Potential applications of bio-ligninolytic systems

Members of various fungal taxa and actinomycetes have been shown to degrade lignin at least partially. The white-rot wood-decomposing basidiomycetes completely metabolize the complex polymer, exhibit the highest reported rates, and are the most studied. Evidence indicates that their degradation of lignin involves oxidative, non-specific reactions, but the nature of the catalysts and the reactive species remain undefined; the catalysts have not been separated from living cells. Culture conditions optimal for lignin metabolism by white-rot fungi have been described, and several potential applications of whole ligninolytic cultures have been explored preliminarily: (a) partial delignification for the production of cellulosic products (bio-mechanical pulping, bio-bleaching); (b) conversion of lignocellulosics (improving ruminant digestibility, cultivating edible mushrooms) into feed and food; and (c) treatment of lignin-derived wastes (decolorizing, removing BOD, COD). The possibility to biomodify by-product lignins to yield valuable polymeric or low molecular weight chemicals has not been approached experimentally, but is another area of potential application. Improved waste treatment processes might well be the first intentional application of bioligninolytic systems.     

Chemistry of softwood lignin degradation byStreptomyces viridosporus

Polymeric lignin isolated from ground spruce phloem/bark tissue following decay by the actinomyceteStreptomyces viridosporus (T7A) was characterized chemically and compared to undergraded lignin from the same source. The chemical transformations resulting from degradation were compared to those that result from fungal degradation of softwood lignins by brown- and white-rot fungi. Degradative chemical analyses showed thatS. viridosporus-degraded lignin was significantly altered in structure. Much of the integrity of the basic 4-hydroxy-3-methoxyphenylpropane subunit structure was lost. Actinomycete-decayed lignin was decreased in carbon and enriched in oxygen and hydrogen contents. It also had been extensively demethylated. Chemical analysed indicated that phenylpropanoid side-chains had been oxidized by introduction of -carbonyls and by side-chain shortening reactions. Although the degraded lignin remained polymeric, it was significantly dearomatized. These changes are similar to those previously reported for white-rotted lignins, except for the increased hydrogen content. The evidence indicated that lignin degradation byS. viridosporus is oxidative and involves demethylations, ring cleavage reactions, and oxidative attack on phenylpropanoid side-chains. Also, some reduced structures accumulate in the polymer and some low molecular weight intermediates are released into the growth medium.Abbreviations MWL milled wood lignin - TMS trimethylsyily - PCA protocatechuic acid Paper nunfber 81512 of the Idaho Agricultural Experiment Station     

Lignin degradation by microorganisms: A review

Lignin is an abundant plant-based biopolymer that has found applications in a variety of industries from construction to bioethanol production. This recalcitrant branched polymer is naturally degraded by many different species of microorganisms, including fungi and bacteria. These microbial lignin degradation mechanisms provide a host of possibilities to overcome the challenges of using harmful chemicals to degrade lignin biowaste in many industries. The classes and mechanisms of different microbial lignin degradation options available in nature form the primary focus of the present review. This review first discusses the chemical building blocks of lignin and the industrial sources and applications of this multifaceted polymer. The review further places emphasis on the degradation of lignin by natural means, discussing in detail the lignin degradation activities of various fungal and bacterial species. The lignin-degrading enzymes produced by various microbial species, specifically white-rot fungi, brown-rot fungi, and bacteria, are described. In the end, possible directions for future lignin biodegradation applications and research investigations have been provided.     

微生物法降解木质素的研究进展

生物质是代替石化资源生产能源和化学品的关键资源,木质素作为植物细胞壁的主要成分已经在很多行业中得到了广泛的应用。然而,由于木质素结构复杂且难以降解,成为生物质资源利用的最大障碍,因此,去除或者降解木质素是利用细胞壁中其他成分的关键步骤。许多行业使用有害化学物质降解木质素,严重危害了生态环境,自然界中木质素经常被包括真菌和细菌在内的微生物降解,因此,研究微生物降解木质素的机制为解决这一问题提供了可能性。本文讨论了木质素的化学组成成分,重点讨论了自然界降解木质素的微生物种类及其降解机制,包括各种真菌和细菌的木质素降解活性,描述了由各种微生物特别是白腐真菌、褐腐真菌和细菌产生的木质素降解酶,并展望了今后木质素生物降解的研究和应用的可能方向。     

