Annele Hatakka

Biodegradation of lignin in a compost environment: a review

Abstract Composting is nowadays a general treatment method for municipal solid waste. Compostable household waste contains, together with vegetable material, varying amounts of papers and boards. In the European Union composting is regarded as one recycling method for packages and this will probably favour compostable packages, like papers and boards, in the future. Paper is made up of lignocellulose and it may contain up to 20% of lignin. Efficient degradation of papers in composting plants means that biodegradation of lignin is also needed. However, very little is known about lignin degradation by mixed microbial compost populations, although lignin degradation by white-rot fungi has been extensively studied in recent years. Organic material is converted to carbon dioxide, humus, and heat by compost microorganisms. It is assumed that humus is formed mainly from lignin. Thus, lignin is not totally mineralized during composting. The elevated temperatures found during the thermophilic phase are essential for rapid degradation of lignocellulose. Complex organic compounds like lignin are mainly degraded by thermophilic microfungi and actinomycetes. The optimum temperature for thermophilic fungi is 40–50°C which is also the optimum temperature for lignin degradation in compost.

Pretreatment of wheat straw by white-rot fungi for enzymic saccharification of cellulose

SummaryOf 19 white-rot fungi tested, Pleurotus ostreatus, Pleurotus sp. 535, Pycnoporus cinnabarinus 115 and Ischnoderma benzoinum 108 increased the susceptibility of straw to enzymic saccharification, thus indicating that these organisms degraded or modified the lignin component. After pretreatment cultivation with Pycnoporus cinnabarinus 115, as much as 54.6% of the residue was converted to reducing sugars in the enzymic saccharification process. Phanerochaete sordida 37, Phlebia radiata 79 and two unidentified fungi also gave better results than Polyporus versicolor, a non-selective reference fungus. After 5 weeks pretreatment with Pleurotus ostreatus, 35% of the original straw was convertable to reducing sugars, 74% of which was glucose; compared with this, only 12% of the untreated control straw was convertable to reducing sugars, 42% of which was glucose. After an alkali pretreatment (2% NaOH, 0.4 g NaOH/g straw, 10 min at 115°C) enzymic saccharification converted 41% of the straw to reducing sugars, of which only 50% was glucose. In the best cases the efficiency of biological pretreatment was comparable with that of alkali treatment, but resulted in a higher proportion of glucose in the hydrolysates. Pretreatment by the fungi Phanerochaete sordida 37 and Pycnoporus cinnabarinus 115 in an oxygen atmosphere reduced the treatment time by approximately 1 week. However, the economic feasibility of a non-optimized biological pretreatment process is still poor due to the long cultivation times required.

Mineralisation of 14C-labelled synthetic lignin and ligninolytic enzyme activities of litter-decomposing basidiomycetous fungi.

Abstract Within a screening program, 27 soil litter-decomposing basidiomycetes were tested for ligninolytic enzyme activities using agar-media containing 2,2′-azinobis(3-ethylbenzthiazoline-6-sulphonate), a humic acid or Mn2+ ions as indicator substrates. Most active species were found within the family Strophariaceae (Agrocybe praecox, Stropharia coronilla, S. rugosoannulata) and used for mineralisation experiments with a 14C-ring-labelled synthetic lignin (14C-DHP). The fungi mineralised around 25% of the lignin to 14CO2 within 12 weeks of incubation in a straw environment; about 20% of the lignin was converted to water-soluble fragments. Mn-peroxidase was found to be the predominant ligninolytic enzyme of all three fungi in liquid culture and its production was strongly enhanced in the presence of Mn2+ ions. The results of this study demonstrate that certain ubiquitous litter-decomposing basidiomycetes possess ligninolytic activities similar to the wood-decaying white-rot fungi, the most efficient lignin degraders in nature.

