Progress in biopulping of non-woody materials: Chemical, enzymatic and ultrastructural aspects of wheat straw delignification with ligninolytic fungi from the genus Pleurotus

Abstract: During screening of basidiomycetes for wheat straw delignification, considerable lignin degradation with a limited attack to cellulose was attained with Pleurotus eryngii . Straw solid-state fermentation (SSF) was optimized, and the enzymatic mechanisms for lignin degradation were investigated. No lignin peroxidase was detected under liquid or SSF conditions, but high laccase and aryl-alcohol oxidase levels were found. The latter enzyme has been fully characterized in PI. eryngii and it seems to be involved in a cyclic redox system for H₂0₂ generation from aromatic compounds. Results obtained using homoveratric acid suggest that Pleurotus laccase could be involved in degradation of phenolic and non-phenolic lignin moieties. Histological and ultrastructural studies provided some general morphological characteristics of the fungal attack on wheat straw. Whereas a simultaneous degradation pattern was observed in straw treated with Phanerochaete chrysosporium , PI. eryngii caused partial degradation of middle lamella and separation of individual sclerenchymatic fibers. When these straw samples were subjected to refining tests, energy saving after biological treatment was the highest in the case of straw treated with PI. eryngii , which also produced the lowest substrate loss. From these results, a correlation between preferential removal of lignin, separation of sclerenchymatic fibers and pulping properties was provided during fungal treatment of wheat straw.     

Effect of culture conditions on manganese peroxidase production and activity by some white rot fungi

The ligninolytic system of white rot fungi is primarily composed of lignin peroxidase, manganese peroxidase (MnP) and laccase. The present work was carried out to determine the best culture conditions for production of MnP and its activity in the relatively little-explored cultures of Dichomitus squalens, Irpex flavus and Polyporus sanguineus, as compared with conditions for Phanerochaete chrysosporium and Coriolus versicolor. Studies on enzyme production under different nutritional conditions revealed veratryl alcohol, guaiacol, Reax 80 and Polyfon H to be excellent MnP inducers. Electronic Publication     

Relationship of nitrogen to the onset and suppression of ligninolytic activity and secondary metabolism in Phanerochaete chrysosporium

Ligninolytic activity in the white-rot fungus Phanerochaete chrysosporium was previously found not to be induced by lignin, but to develop in cultures in response to nitrogen starvation. Added NH ₄ ⁺ suppressed existing activity. The present study examined amino acid profiles and protein concentrations during onset of ligninolytic activity (synthetic ¹⁴C-lignin¹⁴CO₂) in nitrogen-limited cultures, and defined some characteristics of subsequent suppression by added nutrient nitrogen. During the transition between depletion of medium nitrogen and the onset of ligninolytic activity, total free intracellular amino acids increased, then rapidly decreased; changes in glutamate concentration played a major role. Intracellular protein concentration fluctuated in a manner roughly converse to that of the concentration of free amino acids. Protein turnover was rapid (5–7%/h) during the transition period. Glutamate, glutamine, and histidine were the most effective of 14 nitrogenous compounds in suppressing ligninolytic activity after its onset. The suppressive effect was not mediated through carbon (glucose)-catabolite repression or by alterations in culture pH. Activities responsible for oxidation of lignin and the ligninrelated phenol, 4-hydroxy-3-methoxyacetophenone, responded similarly to added nitrogen. Synthesis of a secondary metabolite, veratryl alcohol, like lignin oxidation, was suppressed quite sharply by glutamate and significantly by NH ₄ ⁺ . Results indicate that nitrogen metabolism affects ligninolytic activity as a part of secondary metabolism, and suggest a role for glutamate metabolism in regulating this phase of culture development.Non-Standard Abbreviations DMS 2,2-dimethylsuccinate - GLC gas-liquid chromatography - TCA trichloroacetic acid     

Polymerisation of lignins by ligninases from Phanerochaete chrysosporium

Abstract Four major hemoproteins were purified by isoelectric focusing from an extracellular crude enzyme preparation, produced by the white rot fungus Phanerochaete chrysosporium under carbon-limited conditions. Both the crude enzyme and the purified proteins oxidised milled wood lignin, HCl-dioxane-extracted straw lignin and alkali straw lignin in the presence of hydrogen peroxide. The oxidation resulted mainly in further polymerisation of the lignins and was enhanced by addition of veratryl alcohol to the reaction mixture. Alkali straw lignin was also polymerised by horseradish peroxidase, although veratryl alcohol had no influence on this reaction.     