Enzymatic demethylation of lignin for potential biobased polymer applications

Lignin is a highly methylated, recalcitrant biopolymer available aplenty in nature, and is highly heteropolymer in nature, but yet it has been an under-utilized biopolymer. Modifying it chemically, biologically or enzymatically could render it a good candidate for phenol formaldehyde resin or into fine chemicals, fuels, and plastics applications. Lignin demethylation is facilitated by the enzymes called the O-demethylases, which are able to strip-off of the –OCH₃ group in lignin, that give rise to the more widely accessible phenolic hydroxyls groups. Biological demethylation of lignins can be accomplished by means of the microorganisms, such as the white-rot, soft-rot and brown-rot fungi, besides some species of bacteria. Although the enzymes responsible for the lignin demethylation process have not been identified and purified adequately, it is perhaps possible that the O-demethylases, which have the ability to remove the O-methyl groups at the C-3 and (or) C-4 positions of the benzyl ring of low molecular weight lignin-like model compounds (LMCs) and lignin makes them the suitable candidate. These LMCs resemble the aromatic moieties inherent in the molecular structure of lignins, such as the vanillate, syringate, and veratrate. Thus, these enzymes are known as vanillate-O-demethylases, syringate O-demethylases, veratrate O-demethylases and Tetrahydrofolate (THF)-dependent O-demethylase (LigM), respectively. Whereas, some ligninolytic enzymes are known to cause damage to the structure of lignins (e.g., laccases, manganese-dependent peroxidase and lignin peroxidases). The O-demethylase enzymes are believed to be capable of removing the O-methyl groups from the lignins without affecting the complex backbone structure of the lignins. The mechanism of action of O-demethylases on lignin degradation is still largely unexplored, and their ability to remove the O-methyl groups from lignins has not been elucidated sufficiently. In this review, the recent advances made on the molecular approaches in the lignin demethylation (O-demethylases and ligninolytic enzymes), degradation and the probable strategies to tone up the lignin quality have been discussed in detail. The demethylation process of lignins by means of enzymes is envisaged to open up new vistas for its application as a biopolymer in various bioprocess and biorefinery process.     

The emerging role for bacteria in lignin degradation and bio-product formation

The microbial degradation of lignin has been well studied in white-rot and brown-rot fungi, but is much less well studied in bacteria. Recent published work suggests that a range of soil bacteria, often aromatic-degrading bacteria, are able to break down lignin. The enzymology of bacterial lignin breakdown is currently not well understood, but extracellular peroxidase and laccase enzymes appear to be involved. There are also reports of aromatic-degrading bacteria isolated from termite guts, though there are conflicting reports on the ability of termite gut micro-organisms to break down lignin. If biocatalytic routes for lignin breakdown could be developed, then lignin represents a potentially rich source of renewable aromatic chemicals.     

Lignin degrading system of white-rot fungi and its exploitation for dye decolorization

With global attention and research now focused on looking for the abatement of pollution, white-rot fungi is one of the hopes of the future. The lignin-degrading ability of these fungi have been the focus of attention for many years and have been exploited for a wide array of human benefits. This review highlights the various enzymes produced by white-rot fungi for lignin degradation, namely laccases, peroxidases, aryl alcohol oxidase, glyoxal oxidase, and pyranose oxidase. Also discussed are the various radicals and low molecular weight compounds that are being produced by white-rot fungi and its role in lignin degradation. A brief summary on the developments in research of decolorization of dyes using white-rot fungi has been made.     

Phenomenological principles of microbial lignin degradation

This paper intends to focus the attention to characteristic features of microbial lignin degradation from the phenomenological point of view. Six fundamental principles are discussed under special consideration of white-rot fungi. The necessity of mycelial growth and the formation, secretion, and extracellular action of peroxidases are main requirements for a successful microbial attack on polymeric lignin.     

