Lignin Breakdown Products Research Articles

Increased triacylglycerol production in Rhodococcus opacus by overexpressing transcriptional regulators

Lignocellulosic biomass is currently underutilized, but it offers promise as a resource for the generation of commercial end-products, such as biofuels, detergents, and other oleochemicals. Rhodococcus opacus PD630 is an oleaginous, Gram-positive bacterium with an exceptional ability to utilize recalcitrant aromatic lignin breakdown products to produce lipid molecules such as triacylglycerols (TAGs), which are an important biofuel precursor. Lipid carbon storage molecules accumulate only under growth-limiting low nitrogen conditions, representing a significant challenge toward using bacterial biorefineries for fuel precursor production. In this work, we screened overexpression of 27 native transcriptional regulators for their abilities to improve lipid accumulation under nitrogen-rich conditions, resulting in three strains that accumulate increased lipids, unconstrained by nitrogen availability when grown in phenol or glucose. Transcriptomic analyses revealed that the best strain (#13) enhanced FA production via activation of the β-ketoadipate pathway. Gene deletion experiments confirm that lipid accumulation in nitrogen-replete conditions requires reprogramming of phenylalanine metabolism. By generating mutants decoupling carbon storage from low nitrogen environments, we move closer toward optimizing R. opacus for efficient bioproduction on lignocellulosic biomass.

Unraveling interspecies cross-feeding during anaerobic lignin degradation for bioenergy applications

Lignin, a major component of plant biomass, remains underutilized for renewable biofuels due to its complex and heterogeneous structure. Although investigations into depolymerizing lignin using fungi are well-established, studies of microbial pathways that enable anaerobic lignin breakdown linked with methanogenesis are limited. Through an enrichment cultivation approach with inoculation of freshwater sediment, we enriched a microbial community capable of producing methane during anaerobic lignin degradation. We reconstructed the near-complete population genomes of key lignin degraders and methanogens using metagenome-assembled genomes finally selected in this study (MAGs; 92 bacterial and 4 archaeal MAGs affiliated into 45 and 2 taxonomic groups, respectively). This study provides genetic evidence of microbial interdependence in conversion of lignin to methane in a syntrophic community. Metagenomic analysis revealed metabolic linkages, with lignin-hydrolyzing and/or fermentative bacteria such as the genera Alkalibaculum and Propionispora transforming lignin breakdown products into compounds such as acetate to feed methanogens (two archaeal MAGs classified into the genus Methanosarcina or UBA6 of the family Methanomassiliicoccaceae). Understanding the synergistic relationships between microbes that convert lignin could inform strategies for producing renewable bioenergy and treating aromatic-contaminated environments through anaerobic biodegradation processes. Overall, this study offers fundamental insights into complex community-level anaerobic lignin metabolism, highlighting hitherto unknown players, interactions, and pathways in this biotechnologically valuable process.

Organic compounds in the char deposits characterising the combustion of unauthorised fuels in residential boilers

The paper aims to find a way of simply identifying the addition of unauthorised waste (plastics) in residential boilers burning wood as the main fuel. The burning of plastics leaves a trace in char deposits, which are otherwise made up mostly of lignin breakdown products. Identification of combusted plastics in char deposits is made possible by determining unique organic compounds formed during the pyrolysis phase of combustion by pyrolysis gas chromatography. At temperatures above 800 °C, the amount of charring product from the incineration of PET is 19.5 wt%, while for PP, it is 5 wt%, and LDPE with HDPE 2.1 wt%. Combustion of polypropylene with wood has been verified by the presence of the compounds 2,4-dimethyl-1-heptene, 2-methyl-1-pentene, and 2,4,6-trimethyl-1-nonene. The thermal degradation of PE has been demonstrated by the presence of triplets of alkanes, alkenes, and alkadienes. Combustion of PET in domestic waste can be demonstrated by the presence of benzoic acid and its esters, benzoate compounds, and terephthalic acid. In summarising, sampling char in boilers and analysing samples for markers of plastics incineration could be used as a method for identification of unauthorised waste combustion by authorities.