The mediation of veratryl alcohol in oxidations promoted by lignin peroxidase: the lifetime of veratryl alcohol radical cation

The kinetics of decay of veratryl alcohol radical cation, generated by cerium(IV) ammonium nitrate induced oxidation of veratryl alcohol, have been followed spectrophotometrically in a stopped-flow apparatus. In acidic aqueous acetonitrile the radical cation was found to decay by a first-order process, due to deprotonation from the alpha-carbon leading to an alpha-hydroxybenzyl radical with the rate constant of 17.1+/-0.5 s(-1). This value is in full agreement with those obtained by pulse radiolysis studies but much lower than the value (1.2x10(3) s(-1)) indirectly determined by EPR experiments. The implications of these results with respect to the possible role of veratryl alcohol as a mediator in the oxidative biodegradation of lignin catalysed by lignin peroxidase are discussed.     

A Phanerochaete chrysosporium mutant defective in lignin degradation as well as several other secondary metabolic functions

A pleiotropic mutant of Phanerochaete chrysosporium 104-2 lacking phenol oxidase and unable to form fruit bodies and a revertant strain 424-2 were isolated after UV mutagenesis. Strains 104-2 and 424-2 had no apparent dysfunction in primary metabolism with glucose as a carbon source. Unlike the wild type strain and strain 424-2, strain 104-2 was unable to evolve ¹⁴CO₂ from ¹⁴C ring, side chain and 3-O-¹⁴C-methoxy labeled lignin. In addition, strain 104-2 was unable to evolve ¹⁴CO₂ from a variety of lignin model compounds including ¹⁴C-4-methoxy labeled veratrylglycerol--guaiacyl (V) ether, -¹⁴C-guaiacylglycerol--guaiacyl ether (VI), as well as 1-(¹⁴C-4-methoxy, 3-methoxyphenyl)1,2 propene (III) and 1-(¹⁴C-4-methoxy-3-methoxyphenyl) 1,2 dihydroxypropane (IV). The addition of peroxidase/H₂O₂ to cultures of strain 104-2 did not alter its capacity to degrade the labeled lignins. A variety of unlabeled lignin model compounds previously shown to be degraded by the wild type organism including -aryl ether dimers and diaryl propane dimers were also not degraded by the mutant 104-2. The revertant strain 424-2 regained the capacity to degrade these compounds. The substrates described are degraded by oxygen requiring system(s) expressed during the secondary phase of growth, suggesting this pleiotropic mutant is possibly defective in the onset of postprimary metabolism. The inability of the mutant to produce the secondary metabolite veratryl alcohol and to elaborate enzymes in the veratryl alcohol biosynthetic pathway supports this hypothesis.Abbreviations GLC gas liquid chromatography - TMSi trimethylsilyl - MS mass spectrometry - LDS lignin degrading system     

Synthesis of veratraldehyde from veratryl alcohol by lignin peroxidase with in situ electrogeneration of hydrogen peroxide in an electrochemical reactor

We report the synthesis of veratraldehyde from veratryl alcohol by Phanerochaete chrysosporium lignin peroxidase with in situ electrogeneration of hydrogen peroxide in an electroenzymatic reactor. The effects of operating parameters such as enzyme level, pH, and electrical potential on the efficiency of veratryl alcohol oxidation were investigated. Furthermore, we compared direct addition of hydrogen peroxide with electrogeneration of the material during enzymatic oxidation of veratryl alcohol. The electroenzymatic method using in situ-generated hydrogen peroxide was found to be effective for oxidation of veratryl alcohol by lignin peroxidase. The new method may be easily applied to biodegradation systems.     

Effect of veratryl alcohol and manganese (IV) oxide on ligninolytic activity in semi solid cultures of Phanerochaete chrysosporium

An inert carrier (nylon sponge), a non-inert carrier (barley straw) and the addition of veratryl alcohol or manganese (IV) oxide to the cultures were used to study the production of ligninolytic enzymes by Phanerochaete chrysosporium BKM-F-1767 (ATCC 24725) during semi solid state fermentation conditions. By supplementing the medium with these compounds we could stimulate the ligninolytic system of this fungus. The different carriers employed and the effect of adding veratryl alcohol or manganese (IV) oxide to the cultures were compared in order to determine the best system to produce high activities of ligninolytic enzymes. Lignin peroxidase (LiP) activities higher than 500 U/L and manganese-dependent peroxidase (MnP) activities about 1100 U/L were achieved.     