血红密孔菌高产漆酶菌株的筛选及其对烟梗的生物降解

白腐菌是目前已知的唯一能将木质素彻底降解的微生物,而漆酶在木质素分解过程中起着重要的作用,被广泛应用于农作物秸秆或甘蔗渣等多种类型生物质的生物预处理和生物降解。本研究利用白腐菌产漆酶发酵培养基对30株血红密孔菌Pycnoporus sanguineus菌株进行筛选,得到了多株漆酶高产菌株,并研究了血红密孔菌发酵粗酶液和菌丝对烟梗的生物降解条件。研究结果表明:血红密孔菌及其产生的漆酶表现出了对烟梗木质素较强的生物降解能力。在漆酶浓度为40U/mL、温度30℃、pH4.5的条件下处理24h,烟梗中木质素的降解率可达到50.4%,纤维素和半纤维素的降解率分别为17.5%和17.3%;漆酶浓度为5U/mL、温度30℃、pH4.5的条件下处理48h,木质素降解率可达到65.1%。血红密孔菌菌丝也表现出对烟梗较好的生物降解效果,接种培养7d后烟梗中木质素降解率可达30%以上,21d后木质素的降解率可达79.1%,而纤维素和半纤维素的降解率仅为20%和12%左右。本研究不但为生物质材料的生物预处理和生物降解提供了优质的白腐菌及漆酶资源,还为通过烟梗的生物预处理提高烟草梗丝和卷烟品质提供了重要参数,具有一定的应用前景。     

Advances in microbial delignification

Microbial delignification is a new field of applied research. The progress will therefore run parallel to the development of new basic knowledge on the physiological demands of white-rot fungi to degrade lignin and on new knowledge on enzyme mechanisms involved in lignin degradation.In the last few years both basic and applied research on microbial conversion of lignocellulosic materials have vastly expanded. In certain areas, such as microbial delignification, considerable progress has recently been made. Basidiospores from Sporotrichum pulverulentum and some CEL(-) mutants have been obtained. Crossing of mycelium from single basidiospore cultures of wild-type and CEL(-) mutants will eventually give rise to much better CEL(-) mutants than those which have been used in the past. An understanding of which enzymes are the most important for lignin degradation to take place is also beginning to develop. This review discusses present knowledge and future possibilities in this field.     

Influence of Molecular Size and Ligninase Pretreatment on Degradation of Lignins by Xanthomonas sp. Strain 99

The purpose of this study was to examine the relationship between the molecular size of lignin in several preparations and extent of degradation (mineralization) by Xanthomonas sp. strain 99. The influence of ligninase pretreatment was also examined. Five synthetic lignins and one ¹⁴C-methylated spruce lignin were used. The extent of mineralization to ¹⁴CO₂ was greatest for the samples containing the most low-molecular-weight material, and the low-molecular-weight portions were preferentially (or perhaps solely) degraded. Pretreatment of the five synthetic lignins with crude ligninase increased their molecular size and decreased their degradability by the xanthomonad. Pretreatment of the methylated spruce lignin with crude ligninase caused both polymerization and depolymerization but resulted in a net decrease in bacterial degradability. Our results suggest that the xanthomonad can degrade lignins only up to a molecular weight of 600 to 1,000.     

Degradation of lignin in pulp mill wastewaters by white-rot fungi on biofilm

An investigation was conducted to explore the lignin-degrading capacity of attached-growth white-rot fungi. Five white-rot fungi, Phanerochaete chrysosporium, Pleurotus ostreatus, Lentinus edodes, Trametes versicolor and S22, grown on a porous plastic media, were individually used to treat black liquor from a pulp and paper mill. Over 71% of lignin and 48% of chemical oxygen demand (COD) were removed from the wastewater. Several factors, including pH, concentrations of carbon, nitrogen and trace elements in wastewater, all had significant effects on the degradation of lignin and the removal of COD. Three white-rot fungi, P. chrysosporium, P. ostreatus and S22, showed high capacity for lignin degradation at pH 9.0-11.0. The addition of 1 g l-1 glucose and 0.2 g l-1 ammonium tartrate was beneficial for the degradation of lignin by the white-rot fungi studied.     

