Lignin degradation by white rot fungi on spruce wood shavings during short-time solid-state fermentations monitored by near infrared spectroscopy

Due to their outstanding capability of degrading the recalcitrant biomacromolecule lignin, white rot fungi have been attracting interest for several technological applications in mechanical pulping and wood surface modification. However, little is known about the time course of delignification in early stages of colonisation of wood by these fungi. Using a Fourier transform near infrared (FT-NIR) spectroscopic technique, lignin loss of sterilised spruce wood shavings (0.4–2.0 mm particle size) that had been degraded by various species of white rot fungi could be monitored already during the first 2 weeks. The delignification kinetics of Dichomitus squalens, three Phlebia species (Phlebia brevispora, Phlebia radiata and Phlebia tremellosa), three strains of Ceriporiopsis subvermispora as well as the white rot ascomycete Hypoxylon fragiforme and the basidiomycete Oxyporus latemarginatus were determined. Each of the fungi tested was able to reduce the lignin content of spruce wood significantly during the first week. The amount of delignification achieved by the selected white rot fungi after 2 weeks ranged from 7.2% for C. subvermispora (FPL 105.752) to 2.5% for P. radiata. Delignification was significant (P = 95%) already after 3 days treatment with C. subvermispora and P. tremellosa. Activities of extracellular ligninolytic enzymes (laccase, manganese peroxidase and/or lignin peroxidase), expressed by each of the tested fungi, were determined. Lignin was degraded when peroxidase activity was detected in the fungal cultures, but only a low level of correlation between enzyme activities and the extent of delignification was found.     

Non-Ligninolytic TNT Mineralization in Contaminated Soil by Phanerochaete chrysosporium

The explosive 2,4,6-trinitrotoluene (TNT) is widely used and results in widespread soil contamination. The white-rot fungus Phanerochaete chrysosporium has been shown to degrade TNT, using the peroxidase enzyme. In this study, we report peroxidase-independent degradation of TNT by non-ligninolytic P. chrysosporium. Significant disappearance of TNT from highly contaminated soil using P. chrysosporium has been observed. Soil highly contaminated with TNT (2270 ppm [10 mM]) was diluted to 100 ppm (0.44 mM) with malt extract medium. Pregrown (48 hours) mycelial pellets of P. chrysosporium were added in 100 mL malt extract medium and incubated in Gledhill flasks. Analysis by high-performance liquid chromatography (HPLC) was conducted on soil extracts at specific time points to estimate the disappearance of TNT from contaminated soil incubated with P. chrysosporium. When the pregrown mycelial pellets were added, TNT disappeared within 48 hours. The dissolved concentration of 2-amino-4,6-dinitrotoluene (2Am-DNT) increased up to the third day, then declined before its final disappearance by day 10. Results show that the pregrown mycelial pellets of P. chrysosporium mineralized up to 17.3±6.3% [¹⁴C]-TNT within 30 days.     

Biopreparation of cotton fabric with enzymes produced by solid-state fermentation

Fungal enzyme preparations from Phanerochaete chrysosporium, Aspergillus oryzae, Aspergillus giganteus and Trichoderma virens, produced by solid-state fermentation (SSF) on cotton seed-coat fragment waste as substrate and enzyme inducer were investigated in biopreparation of cotton fabric. Cotton seed-coat fragment is rich in lignin, cellulose and hemicelluloses, therefore enzyme complexes produced by target fungi on such a substrate can be used effectively to degrade impurities in cotton fabrics during biopreparation. Activities of extracellular hydrolytic and ligninolytic enzymes were determined from the SSF extract materials. The potential of the hydrolytic and accompanying oxidative enzymes in the whole SSF cultures was exploited in degradation of seed-coat fragments and other coloring materials of greige cotton fabric. Enzyme assays indicated that many extracellular enzymes have been produced under these conditions including both hydrolytic and oxidative enzymes. A. oryzae NRRL 3485 produced significantly higher amounts of both hydrolytic and oxidative enzymes than other tested fungi. Best results in removal of seed-coat fragments from cotton fabric were obtained by P. chrysosporium NCAIM (=ATCC 34541), P. chrysosporium VKM F-1767 and A. oryzae NRRL 3485 SSF enzyme complexes.     

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) Biodegradation in Liquid and Solid-State Matrices by Phanerochaete chrysosporium

Extensive biodegradation of hexahydro-1,3,5 -trinitro-1,3,5 -triazine (RDX) by the white-rot fungus Phanerochaete chrysosporium in liquid and solid matrices was observed. Some degradation in liquid occurred under nonligninolytic conditions, but was approximately 10 times higher under ligninolytic conditions. Moreover, elimination was accounted for almost completely as carbon dioxide. No RDX metabolites were detected. The degradation rates in liquid appeared to be limited to RDX concentration in solution (approximately 80 mg/L), but degradation rates in soil were nonsaturable to 250 mg/kg. Manganese-dependent peroxidase (MnP) and cellobiose dehydrogenase (CDH) from P. chrysosporium, but not lignin peroxidase, were able to degrade RDX. MnP degradation of RDX required addition of manganese, but CDH degraded RDX anaerobically without addition of mediators. Attempts to improve biodegradation by supplementing cultures with micronutrients showed that addition of manganese and oxalate stimulated degradation rates in liquid, sawdust, and sand by the fungus, but not in loam soil. RDX degradation by P. chrysosporium in sawdust and sand was better than observed in liquid. However, degradation in solid matrices by the fungus only began after a lag period of 2 to 3 weeks, during which time extractable metabolites from wood were degraded.     