Characterizing the Interplay of Rubisco and Nitrogenase Enzymes in Anaerobic-Photoheterotrophically Grown Rhodopseudomonas palustris CGA009 through a Genome-Scale Metabolic and Expression Model.

ABSTRACTRhodopseudomonas palustris CGA009 is a Gram-negative purple nonsulfur bacterium that grows phototrophically by fixing carbon dioxide and nitrogen or chemotrophically by fixing or catabolizing a wide array of substrates, including lignin breakdown products for its carbon and fixing nitrogen for its nitrogen requirements. It can grow aerobically or anaerobically and can use light, inorganic, and organic compounds for energy production. Due to its ability to convert different carbon sources into useful products during anaerobic growth, this study reconstructed a metabolic and expression (ME) model of R. palustris to investigate its anaerobic-photoheterotrophic growth. Unlike metabolic (M) models, ME models include transcription and translation reactions along with macromolecules synthesis and couple these reactions with growth rate. This unique feature of the ME model led to nonlinear growth curve predictions, which matched closely with experimental growth rate data. At the theoretical maximum growth rate, the ME model suggested a diminishing rate of carbon fixation and predicted malate dehydrogenase and glycerol-3 phosphate dehydrogenase as alternate electron sinks. Moreover, the ME model also identified ferredoxin as a key regulator in distributing electrons between major redox balancing pathways. Because ME models include the turnover rate for each metabolic reaction, it was used to successfully capture experimentally observed temperature regulation of different nitrogenases. Overall, these unique features of the ME model demonstrated the influence of nitrogenases and rubiscos on R. palustris growth and predicted a key regulator in distributing electrons between major redox balancing pathways, thus establishing a platform for in silico investigation of R. palustris metabolism from a multiomics perspective.IMPORTANCE In this work, we reconstructed the first ME model for a purple nonsulfur bacterium (PNSB). Using the ME model, different aspects of R. palustris metabolism were examined. First, the ME model was used to analyze how reducing power entering the R. palustris cell through organic carbon sources gets partitioned into biomass, carbon dioxide fixation, and nitrogen fixation. Furthermore, the ME model predicted electron flux through ferredoxin as a major bottleneck in distributing electrons to nitrogenase enzymes. Next, the ME model characterized different nitrogenase enzymes and successfully recapitulated experimentally observed temperature regulations of those enzymes. Identifying the bottleneck responsible for transferring an electron to nitrogenase enzymes and recapitulating the temperature regulation of different nitrogenase enzymes can have profound implications in metabolic engineering, such as hydrogen production from R. palustris. Another interesting application of this ME model can be to take advantage of its redox balancing strategy to gain an understanding of the regulatory mechanism of biodegradable plastic production precursors, such as polyhydroxybutyrate (PHB).

Heterologous phasin expression in Rhodopseudomonas palustris CGA009 for bioplastic production from lignocellulosic biomass

Rhodopseudomonas palustris CGA009 is a metabolically robust microbe that can utilize lignin breakdown products to produce polyhydroxyalkanoates (PHAs), biopolymers with the potential to replace conventional plastics. Our recent efforts suggest PHA granule formation is a limiting factor for maximum production of the bioplastic poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) by R. palustris. The Phap1 phasin (phaP1) from the PHB-producing model bacterium Cupriavidus necator H16 was expressed in R. palustris with the aim of overproducing PHBV from the lignin breakdown product p-coumarate by fostering smaller and more abundant granules. Expression of phaP1 yielded PHBV production from R. palustris aerobically (0.7 g/L), which does not occur in the wild-type strain, and led to a significantly higher PHBV titer than wild-type anaerobic production (0.41 g/L). The 3HV fractions were also significantly increased under both anaerobic and aerobic conditions, which boosts thermomechanical properties and potential for application. Thus, heterologous phasin expression in R. palustris provides flexibility for industrial processing and could foster compositional changes in copolymers with better thermomechanical properties compared to PHB alone.