Production of organic acids and oxalate decarboxylase in lignin-degrading white rot fungi

Intracellular oxalate decarboxylase (ODC, EC 4.1.1.2) activity was screened in the mycelium of 12 white rot fungi. ODC activity was detected in the mycelial extracts of Dichomitus squalens, Phanerochaete sanguinea, Trametes ochracea, and Trametes versicolor (strain R/7) after addition of 5-mM oxalic acid to the liquid culture medium. In D. squalens, intracellular ODC activity increased six-fold with addition of oxalic acid. Production of extracellular organic acids by the four ODC-positive fungi was followed in liquid cultures and in solid state cultures of spruce wood chips by using HPLC and capillary zone electrophoresis (CZE). The four ODC-positive fungi secreted oxalic acid both in liquid and solid state cultures showing different production patterns until the end of growth (31 days). Upon cultivation on solid spruce wood chips, manganese peroxidase (MnP) activity peaked simultaneously in these fungi with the accumulation of extracellular oxalic acid. In addition to oxalic acid, glyoxylic and formic acids were detected in the cultures of D. squalens.

Degradation of 14C-labelled poplar wood lignin by selected white-rot fungi

SummaryOf eight white-rot fungi examined, seven fungi grew on nitrogen-limited poplar wood meal medium and degraded 14C-lignin in wood meal to 14CO2. Increased oxygen enhanced both the rate and extent of degradation. However, whereas Pleurotus ostreatus, Pycnoporus cinnabarinus 115 and Pycnoporus cinnabarinus A-360 degraded 12–17% of 14C-(U)-lignin of poplar wood to 14CO2 also in an air atmosphere, Sporotrichum pulverulentum, Phlebia radiata 79 and Phanerochaete sordida 37 degraded only 1–5% under these conditions. Addition of cellulose and glucose to the poplar wood medium stimulated degradation of 14C-(RING)-lignin of poplar wood by Phlebia radiata 79 but repressed degradation by Polyporus versicolor and Pleurotus ostreatus. Cellulose added to the wood meal medium had no effect on the degradation of lignin by Phanerochaete sordida 37 and Sporotrichum pulverulentum but glucose slightly repressed lignin degradation by these fungi. Those white-rot fungi which were considered as preferentially lignin attacking fungi could degrade 14C-(RING)-lignin of poplar wood efficiently under 100% oxygen. They did not require an extra energy source in addition to wood meal polysaccharides for rapid ring cleavage and they degraded up to 50–60% of the 14C-lignin to 14CO2 in 6–7 weeks at a maximum rate of 3–4% per day.

Degradation of Humic Acids by the Litter-Decomposing Basidiomycete Collybia dryophila

ABSTRACT The basidiomycete Collybia dryophila K209, which colonizes forest soil, was found to decompose a natural humic acid isolated from pine-forest litter (LHA) and a synthetic 14C-labeled humic acid (14C-HA) prepared from [U-14C]catechol in liquid culture. Degradation resulted in the formation of polar, lower-molecular-mass fulvic acid (FA) and carbon dioxide. HA decomposition was considerably enhanced in the presence of Mn2+ (200 μM), leading to 75% conversion of LHA and 50% mineralization of 14C-HA (compared to 60% and 20%, respectively, in the absence of Mn2+). There was a strong indication that manganese peroxidase (MnP), the production of which was noticeably increased in Mn2+-supplemented cultures, was responsible for this effect. The enzyme was produced as a single protein with a pI of 4.7 and a molecular mass of 44 kDa. During solid-state cultivation, C. dryophila released substantial amounts of water-soluble FA (predominantly of 0.9 kDa molecular mass) from insoluble litter material. The results indicate that basidiomycetes such as C. dryophila which colonize forest litter and soil are involved in humus turnover by their recycling of high-molecular-mass humic substances. Extracellular MnP seems to be a key enzyme in the conversion process.

Fungal Biodegradation of Lignocelluloses

Many fungi degrade cellulose and hemicelluloses using extracellular hydrolytic enzymes, but fungi that degrade woody biomass are the only ones to efficiently degrade polysaccharides encased in lignin. White-rot basidiomycetes begin by mineralizing the lignin, using extracellular oxidative enzymes to cleave this recalcitrant biopolymer. Enzymes with likely roles include lignin peroxidases, manganese peroxidases, versatile peroxidases and laccases. In some cases the enzyme may attack the lignin polymer directly; in others the ligninolytic agent is likely a small molecule that one of the enzymes has oxidized to a reactive form. So far, all white rot fungi appear to secrete manganese peroxidases, and most produce laccases, whereas the other two enzymes are less common. After ligninolysis, white-rot fungi assimilate the remaining polysaccharides using conventional glycosylhydrolase systems that contain both endo- and exo-acting enzymes. Brown rot basidiomycetes also degrade lignocellulose efficiently, but their biodegradative systems are less comprehensive. These fungi generally lack ligninolytic enzymes, initiating decay instead with reactive oxygen species generated from the reaction between Fe2+ and H2O2. The limited disruption caused by these oxidants apparently allows a limited set of endo-acting glycosylhydrolases to depolymerize the remaining polysaccharides. Biological wood pulping is one promising application of ligninolytic fungi.

