Biodegradation Of Lignin By Fungi, Bacteria And Laccases

Lignin, a heterogeneous three-dimensional biopolymer, is one of the building blocks of lignocellulosic biomass. Due to its limited chemical reactivity, lignin is currently processed as a low value by-product in pulp and paper mills. Therefore, valorization of lignin holds great potential as this would provide a readily available source of aromatic compounds for various industrial applications. In this study, indulin AT biodegradation was assessed by comparing the effects of basidiomycetous fungi (Coriolus versicolor and Trametes gallica) and actinobacteria (Mycobacterium sp. and Streptomyces sp.) to those of two commercial laccases, those from T. versicolor (≥ 10 U/mg) and C. versicolor (≥ 0.3 U/mg) while using a suite of chemical analysis methods. The research results showed that after 54 days of cultivation, microbial (especially fungal) lignin biodegradation is significantly greater than that caused by commercial laccases, reaching a maximum of 20 wt. % degradation for C. versicolor by gravimetric analysis. The extent of microbial degradation was further confirmed by thermal carbon analysis (TCA), as all treatments led to changes of the thermal carbon elution profile in the supernatants. However, laccase treatments resulted in only minor changes with increases occurring in the 850 °C and char fractions, thus evidencing the formation of cross-linked polymers. The fungally treated lignin showed a similar change in the thermal carbon elution profile, along with a gradual decrease of the total carbon in the supernatant, indicating significant lignin mineralization. Bacterial treatment, on the other hand, mainly led to carbon solubilization instead of mineralization. Chemical characterization of lignin degradation products performed by Thermal Desorption-Pyrolysis-Gas Chromatography-Mass Spectrometry (TD-Pyrolysis-GC-MS) corroborated the carbon fractionation obtained by TCA. The laccase treatments yielded more phenol-based compounds and aromatic hydrocarbons eluting only at higher pyrolytic temperatures, i.e., 700 °C at the expense of monomers eluting at lower temperatures, thus confirming extensive lignin polymerization. The fungal treatments led to similar changes, with a significant consumption of low molecular weight phenolics whereas the bacterial treatments, conversely, facilitated the production of phenolic monomers eluting at low temperatures. Thus, fungi appear to mostly cause significant lignin mineralization combined with polymerization whereas bacteria instead tend to produce phenolic monomers without their further catabolism

Electrospray Ionization with High-Resolution Mass Spectrometry as a Tool for Lignomics: Lignin Mass Spectrum Deconvolution

Capability to characterize lignin, lignocellulose, and their degradation products is essential for development of new renewable feedstocks. Electrospray ionization high-resolution time-offlight mass spectrometry (ESI HR TOF MS) method was developed expanding the lignomics toolkit while targeting the simultaneous detection of low and high molecular weight (MW) lignin species. The effect of a broad range of electrolytes and various ionization conditions on ion formation and ionization effectiveness was studied using a suite of mono-, di- and triarene lignin model compounds as well as intact lignin. Contrary to the previous studies, the positive ionization mode was found to be more effective for methoxy-substituted arenes and polyphenols, i.e., species of a broadly varied MW structurally similar to the native lignin. For the first time, we report an effective formation of multiply charged species of lignin with the subsequent mass spectrum deconvolution in the presence of 100 mmol·L-1 formic acid in the positive ESI mode. The developed method enabled the detection of lignin species with an MW between 150 and 9,000 Da or higher, depending on the mass analyzer. The obtained Mn and Mw values of 1,500 and 2,500 Da, respectively, were in good agreement with those determined by gel permeation chromatography. Furthermore, the deconvoluted ESI mass spectrum was similar to that obtained with matrixassisted laser desorption/ionization (MALDI) TOF MS, yet featuring a higher signal-to-noise ratio. The formation of multiply charged species was confirmed with ESI ion mobility HR Q-TOF MS

Fungal Biotransformation of Insoluble Kraft Lignin into a Water Soluble Polymer

Low substrate solubility and slow decomposition/biotransformation rate are among the main impediments for industrial scale lignin biotreatment. The outcome and dynamics of kraft lignin biomodification by basidiomycetous fungi, <i>Coriolus versicolor</i>, were investigated in the presence of dimethyl sulfoxide (DMSO). The addition of 2 vol % DMSO to aqueous media increased the lignin solubility up to 70%, while the quasi-immobilized fungi (pregrown on agar containing kenaf biomass) maintained their ability to produce lignolytic enzymes. Basidiomycetous fungi were able to grow on solid media containing both 5–25 g/L lignin and up to 5 vol % DMSO, in contrast to no growth in liquid media as a free suspended culture. When a fungal culture pregrown on agar was used for lignin treatment in an aqueous medium containing 2–5% DMSO with up to 25 g/L lignin, significant lignin modification was observed in 1–6 days. The product analysis suggests that lignin was biotransformed, rather than biodegraded, into an oxygenated and cross-linked phenolic polymer. The resulting product showed the removal of phenolic monomers and/or their immediate precursors based on gas chromatography and thermal desorption–pyrolysis–gas chromatography–mass spectrometry analyses. Significant intramolecular cross-linking among the reaction products was shown by thermal carbon analysis and ¹H NMR spectroscopy. An increase in polarity, presumably due to oxygenation, and a decrease in polydispersity of the lignin treatment product compared to untreated lignin were observed while using liquid chromatography