Emission factors and chemical characterization of particulate emissions from garden green waste burning

This work provides an evaluation of the emission factors (EFs) of typical garden waste burning (fallen leaves and hedge trimming) in terms of particulate matter (PM), elemental and organic carbon (EC-OC) together with a detailed chemical characterization of 88 particle-bound organic species including polycyclic aromatic hydrocarbons (PAHs), levoglucosan and its isomers, lignin breakdown products (methoxyphenols), cholesterol, alkanes, polyols and sugars. Furthermore, wood-log based burning experiments have been performed to highlight key indicators or chemical patterns of both, green waste and wood burning (residential heating) sources, that may be used for PM source apportionment purposes. Two residential log wood combustion appliances, wood stove (RWS) and fireplace, under different output conditions (nominal and reduced) and wood log moisture content (mix of beech, oak and hornbeam), have been tested. Open wood burning experiments using wood logs were also performed. Green waste burning EFs obtained were comparable to the available literature data for open-air biomass burning. For PM and for most of the organic species studied, they were about 2 to 30 times higher than those observed for wood log combustion experiments. Though, poor performance wood combustions (open-air wood log burning, fireplace and RWS in reduced output) showed comparable EFs for levoglucosan and its isomers, methoxyphenols, polyols, PAHs and sugars. Toxic PAH equivalent benzo[a]pyrene EFs were even 3–10 times higher for the fireplace and open-air wood log burning. These results highlighted the impact of the nature of the fuel burnt and the combustion performances on the emissions. Different chemical fingerprints between both biomass burning sources were highlighted with notably a predominance of odd high-molecular weight n-alkanes (higher carbon preference index, CPI), lower levoglucosan/mannosan ratio and lower sinapylaldehyde abundance for green waste burning. However, the use of such indicators seems limited, especially if applied alone, for a clear discrimination of both sources in ambient air.

Enhancement of polyhydroxyalkanoate production by co-feeding lignin derivatives with glycerol in Pseudomonas putida KT2440

BackgroundEfficient utilization of all available carbons from lignocellulosic biomass is critical for economic efficiency of a bioconversion process to produce renewable bioproducts. However, the metabolic responses that enable Pseudomonas putida to utilize mixed carbon sources to generate reducing power and polyhydroxyalkanoate (PHA) remain unclear. Previous research has mainly focused on different fermentation strategies, including the sequential feeding of xylose as the growth stage substrate and octanoic acid as the PHA-producing substrate, feeding glycerol as the sole carbon substrate, and co-feeding of lignin and glucose. This study developed a new strategy—co-feeding glycerol and lignin derivatives such as benzoate, vanillin, and vanillic acid in Pseudomonas putida KT2440—for the first time, which simultaneously improved both cell biomass and PHA production.ResultsCo-feeding lignin derivatives (i.e. benzoate, vanillin, and vanillic acid) and glycerol to P. putida KT2440 was shown for the first time to simultaneously increase cell dry weight (CDW) by 9.4–16.1% and PHA content by 29.0–63.2%, respectively, compared with feeding glycerol alone. GC–MS results revealed that the addition of lignin derivatives to glycerol decreased the distribution of long-chain monomers (C10 and C12) by 0.4–4.4% and increased the distribution of short-chain monomers (C6 and C8) by 0.8–3.5%. The 1H–13C HMBC, 1H–13C HSQC, and 1H–1H COSY NMR analysis confirmed that the PHA monomers (C6–C14) were produced when glycerol was fed to the bacteria alone or together with lignin derivatives. Moreover, investigation of the glycerol/benzoate/nitrogen ratios showed that benzoate acted as an independent factor in PHA synthesis. Furthermore, 1H, 13C and 31P NMR metabolite analysis and mass spectrometry-based quantitative proteomics measurements suggested that the addition of benzoate stimulated oxidative-stress responses, enhanced glycerol consumption, and altered the intracellular NAD+/NADH and NADPH/NADP+ ratios by up-regulating the proteins involved in energy generation and storage processes, including the Entner–Doudoroff (ED) pathway, the reductive TCA route, trehalose degradation, fatty acid β-oxidation, and PHA biosynthesis.ConclusionsThis work demonstrated an effective co-carbon feeding strategy to improve PHA content/yield and convert lignin derivatives into value-added products in P. putida KT2440. Co-feeding lignin break-down products with other carbon sources, such as glycerol, has been demonstrated as an efficient way to utilize biomass to increase PHA production in P. putida KT2440. Moreover, the involvement of aromatic degradation favours further lignin utilization, and the combination of proteomics and metabolomics with NMR sheds light on the metabolic and regulatory mechanisms for cellular redox balance and potential genetic targets for a higher biomass carbon conversion efficiency.

