC2) recovered after each alkali treatment. LC1 and LC2 contained 39% and 8% of the total lignin amount, respectively. These two fractions contained structurally distinct lignin molecules. While the content of resistant interunit bonds in lignin was 77% in LC1, it was increased up to 98% in LC2. Not unexpectedly, both alkali-soluble fractions contained substantial amount of p-coumaric and ferulic acids ether-linked to lignins. These results outline heterogeneity of maize stem lignins related to fractionation of grass materials.

Adaptation of the Carrez procedure for the purification of ferulic and p-coumaric acids released from lignocellulosic biomass prior to LC/MS analysis.

The objective of this study was to adapt and improve an environmentally friendly and fast routine method for the analysis of ferulic and p-coumaric acids released from grass cell-walls by alkaline hydrolysis. This methodological development was performed on maize samples selected for their contrasted contents in ferulic and p-coumaric acids as a consequence of their different maturity stages (from stage of 7th leaf with visible ligule to stage of silage harvest). We demonstrate that the Carrez method is an efficient substitute to the common solvent-consuming extraction by ethyl acetate for the preparation of samples suitable for HPLC-ESI-MS analysis. We prove that it is possible to replace methanol by ethanol in the Carrez step and at last we propose a scale reduction of this procedure that offer a first step towards high throughput determinations. The new method leads to a solvent consumption reduced by a factor 100 and only requires ethanol as organic solvent.

Impact of lignin structure and cell wall reticulation on maize cell wall degradability.

In this study, eight maize recombinant inbred lines were selected to assess both the impact of lignin structure and the impact of cell wall reticulation by p-hydroxycinnamic acids on cell wall degradability independently of the main "lignin content" factor. These recombinant lines and their parents were analyzed for in vitro degradability, cell wall residue content, esterified and etherified p-hydroxycinnamic acid content, and lignin content and structure. Lignin structure and esterified p-coumaric acid content showed significantly high correlation with in vitro degradability (r=-0.82 and r=-0.72, respectively). A multiple regression analysis showed that more than 80% of cell wall degradability variations within these 10 lines (eight recombinant inbred lines and their two parents) were explained by a regression model including two main explanatory factors: lignin content and estimated proportion of syringyl lignin units esterified by p-coumaric acid. This study revealed new biochemical parameters of interest to improve cell wall degradability and promote lignocellulose valorization.

Effect of pretreatment and enzymatic hydrolysis of wheat straw on cell wall composition, hydrophobicity and cellulase adsorption.

The present study aimed to determine the impact of cell wall composition and lignin content on enzyme adsorption and degradability. Thioacidolysis analysis of residual lignins in wheat straw after steam-explosion or organosolv pretreatment revealed an increase in lignin condensation degree of 27% and 33%, respectively. Surface hydrophobicity assessed through wettability tests decreased after the pretreatments (contact angle decrease of 20-50%), but increased with enzymatic conversion (30% maximum contact angle increase) and correlatively to lignin content. Adsorption of the three major cellulases Cel7A, Cel6A and Cel7B from Trichoderma reesei decreased with increasing hydrolysis time, down to 7%, 31% and 70% on the sample with the highest lignin content, respectively. The fraction of unspecifically bound enzymes was dependent both on the enzyme and the lignin content. Adsorption and specific activity were shown to be inversely proportional to lignin content and hydrophobicity, suggesting that lignin is one of the factors restricting enzymatic hydrolysis.

Study of a specific lignin model: γ-oxidation and how it influences the hydrolysis efficiency of alcohol-aldehyde dehydrogenation copolymers.

Six coniferyl alcohol-coniferaldehyde dehydrogenation copolymers (DHcoPs) were synthesized in order to determine the influence of an increased number of aldehyde functions on hydrolysis. After heterogeneous hydrolysis using acidic Montmorillonite K10 clay, the DHcoPs were thioacidolyzed and analyzed by gel permeation chromatography (GPC). Comparison of the thioacidolyzed products, with and without the hydrolysis step, showed that there was a greater proportion of condensation reaction in the absence of aldehyde. When the coniferaldehyde content in the initial synthetic mixture was more than 30% (w/w), only a low fraction of condensed products was generated during the K10 clay hydrolysis step. This suggests that condensation pathways are mainly due to the alcohol present in the γ-position in the DHcoPs. Investigation of the reactivity and the potential condensation of aldehyde and alcohol monomers under hydrolysis conditions showed the important conversion of coniferyl alcohol and conversely the stability of coniferaldehyde.