The effect of culture conditions on the production of lignin modifying enzymes by the white-rot fungus Phlebia radiata

The extracellular enzymes synthesized by Phlebia radiata Fr. 79 (ATCC 64658) under various cultivation conditions were studied in order to find out suitable enzyme combinations for the modification of lignin. The fungus produced lignin peroxidase (LiP, ligninase), manganese-dependent peroxidase (MnP) and laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) in various proportions depending on the cultivation conditions. Addition of veratryl alcohol increased lignin peroxidase and manganese-dependent peroxidase activities both in agitated and non-agitated flask cultures. Laccase production was more enhanced by the addition of benzyl alcohol and veratric acid. However, the highest lignin peroxidase activities were obtained using a non-phenolic dimeric β-O-4 model compound. Pressure ground wood (PGW), chemithermomechanical pulp (CTMP) or spruce shavings in the bioreactors decreased the amount of lignin peroxidase, whereas laccase and manganese-dependent peroxidase activities increased. Lignin peroxidase and partly manganese-dependent peroxidase was adsorbed on the lignocellulose substrate, but laccase remained in the culture liquid. Here the lignin peroxidase activity was due to isozyme LiP3. In flask cultures more lignin peroxidase isozymes were secreted, the major component of which was the isozyme LiP2.

Vanillic acid metabolism by the white-rot fungus Sporotrichum pulverulentum

Vanillic acid metabolism was studied in wild-type Sporotrichum pulverulentum and three different mutants. Vanillic acid was found to be oxidatively decarboxylated to methoxyhydroquinone (MHQ) and simultaneously reduced to vanillin and vanillyl alcohol to different degrees depending upon the cultivation conditions. The reducing pathway cannot be utilized unless the fungus has access to an easily metabolized carbon source such as glucose or cellobiose, while decarboxylation takes place in cultures with only vanillic acid present. Polymerization reactions also occurred in the culture solutions. Some evidence for reoxidation of vanillin and vanillyl alcohol was obtained in vivo, and in vitro experiments using horseradish peroxidase.Using vanillic acids labelled in the carboxyl, methoxyl and the aromatic ring it was shown that decarboxylation occures before ring-cleavage, which in turn takes place earlier than the release of 14CO2 from O14CH3-vanillate. The 14CO2 evolution from the methoxyl group is repressed by 1% cellobiose as compared to 0.25% cellobiose, but is stimulated by 26 mM nitrogen (as asparagine plus NH4NO3) compared to 2.6 mM nitrogen. Since S. pulverulentum appears to require three hydroxyl groups attached to the benzene ring before ring-cleavage can occur, preparation for ring-cleavage is apparently achieved by hydroxylation rather than by demethylation.A scheme for metabolism of vanillic acid by S. pulverulentum based upon these results is proposed.

Plant-Polysaccharide-Degrading Enzymes from Basidiomycetes

SUMMARY Basidiomycete fungi subsist on various types of plant material in diverse environments, from living and dead trees and forest litter to crops and grasses and to decaying plant matter in soils. Due to the variation in their natural carbon sources, basidiomycetes have highly varied plant-polysaccharide-degrading capabilities. This topic is not as well studied for basidiomycetes as for ascomycete fungi, which are the main sources of knowledge on fungal plant polysaccharide degradation. Research on plant-biomass-decaying fungi has focused on isolating enzymes for current and future applications, such as for the production of fuels, the food industry, and waste treatment. More recently, genomic studies of basidiomycete fungi have provided a profound view of the plant-biomass-degrading potential of wood-rotting, litter-decomposing, plant-pathogenic, and ectomycorrhizal (ECM) basidiomycetes. This review summarizes the current knowledge on plant polysaccharide depolymerization by basidiomycete species from diverse habitats. In addition, these data are compared to those for the most broadly studied ascomycete genus, Aspergillus, to provide insight into specific features of basidiomycetes with respect to plant polysaccharide degradation.