Internalization and accumulation of model lignin breakdown products in bacteria and fungi

BackgroundValorization of lignin has the potential to significantly improve the economics of lignocellulosic biorefineries. However, its complex structure makes conversion to useful products elusive. One promising approach is depolymerization of lignin and subsequent bioconversion of breakdown products into value-added compounds. Optimizing transport of these depolymerization products into one or more organism(s) for biological conversion is important to maximize carbon utilization and minimize toxicity. Current methods assess internalization of depolymerization products indirectly—for example, growth on, or toxicity of, a substrate. Furthermore, no method has been shown to provide visualization of depolymerization products in individual cells.ResultsWe applied mass spectrometry to provide direct measurements of relative internalized concentrations of several lignin depolymerization compounds and single-cell microscopy methods to visualize cell-to-cell differences in internalized amounts of two lignin depolymerization compounds. We characterized internalization of 4-hydroxybenzoic acid, vanillic acid, p-coumaric acid, syringic acid, and the model dimer guaiacylglycerol-beta-guaiacyl ether (GGE) in the lignolytic organisms Phanerochaete chrysosporium and Enterobacter lignolyticus and in the non-lignolytic but genetically tractable organisms Saccharomyces cerevisiae and Escherichia coli. The results show varying degrees of internalization in all organisms for all the tested compounds, including the model dimer, GGE. Phanerochaete chrysosporium internalizes all compounds in non-lignolytic and lignolytic conditions at comparable levels, indicating that the transporters for these compounds are not specific to the lignolytic secondary metabolic system. Single-cell microscopy shows that internalization of vanillic acid and 4-hydroxybenzoic acid analogs varies greatly among individual fungal and bacterial cells in a given population. Glucose starvation and chemical inhibition of ATP hydrolysis during internalization significantly reduced the internalized amount of vanillic acid in bacteria.ConclusionsMass spectrometry and single-cell microscopy methods were developed to establish a toolset for providing direct measurement and visualization of relative internal concentrations of mono- and di-aryl compounds in microbes. Utilizing these methods, we observed broad variation in intracellular concentration between organisms and within populations and this may have important consequences for the efficiency and productivity of an industrial process for bioconversion. Subsequent application of this toolset will be useful in identifying and characterizing specific transporters for lignin-derived mono- and di-aryl compounds.

Enhanced removal of bio-refractory dissolved organic matter from cassava distillery wastewater by powdered activated carbon-ballasted coagulation: Detailed study of separation characteristics and mechanisms

Efficient removal of bio-refractory dissolved organic matter (DOM) and colorants is essential for discharging or reusing the distillery wastewater. An important part of recalcitrant DOM still exists in the effluent of regular coagulation though the ferric coagulant has been found to be effective in decoloration. The present work adopted powdered activated carbon (PAC) as ballasting agent to achieve robust separation effect and efficiency of bio-refractory DOM from the bio-chemically treated cassava distillery wastewater (BTDWW). More than 90% of DOC could be removed at the PAC and Fe(III)-coagulant dosage of 1.40 g/L and 0.84 g/L as Fe when the BTDWW was neutral. PAC should be dosed before coagulant in order to mix well with the DOM in the BTDWW. The analyses of DOM in effluent reveal that PAC facilitated the removal of lignin breakdown products which could not be well eliminated by regular coagulation; the removal of DOM with MW < 5 kDa was mostly enhanced. The characteristics of flocs demonstrate that PAC reinforced the interaction between Fe(III) species and DOM by providing more reaction sites. The sedimentation could be completed within the initial 5 min, and the highest settling velocity was almost 8 times higher than that of the only Fe(III)-involved flocs. The large size and favorable robustness of PAC-involved flocs enabled decent sedimentation even though their stretched structure might not be desirable in regular coagulation. The PAC-ballasted coagulation is recommended as tertiary treatment of BTDWW considering its high efficiency and sound economic feasibility.