Selective Degradation of Wood Components by White-Rot Fungi

In order to find naturally occurring white-rot fungi which preferentially degrade lignin. 25 different species of such fungi were cultivated on pine wood blocks and on kraft lignin agar plates with and without cellulose. Due to differences in phenol oxidase reactions on the kraft lignin agar plates, the 25 fungi could be divided into two groups, 1 and 2, which also differed in other properties. The three Group I fungi Sporotrichum pulverulentum, Phanerochaete sp. L1 and Polyporus dichrous produced high levels of endo-l,4-β-glucanase and cellobiose:quinone oxidoreductase in shaking cellulose flasks and a low level of phenol oxidase in standing wood meal flasks, The four fungi Merulius tremellosus, Phlebia radiata, Pycuoporus cinnabarinus and Pleurotus ostreatus from Group 2, on the other hand, produced low levels of endo-1,4-β-glucanase and cellobiose:.quinone oxidoreductase in the cellulose. flasks and a high level of phenol oxidase in the wood meal flasks. Analyses of pine wood blocks degraded by the above-mentioned fungi in the presence of either malt extract, asparagine or NH₄H₂PO₄ revealed that malt extract gave good lignin degradation. In the presence of this nutrient source. P. cinnabarinus, at 3.4% weight loss, even degraded 12.5% lignin without loss of cellulose or mannan. No common degradation pattern was, however, obtained using mall extract, asparagine or NH₄H₂PO₄, It is suggested that while-rot fungi, which preferentially degrade lignin, may be found among Group 2 fungi producing large amounts of phenol oxidases.     

Antioxidant activity in fungi degrading lignocellulose substrates

Considerable differences in lignin degradation by fungi of two ecological groups have been revealed. Xylotrophs cause a twofold decrease in the molecular weight of lignin. The degrading activity of saprotrophs is insignificant. Xylotrophs demethoxylate and oxidize lignin more rapidly than saprotrophs, showing a higher level of antioxidant activity. As follows from the comparison of the degrading and antioxidative effects, measurement of the antioxidant activity can be used in screening of fungi for the ability to degrade lignocellulose substrates.     

Biological Pretreatment of Lignocellulosic Substrates for Enhanced Delignification and Enzymatic Digestibility

Sheer enormity of lignocellulosics makes them potential feedstock for biofuel production but, their conversion into fermentable sugars is a major hurdle. They have to be pretreated physically, chemically, or biologically to be used by fermenting organisms for production of ethanol. Each lignocellulosic substrate is a complex mix of cellulose, hemicellulose and lignin, bound in a matrix. While cellulose and hemicellulose yield fermentable sugars, lignin is the most recalcitrant polymer, consisting of phenyl-propanoid units. Many microorganisms in nature are able to attack and degrade lignin, thus making access to cellulose easy. Such organisms are abundantly found in forest leaf litter/composts and especially include the wood rotting fungi, actinomycetes and bacteria. These microorganisms possess enzyme systems to attack, depolymerize and degrade the polymers in lignocellulosic substrates. Current pretreatment research is targeted towards developing processes which are mild, economical and environment friendly facilitating subsequent saccharification of cellulose and its fermentation to ethanol. Besides being the critical step, pretreatment is also cost intensive. Biological treatments with white rot fungi and Streptomyces have been studied for delignification of pulp, increasing digestibility of lignocellulosics for animal feed and for bioremediation of paper mill effluents. Such lignocellulolytic organisms can prove extremely useful in production of bioethanol when used for removal of lignin from lignocellulosic substrate and also for cellulase production. Our studies on treatment of hardwood and softwood residues with Streptomyces griseus isolated from leaf litter showed that it enhanced the mild alkaline solubilisation of lignins and also produced high levels of the cellulase complex when growing on wood substrates. Lignin loss (Klason lignin) observed was 10.5 and 23.5% in case of soft wood and hard wood, respectively. Thus, biological pretreatment process for lignocellulosic substrate using lignolytic organisms such as actinomycetes and white rot fungi can be developed for facilitating efficient enzymatic digestibility of cellulose.     