黄孢原毛平革菌木素降解酶系的研究进展

黄孢原毛平革菌木素降解酶系主要由木素过氧化物酶、锰过氧化物酶和乙二醛氧化酶组成。由于该酶系特殊的降解机制,除了木质素,它能降解许多种类的有机污染物,因此在环保方面有巨大的应用前景。本文主要综述了国内外对该酶系的研究进展。     

Extracellular proteases produced by the wood-degrading fungus Phanerochaete chrysosporium under ligninolytic and non-ligninolytic conditions

When subjected to nitrogen limitation, the wood-degrading fungus Phanerochaete chrysosporium produces two groups of secondary metabolic, extracellular isoenzymes that depolymerize lignin in wood: lignin peroxidases and manganese peroxidases. We have shown earlier the turnover in activity of the lignin peroxidases to be due in part to extracellular proteolytic activity. This paper reports the electrophoretic characterization of two sets of acidic extracellular proteases produced by submerged cultures of P. chrysosporium. The protease activity seen on day 2 of incubation, during primary growth when nitrogen levels are not known to be limiting, consisted of at least six proteolytic bands ranging in size from 82 to 22 kDa. The activity of this primary protease was strongly reduced in the presence of SDS. Following the day 2, when nitrogen levels are known to become limiting and cultures become ligninolytic, the main protease activity (secondary protease) consisted of a major proteolytic band of 76 kDa and a minor band of 25 kDa. The major and minor secondary protease activities were inhibited by phenylmethylsulfonyl fluoride and pepstatin A, respectively. When cultures were grown in the presence of excess nitrogen (non-ligninolytic condition), the primary protease remained the principal protease throughout the culture period. These results identify and characterize a specific proteolytic activity associated with conditions that promote lignin degradation.     

Biopulping: An overview of developments in an environmentally safe paper-making technology

Abstract: Treatment of wood chips with lignin-degrading fungi prior to pulping has been shown to have great potential for mechanical as well as chemical pulping on a laboratory scale. Ceriporiopsis subvermispora , when grown on aspen or loblolly pine for 4 weeks, was found to be superior to other fungi. On aspen there was an energy savings of 47%, and an increase in burst and tear indices of 22% and 119%, respectively. With loblolly pine, energy savings amounted to 37%, while burst and tear indices increased by 41% and 54%, respectively. The weight loss was only 6%, but a decrease in optical properties had to be accepted. After sulfite cooking of wood chips pretreated for 2 weeks, the Kappa number decreased by 30% with hard- and softwood. Tensile and tear indices decreased by only 10%, while the brightness of unbleached pulp increased by 4% with birch. Information obtained by immunoelectron microscopy and differential staining led to the conclusion that the biopulping effect obtained after 2 weeks of incubation cannot be explained by the direct action of enzymes on lignin or polysaccharides. Instead, a low molecular mass agent is considered to be responsible for the biopulping effect. These results have changed the aims of biopulping from an emphasis on removing the bulk of lignin to an emphasis on a short-term process, lasting 2 weeks and yielding a low mass loss. Data on these kinetics of fungal development and the degree of asepsis will help to scale-up the process. An advanced chip pile is assumed to be the most feasible process design, rather than a controlled enclosed reactor.     

Degradation of anthracene by selected white rot fungi

Abstract Approximately 60% of the originally supplied anthracene (AC) was degraded in ligninolytic stationary cultures of selected white rot fungi within 21 days. All the white rot fungi tested oxidized AC to anthraquinone (AQ). Unlike Phanerochaete chrysosporium and strain Px, with Pleurotus ostreatus, Coriolopsis polyzona and Trametes versicolor , AQ did not accumulate in the cultures, indicating that AQ was degraded further and its degradation did not appear to be a rate-limiting step. However, P. ostreatus and C. polyzona failed to degrade AQ in the absence of AC. P. ostreatus, T. versicolor and strain Px did not produce lignin peroxidase (ligninase) (LIP) under the test conditions but oxidized AC to AQ suggesting that white rot fungi produce enzyme(s) other than LIP capable of oxidizing compounds with high ionization potential like AC. Moreover, in the case of Ph. chrysosporium and C. polyzona , AC degradation started earlier than the production of LIP. Veratryl alcohol (VA) seemed to be playing a role in AC oxidation catalyzed by LIP in Ph. chrysosporium .     