Molecular-Weight-Fractionation Characteristics and Coagulation Behaviors of Biorecalcitrant Dissolved Organic Matter and Colorants in Cassava Distillery Wastewater

The biorecalcitrant dissolved organic matter (DOM) and colorants in biochemically treated effluent, known for their complex and indefinite composition, are drawing increasing environmental concern in the distillery industry. The current study focuses on the evaluation of the hard DOM and colorants based on their molecular weight fractionation, which facilitates the colorant identification and the mechanism exploration in the subsequent coagulation process. It was found that the DOM was distributed most in the molecular weight (MW) range of 1–5 kDa and least in the MW range below 1 kDa; the fluorescent components in the biochemically treated distillery wastewater mainly distributed in low MW (<5 kDa, 73%). As the primary cassava-based biorecalcitrant colorants, the colorants with MWs of 1–5 kDa were probably lignin breakdown products with aromaticity. In the coagulation process, the MW of DOM was among the most significant factors influencing the floc size which decreased with the MW reduction.

Transport of Lignin-Breakdown Products by Genetically Engineered Non-Lignolytic Bacteria and Fungi

Transport of Lignin-Breakdown Products by Genetically Engineered Non-Lignolytic Bacteria and Fungi

Bio-refractory dissolved organic matter and colorants in cassava distillery wastewater: Characterization, coagulation treatment and mechanisms

An important portion of organic matter and colorants still remain in the biologically treated distillery wastewater, leaving the dark brown and odorous downstream with the heavy loading of chemical oxygen demand and the potential of forming disinfection byproducts. However, those bio-recalcitrant colorants have not been clearly recognized. The current study investigated the features of the bio-refractory organic matter and colorants in a typical distillery effluent, cassava distillery wastewater; special attention was paid to their change and behaviors in the coagulation treatment following the bio-processes. The wastewater analyses denoted that the fraction of high molecular weight (1–50 kDa and >50 kDa) became predominant after the anaerobic-aerobic processes. Importantly, the lignin breakdown products, melanoidins and lignin phenols were confirmed to be the leading colored components, according to the parallel factor analysis of fluorescence excitation-emission matrixes results. Compared with lignin phenols, the former two types of colorants exhibited stronger bio-refractory activity and resulted in smaller color reduction after the aerobic treatment. Neither advanced oxidation nor adsorption could perform efficiently as post-treatment for decolorization in this study. Nevertheless, high removal of color and dissolved organic matter (∼94.0% and ∼78.3%, respectively) could be achieved by the FeCl3-involved coagulation under the optimal conditions. The ferric coagulant was found to preferably interact with the aromatic compounds (such as lignin derivatives) and melanoidins via either surface complexation or electric charge neutralization, or both. The findings presented herein might provide an insight into the evaluation of bio-refractory organic colorants and the Fe(III)-involved decolorization mechanisms of ethanol production wastewaters.

Novel Mixed‐Mode Stationary Phases for Chromatographic Separation of Complex Mixtures of Decomposed Lignin

AbstractElectrochemical degradation of lignin results in a large number of unknown products, formed by both oxidative and reductive decomposition pathways, which have previously been studied by direct mass spectrometry experiments without prior liquid chromatography separation, revealing the presence of many isobaric molecules. In this study, novel phosphonium‐based ionic liquid stationary phases were synthesized and applied in conjunction with high resolution mass spectrometry, to further increase the resolving power of the analytical methodology. The new stationary phases exhibited a wide range of interaction mechanisms in comparison to reversed‐phase materials, and the preliminary results of this proof‐of‐concept study illustrated class‐specific isolation and separation of the major chemical classes of the lignin breakdown products members into very specific retention time windows. For the investigated electrochemically decomposed lignin, the separations revealed predominantly aldehydes and alcohols, and also confirmed the presence of a significant number of isobars.