Lignin peroxidase: toward a clarification of its role in vivo

The extracellular lignin peroxidase from the white-rot basidiomycete Phanerochaete chrysosporium is thought to play an important role in lignin biodegradation. However, the majority of lignin-derived preparations actually experience overall polymerization at the hands of the enzyme in vitro. It has now been found that, in the presence of H2O2 at pH 4.0, the monomeric lignin precursor coniferyl alcohol is polymerized quantitatively by a lignin peroxidase preparation which is uncontaminated with MnII-dependent peroxidases. 13C NMR spectrometry of the resulting dehydropolymerisates from 13C-labeled monolignols confirms that the frequencies of different interunit linkages are very similar to those engendered through the action of horseradish peroxidase with H2O2. Indeed, lignin peroxidase does not ultimately seem to be a prerequisite for lignin degradation in vivo, yet its activity can still accelerate the conversion of lignin-derived preparations by P. chrysosporium to CO2. Consequently, lignin peroxidase can provisionally be expected to fulfill two important functions. On the one hand, the enzyme may detoxify lower molecular weight phenolic compounds released from lignins during their fungal decomposition. On the other hand, through the introduction of suitable functional groups, lignin peroxidase could indirectly enhance the susceptibility of macromolecular lignin structures toward depolymerization by another enzyme.     

Temporal Alterations in the Secretome of the Selective Ligninolytic Fungus Ceriporiopsis subvermispora during Growth on Aspen Wood Reveal This Organism's Strategy for Degrading Lignocellulose

The white-rot basidiomycetes efficiently degrade all wood cell wall polymers. Generally, these fungi simultaneously degrade cellulose and lignin, but certain organisms, such as Ceriporiopsis subvermispora, selectively remove lignin in advance of cellulose degradation. However, relatively little is known about the mechanism of selective ligninolysis. To address this issue, C. subvermispora was grown in liquid medium containing ball-milled aspen, and nano-liquid chromatography-tandem mass spectrometry was used to identify and estimate extracellular protein abundance over time. Several manganese peroxidases and an aryl alcohol oxidase, both associated with lignin degradation, were identified after 3 days of incubation. A glycoside hydrolase (GH) family 51 arabinofuranosidase was also identified after 3 days but then successively decreased in later samples. Several enzymes related to cellulose and xylan degradation, such as GH10 endoxylanase, GH5_5 endoglucanase, and GH7 cellobiohydrolase, were detected after 5 days. Peptides corresponding to potential cellulose-degrading enzymes GH12, GH45, lytic polysaccharide monooxygenase, and cellobiose dehydrogenase were most abundant after 7 days. This sequential production of enzymes provides a mechanism consistent with selective ligninolysis by C. subvermispora.     

Antioxidant Activity in Fungi Degrading Lignocellulose Substrates

Considerable differences in lignin degradation by fungi of two ecological groups have been revealed. Xylotrophs cause a twofold decrease in the molecular weight of lignin. The degrading activity of saprotrophs is insignificant. Xylotrophs demethoxylate and oxidize lignin more rapidly than saprotrophs, showing a higher level of antioxidant activity. As follows from the comparison of the degrading and antioxidative effects, measurement of the antioxidant activity can be used in screening of fungi for the ability to degrade lignocellulose substrates.     

Laccase production and wood degradation by a white-rot fungus Daedalea flavida

A comparative study has been conducted on seven white rot fungi to investigate their abilities to produce laccase and selectively degrade lignin. Laccase was produced constitutively on the different media tested. Of the different lignins, phenolic compounds and sugars involved, the highest laccase yield was obtained on indulin AT. Salicylic acid inhibited enzyme activity. A temperature of 20°C and 0.2% of indulin AT were found to be optimum for enzyme activity. No correlation was found between the amount of enzyme and fungal mass produced. During semisolid degradation of angiospermic wood sawdust, Daedalea flavida caused a total weight loss of 11%, with a lignin loss of 15.77% during two months of decay. Lignin removal was comparatively selective during the first month, during which time laccase production was also higher, indicating its probable role in lignin degradation.