Stimulation of ligninolytic enzyme production in Phanerochaete chrysosporium by polyoxyalkanes

AIMS: Poly(ethylene glycol) (PEG) and some substances similar to PEG in chemical structure were tested as stimulators of ligninolytic enzyme production in shaken culture of Phanerochaete chrysosporium. METHODS AND RESULTS: The substances that caused high enzymatic activity were linear polymers [poly(ethylene glycol), poly(propylene glycol), poly(butylene glycol) and poly(vinyl alcohol)] and cyclic polymers (crown ether). They can have terminal groups other than -OH [PEG (di)methyl ether, PEG sulphate, PEG derivative with the amino group and xanthate]. The maximum lignin peroxidase activities were compared with the surface pressure caused by the stimulator. Addition of polymers composed of charged monomer units did not increase the enzymatic activity and the fungi did not grow at all on addition of polymers having a fixed positive charge. CONCLUSIONS: Lignin peroxidase activity was increased after the addition of polymers with uncharged monomer units. It was higher and its maximum was reached in a shorter time on addition of polymers with higher molecular weights. SIGNIFICANCE AND IMPACT OF STUDY: Beside Tweens there are several polymers that stimulate ligninolytic enzyme production in shaken culture of P. chrysosporium. Their characteristics are: similarity to PEG in chemical structure, having uncharged monomer units and high molecular weight.     

Diuron degradation by Phanerochaete chrysosporium BKM- F-1767 in synthetic and natural media

When incubated in synthetic (N-limited) medium and on ashwood chips, Phanerochaete chrysosporium BKM-F-1767 degraded 14 and 10 mg/l diuron, respectively. The wood chips were used as support and sole nutrient source for the fungus. A higher degradation efficiency was found in ashwood culture as compared to the liquid culture, probably as a result of the synergetic effect of attached fungal growth, presence of limiting-substrate conditions and the microenvironment provided by ashwood, all favorable for production of high extracellular enzyme titres. Diuron degradation occured during the idiophasic growth, in the presence of manganese peroxidase, detected as dominant enzyme in both cultures.     

黄孢原毛皮革菌降解苯胺的工艺研究

通过正交试验优化筛选了适合黄孢原毛皮革菌降解苯胺的适宜培养基和摇瓶培养降解条件。结果表明：其适宜降解的液体培养基组成为：蔗糖20g／L,可溶性淀粉20g／L,(NH4)2SO4l0g／L,Mn^2 lμmol／L,Tween-800．3％,蛋白胨30g／L。适宜降解的摇瓶培养条件为：接种量为20％、pH为7．0、温度为30℃、培养时间为12d．此条件下的苯胺最高降解率可达95．5％。     

The white-rot fungus <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>: conditions for the production of lignin-degrading enzymes

Investigating optimal conditions for lignin-degrading peroxidases production by Phanerochaete chrysosporium (P. chrysosporium) has been a topic for numerous researches. The capability of P. chrysosporium for producing lignin peroxidases (LiPs) and manganese peroxidases (MnPs) makes it a model organism of lignin-degrading enzymes production. Focusing on compiling and identifying the factors that affect LiP and MnP production by P. chrysosporium, this critical review summarized the main findings of about 200 related research articles. The major difficulty in using this organism for enzyme production is the instability of its productivity. This is largely due to the poor understanding of the regulatory mechanisms of P. chrysosporium responding to different nutrient sources in the culture medium, such as metal elements, detergents, lignin materials, etc. In addition to presenting the major conclusions and gaps of the current knowledge on lignin-degrading peroxidases production by P. chrysosporium, this review has also suggested further work, such as correlating the overexpression of the intra and extracellular proteins to the nutrients and other culture conditions to discover the regulatory cascade in the lignin-degrading peroxidases production process, which may contribute to the creation of improved P. chrysosporium strains leading to stable enzyme production.