Combined hemicellulolytic and phenoloxidase activities of Thermobacillus xylanilyticus enable growth on lignin-rich substrates and the release of phenolic molecules.

Major challenge in biorefineries is the use of all lignocellulosic components, particularly lignins. In this study, Thermobacillus xylanilyliticus grew on kraft lignin, steam-exploded and native wheat straws produced different sets of phenoloxidases and xylanases, according to the substrate. After growth, limited lignin structural modifications, mainly accompanied by a decrease in phenolic acids was observed by Nuclear Magnetic Resonance spectroscopy. The depletion of p-coumaric acid, vanillin and p-hydroxybenzaldehyde combined to vanillin production in the culture media indicated that the bacterium can transform some phenolic compounds. Proteomic approaches allowed the identification of 29 to 33 different hemicellulases according to the substrates. Twenty oxidoreductases were differentially expressed between kraft lignin and steam-exploded wheat straw. These oxidoreductases may be involved in lignin and aromatic compound utilization and detoxification. This study highlights the potential value of Thermobacillus xylanilyticus and its enzymes in the simultaneous valorization of hemicellulose and phenolic compounds from lignocelluloses.

Morphological growth pattern of Phanerochaete chrysosporium cultivated on different Miscanthus x giganteus biomass fractions.

BACKGROUND: Solid-state fermentation is a fungal culture technique used to produce compounds and products of industrial interest. The growth behaviour of filamentous fungi on solid media is challenging to study due to the intermixity of the substrate and the growing organism. Several strategies are available to measure indirectly the fungal biomass during the fermentation such as following the biochemical production of mycelium-specific components or microscopic observation. The microscopic observation of the development of the mycelium, on lignocellulosic substrate, has not been reported. In this study, we set up an experimental protocol based on microscopy and image processing through which we investigated the growth pattern of Phanerochaete chrysosporium on different Miscanthus x giganteus biomass fractions. RESULTS: Object coalescence, the occupied surface area, and radial expansion of the colony were measured in time. The substrate was sterilized by autoclaving, which could be considered a type of pre-treatment. The fastest growth rate was measured on the unfractionated biomass, followed by the soluble fraction of the biomass, then the residual solid fractions. The growth rate on the different fractions of the substrate was additive, suggesting that both the solid and soluble fractions were used by the fungus. Based on the FTIR analysis, there were differences in composition between the solid and soluble fractions of the substrate, but the main components for growth were always present. We propose using this novel method for measuring the very initial fungal growth by following the variation of the number of objects over time. Once growth is established, the growth can be followed by measurement of the occupied surface by the mycelium. CONCLUSION: Our data showed that the growth was affected from the very beginning by the nature of the substrate. The most extensive colonization of the surface was observed with the unfractionated substrate containing both soluble and solid components. The methodology was practical and may be applied to investigate the growth of other fungi, including the influence of environmental parameters on the fungal growth.

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.

Oxalic acid: a microbial metabolite of interest for the pulping industry.

Oxalate is a common metabolite produced by almost all plant-pathogenic fungi. The degradation of cell wall from poplar chips and poplar sawdust by oxalate is reviewed here. Oxalate treatments decrease slightly the amount of sugars constituting hemicelluloses, but only in fibres and not in sawdust or wood chips. The examination of the cell wall ultrastructure of wood chips by transmission electron microscopy (TEM) after polysaccharide staining showed a characteristic fading of the staining of the S1/S2 and S2/S3 transition areas, supporting the idea that reactivity and organization of polysaccharides had changed after the oxalate treatments. Finally, all these changes enhanced the ability of the wood chips to be defibrated by a thermomechanical (TMP) process, as well as the further refining of the pulps. Looking at the fiber surface, it became apparent that fracture areas during the TMP pulping had moved toward the S2 layer, explaining why defibrating and refining occurred more easily, with less energy input in the process.

Abiotic degradation of lignified cell walls by carbonate and copper salt.

This study reports on the destructuration of Wheat straw and Spruce wood cell walls after maceration in potassium carbonate or sodium hydroxide at pH = 10 in the presence of copper acetate. The alkaline treatments had a predominant impact on the wheat straw cell wall components over copper acetate. Either K-carbonate or Na-hydroxide extracted from wheat straw a particular lignin fraction rich in condensed C-C linkages, leading to the unmasking of new ether-linked sub-structures in the cell wall. This unmasking was increased in the presence of copper salt but only in the nonextracted Wheat straw sample incubated in carbonate and not in the corresponding extractive-free sample. This difference was related to the leaching of compounds from the nonextracted cell wall, which could sustain oxidative activity of copper by hindering its precipitation into inactive hydroxide and/or carbonate species. In Spruce wood samples, copper salt was the principal factor impacting on the lignin structure over alkali alone. Its effect was, however, only detected at the level of C-C linked dimers. These results confirmed that unmasking of lignin sub-structures also occurred in Spruce wood, but probably through mechanisms different from that evidenced in Wheat straw.

The unmasking of lignin structures in wheat straw by alkali.

This study reports on the structural modifications of wheat straw cell wall promoted by potassium carbonate and sodium hydroxide that lead to the unmasking of some lignin structures. The first impact of the treatments was the extraction of a particular fraction of lignin enriched in C-C linked structures compared to the mean composition in reference wheat straw. Concomitantly, an apparent increase in the amount of lignin monomers released by the cleavage of alkyl-aryl ether bonds was observed in alkali-extracted samples. By summing the amount of ether linked monomers analyzed by thioacidolysis in the solubilized lignin to that found in the extracted wheat straw, an excess of up to 37% is apparent, relative to the corresponding amount in the reference wheat straw. Other modifications of the cell wall were also found. Indeed, a fraction of uronic acids was lost during the treatments and a new fractionation pattern of the lignin-carbohydrate complexes was evidenced. It can thus be concluded that a significant proportion of lignin within the cell wall was unmasked after (i) the selective removal of a particular lignin fraction, (ii) a partial saponification of the esterified fraction of lignin with uronic acids and (iii) a modification of the interactions between the cell wall constituents.