Role of laccases and peroxidases in saprotrophic activities in the lichen Usnea undulata

Abstract Lichens produce various oxidoreductases including heme-containing peroxidases and the copper-containing phenol oxidases tyrosinase and laccase. Our earlier findings suggested that significant oxidoreductase activity occurs mainly in lichens from the order Peltigerales. Here we show that the non-Peltigeralean lichen Usnea can display significant activities of peroxidases and laccases. Strong evidence for the involvement of peroxidases and laccases in saprotrophic activities comes from the observation that their activities are induced by “starvation” due to prolonged dark storage, and also by treatment with soluble cellulose and lignin breakdown products. We also show that, given a quinone and chelated Fe, Usnea can produce hydroxyl radicals; these radicals contribute to the break down of carbohydrates or lignin. However, hydroxyl radical production is independent of laccase and peroxidase activity. Laccases and peroxidases are involved in other aspects of lichen biology; here we show that peroxidases, but not laccases, can break down lichen substances. Reduction in the amounts of lichen substances will reduce photoprotection, which will increase the photosynthetic capacity of thalli during winter when light intensities are low.

Evaluation of CV2025 ω-transaminase for the bioconversion of lignin breakdown products into value-added chemicals: synthesis of vanillylamine from vanillin

Lignin is an essential component of the cell wall of various plants and represents an abundant and renewable natural resource. Both thermo-chemical and biological pre-treatment can be applied to break down the phenylpropanoid polymer subunits present in lignin. These liberate a range of phenolic compounds which represent potential substrates for bioconversion by ω-transaminases. In this work, the CV2025 ω-transaminase (ω-TAm) from Chromobacterium violaceum DSM30191, heterologously expressed in E. coli, was explored for selective amination of lignin breakdown intermediates into value-added products. Eight potential ω-TAm substrates were initially screened using (S)-α-methylbenzylamine (MBA) as the amino donor. Vanillin was identified as the best potential substrate which is converted into vanillylamine; an intermediate in the preparation of pelargonic acid vanillylamide used as a hyperemia inducing active substance in wound dressings. At low vanillin and MBA concentrations (< 10 mM) and with an excess of the amine donor (1:4 mol/mol) 100% w/w conversion of vanillin into vanillylamine was observed within 25 min. At vanillin concentrations above 10 mM, substrate inhibition was observed decreasing the rate and yield of the bioconversion. High concentrations of the reaction product (vanillylamine) and by-product (acetophenone) also limited the conversion due to increased backward reaction rate and inhibition. Vanillylamine synthesis could be carried out by both whole cell and clarified lysate forms of the CV2025 ω-TAm while fed-batch bioconversions (feeding low concentrations of both vanillin and MBA) could help overcome substrate inhibition and double the final product concentrations obtained. These results demonstrate the potential for bioconversion of lignin breakdown products into value-added chemicals but illustrate the need for enzymes with improved substrate range and implementation of techniques to overcome product inhibition and equilibrium constraints.

Analysis of lignin–carbohydrate and lignin–lignin linkages after hydrolase treatment of xylan–lignin, glucomannan–lignin and glucan–lignin complexes from spruce wood

Xylan-lignin (XL), glucomannan-lignin (GML) and glucan-lignin (GL) complexes were isolated from spruce wood, hydrolyzed with xylanase or endoglucanase/β-glucosidase, and analyzed by analytical pyrolysis and 2D-NMR. The enzymatic hydrolysis removed most of the polysaccharide moieties in the complexes, and the lignin content and relative abundance of lignin-carbohydrate linkages increased. Analytical pyrolysis confirmed the action of the enzymatic hydrolysis, with strong decreases of levoglucosane and other carbohydrate-derived products. Unexpectedly it also revealed that the hydrolase treatment alters the pattern of lignin breakdown products, resulting in higher amounts of coniferyl alcohol. From the anomeric carbohydrate signals in the 2D-NMR spectra, phenyl glycoside linkages (undetectable in the original complexes) could be identified in the hydrolyzed GML complex. Lower amounts of glucuronosyl and benzyl ether linkages were also observed after the hydrolysis. From the 2D-NMR spectra of the hydrolyzed complexes, it was concluded that the lignin in GML is less condensed than in XL due to its higher content in β-O-4' ether substructures (62 % of side chains in GML vs 53 % in XL) accompanied by more coniferyl alcohol end units (16 vs 13 %). In contrast, the XL lignin has more pinoresinols (11 vs 6 %) and dibenzodioxocins (9 vs 2 %) than the GML (and both have ~13 % phenylcoumarans and 1 % spirodienones). Direct 2D-NMR analysis of the hydrolyzed GL complex was not possible due to its low solubility. However, after sample acetylation, an even less condensed lignin than in the GML complex was found (with up to 72 % β-O-4' substructures and only 1 % pinoresinols). The study provides evidence for the existence of structurally different lignins associated to hemicelluloses (xylan and glucomannan) and cellulose in spruce wood and, at the same time, offers information on some of the chemical linkages between the above polymers.

Survey of renewable chemicals produced from lignocellulosic biomass during ionic liquid pretreatment

BackgroundLignin is often overlooked in the valorization of lignocellulosic biomass, but lignin-based materials and chemicals represent potential value-added products for biorefineries that could significantly improve the economics of a biorefinery. Fluctuating crude oil prices and changing fuel specifications are some of the driving factors to develop new technologies that could be used to convert polymeric lignin into low molecular weight lignin and or monomeric aromatic feedstocks to assist in the displacement of the current products associated with the conversion of a whole barrel of oil. We present an approach to produce these chemicals based on the selective breakdown of lignin during ionic liquid pretreatment.ResultsThe lignin breakdown products generated are found to be dependent on the starting biomass, and significant levels were generated on dissolution at 160°C for 6 hrs. Guaiacol was produced on dissolution of biomass and technical lignins. Vanillin was produced on dissolution of kraft lignin and eucalytpus. Syringol and allyl guaiacol were the major products observed on dissolution of switchgrass and pine, respectively, whereas syringol and allyl syringol were obtained by dissolution of eucalyptus. Furthermore, it was observed that different lignin-derived products could be generated by tuning the process conditions.ConclusionsWe have developed an ionic liquid based process that depolymerizes lignin and converts the low molecular weight lignin fractions into a variety of renewable chemicals from biomass. The generated chemicals (phenols, guaiacols, syringols, eugenol, catechols), their oxidized products (vanillin, vanillic acid, syringaldehyde) and their easily derivatized hydrocarbons (benzene, toluene, xylene, styrene, biphenyls and cyclohexane) already have relatively high market value as commodity and specialty chemicals, green building materials, nylons, and resins.

Degradation of aromatic compounds through the β-ketoadipate pathway is required for pathogenicity of the tomato wilt pathogen Fusarium oxysporum f. sp. lycopersici.

Plant roots react to pathogen attack by the activation of general and systemic resistance, including the lignification of cell walls and increased release of phenolic compounds in root exudate. Some fungi have the capacity to degrade lignin using ligninolytic extracellular peroxidases and laccases. Aromatic lignin breakdown products are further catabolized via the β-ketoadipate pathway. In this study, we investigated the role of 3-carboxy-cis,cis-muconate lactonizing enzyme (CMLE), an enzyme of the β-ketoadipate pathway, in the pathogenicity of Fusarium oxysporum f. sp. lycopersici towards its host, tomato. As expected, the cmle deletion mutant cannot catabolize phenolic compounds known to be degraded via the β-ketoadipate pathway. In addition, the mutant is impaired in root invasion and is nonpathogenic, even though it shows normal superficial root colonization. We hypothesize that the β-ketoadipate pathway in plant-pathogenic, soil-borne fungi is necessary to degrade phenolic compounds in root exudate and/or inside roots in order to establish disease.

Decomposition of lignin in wheat straw in a sand-dune grassland

The degradation of organic macromolecules, including lignin, in plant-derived soil organic matter, is important to the global carbon cycle. In grasslands, saprotrophic (decomposer) fungi are major decomposers of such organic material. The aim of this study was to characterise lignin degradation, particularly with respect to lignin oxidation typical of white-rot basidiomycete fungi. Lignin breakdown products, analysed by gas chromatography–mass spectrometry (GC–MS) with TMAH thermochemolysis, in initial wheat (Triticum aestivum var. Swatham) straw samples were compared with those in samples which had been buried as a “model” resource for 46 months in a sand-dune grassland at Ainsdale National Nature Reserve, Lancashire, UK.Our results showed that lignin oxidation occurred in the straw over the 46 month period, as there were general increases in the [Ac/Al]S and [Ac/Al]G ratios and a clear decrease in the [S/G] ratio. These data provide tentative support for the theory that white-rot basidiomycete fungi are involved in the degradation of lignin in grasslands.

Size-exclusion chromatographic study of ECF and TCF softwood kraft pulp bleaching liquors

Currently, elemental chlorine-free (ECF) and totally chlorine-free (TCF) bleaching systems are widely used for pulp production. Low and medium molecular weight lignin break-down products are known to have harmful effects on the environment. According to some recent results, also high molecular weight (HMW) material consisting mainly of lignin and carbohydrates may cause toxic effects to the environment. For these reasons, toxicity and structure studies of HMW materials are of great importance. This investigation is a part of a larger project to obtain more structure information of HMW materials and toxicity of ECF and TCF bleaching effluents. Size-exclusion chromatography (SEC) has been commonly used for the characterization of organic macromolecules such as lignin, but to our knowledge, no reports have appeared dealing with the comparison of SEC of ECF and TCF bleaching liquors. The aim of the present study was to get more information about the molecular weight distribution (MWD) of HMW fractions of waste liquors from ECF and TCF bleaching sequences by SEC. The MWDs of organic materials dissolved during different stages of ECF bleaching (O-D-EOP-D-ED) and TCF bleaching (O-Z-Q-P-Z-Q-P-P) of softwood (Pinus sylvestris) kraft pulp were determined and compared by SEC. All effluent samples from the above bleaching stages were ultrafiltrated using a membrane with a cutoff value of 1,000Da. SEC was performed on high and also low molecular weight fractions and non-fractionated effluents. In the SEC experiments, a Superdex 75 column was used with 0.1M NaOH solution as the eluent. Standards used for calibrating the SEC system were albumin, carboanhydrase, cytochrome C, tannic acid, dehydrodiacetovanillone, and vanillin. The chromatograms of liquors from TCF bleaching stages vary more than those from ECF bleaching. Peroxide and chelating stages contained mostly high molecular weight (HMW) matter whereas chlorine dioxide and ozone stages had more low molecular weight compounds. The lignin content in HMW matter was higher than in stages that consisted of low molecular matter. Bleaching effluents contained the highest amounts of HMW material, mainly lignin, in the beginning of the sequences; the amounts decreased towards the end of the bleaching sequence. Determinations of MWD by the SEC method showed that effluents from the TCF sequence contained more HMW material than those from the ECF stage. This might be due to peroxide stages (P) that dissolve HMW lignin effectively. However, the molecular weights of ozone stages (Z) were very low compared to other stages. Chlorine dioxide stages also dissolved mostly low molecular weight lignin. Ultrafiltration of bleaching liquors showed that high molecular weight fraction also included some low molecular weight compounds and vice versa. High polydispersity and high lignin content correlated with the amount of HMW material in ECF and TCF bleaching stages. Our liquor samples were studied by using a UV detector commonly used for lignin preparations; in upcoming investigations, it will be interesting to determine carbohydrates such as hemicelluloses. The results are applicable in papermaking in order to improve commonly used bleaching procedures, to test new potential bleaching systems, and to study chemical behavior of HMW materials in various bleaching liquors. The present results also form a good basis for toxicity measurements of ECF and TCF bleaching effluents and for more comprehensive spectroscopic and chromatographic experiments with samples taken from various bleaching stages. From the behavior of liquors studied, it appears that our other structure investigations by spectroscopic and chromatographic (NMR, Py-GC/MS, etc.) methods mostly correlate well with the present results.