Transformation of 2,4,6-Trinitrotoluene (TNT) Reduction Products by Lignin Peroxidase (H8) from the White-Rot Basidiomycete Phanerochaete chrysosporium

White-rot fungi are known to degrade a wide range of xenobiotic environmental pollutants, including the nitroaromatic explosive 2,4,6-trinitrotoluene (TNT). TNT is first reduced by the fungal mycelium to aminodinitrotoluenes and diaminonitrotoluenes. In a second phase, reduced TNT metabolites are oxidatively transformed and mineralized. The extracellular oxidative enzyme of the ligninolytic system of these fungi includes the lignin peroxidases (LiP) and the manganese-dependent peroxidases (MnP). In the present study, we have shown that a cell-free enzymatic system containing fast protein liquid chromatography (FPLC)-purified LiP (H8) from the white-rot fungus Phanerochaete chrysosporium was able to completely transform 50 mg/L of 2,4-diamino-6-nitrotoluene (2,4-DA-6-NT) and 2-amino-4,6-dinitrotoluene (2-A-4,6-DNT) in 1 and 48 h, respectively. Veratryl alcohol (VA), often described as a mediator in the LiP-catalyzed oxidative depolymerization of lignin, was not required for the enzymatic transformation of 2,4-DA-6-NT or 2-A-4,6-DNT. 2,4-DA-6-NT was also shown to be a competitive inhibitor of the LiP activity measured through the oxidation of VA. Experiments using ¹⁴C-U-ring labeled compounds showed that 2-A-4,6-DNT was converted to 2,2'-azoxy-4,4' ,6,6'-tetranitrotoluene. No significant mineralization, measured by the release of ¹⁴CO2, was observed over 5 d.     

Oxidase of the white rot fungus Panus tigrinus 8/18

Extracellular oxidase of the white rot fungus Panus tigrinus earlier reported as laccase)contains copper but has no absorption spectrum typical of ‘blue’ oxidases. Thioglycolate and sodium azide inhibit the activity of this enzyme at concentrations 2.5–3 orders lower than those needed for fungal laccases. The oxidase of P. tigrinus oxidizes syringaldazine, coniferyl alcohol, ABTS, syringic acid, diaminobenzidine, guaiacol, catechol and vanillylacetone with different efficiencies. Oxygen consumption and no hydrogen peroxide formation were detected during substrate oxidation by P. tigrinus oxidase. It is proposed that P. tigrinus oxidase is a new ligninolytic enzyme.     

Laboratory investigations on the potential of entomopathogenic fungi for biocontrol of Helicoverpa armigera (Lepidoptera: Noctuidae) larvae and pupae

The susceptibility of third instar Helicoverpa armigera to seven strains of three entomopathogenic fungal species, i.e. Metarhizium anisopliae, Beauveria bassiana and Paecilomyces fumosoroseus, was tested under laboratory conditions using the larval immersion method. High efficacies ranging from 68 to 100% corrected mortality were recorded with more profound effects in treatments with B. bassiana and P. fumosoroseus strains. The median lethal concentration (LC₅₀) for L3 was 6.0×10⁵ in M. anisopliae 79, 1.5×10⁵ in B. bassiana 124 and 4.2×10⁴ in P. fumosoroseus 14. These three strains were further used to characterize the age-dependent mortality of different larval stages (L2-L5) and the effect against pupae of H. armigera. Larval stages did not differ in their mortality but differed i in median lethal time, with shorter values recorded in the second instar. Tested fungi also caused a high reduction between 74.4 and 100% in the emergence of pupae using the soil inoculation method and the pupal immersion technique. All three fungal species, especially P. fumosoroseus, have a high potential for biocontrol of H. armigera larvae and also as a soil treatment targeting the pupae.     

生石灰对林下酸化土壤的调控作用及三七生长的影响

土壤酸化是导致林下三七生长不良和根腐病发生的重要因素。本试验利用不同量(0、500、1000、1500和2000 kg·hm^-2)的生石灰改良林下酸化土壤,评价生石灰对土壤理化性质、酚类物质、根际微生物和三七生长的影响。结果表明: 适量的生石灰(500～1000 kg·hm^-2)可显著提高土壤pH值,降低酚类物质(羟基苯甲酸、香草醛、丁香酸、阿魏酸和香草酸)含量,促进三七的生长,并降低根腐病的发生。适量的生石灰(500～1000 kg·hm^-2)能显著降低土壤的真菌/细菌,提高细菌多样性,提高门水平上子囊菌门、变形菌门和属水平上马赛菌属和鞘脂单胞菌属的相对丰度。但是过量的生石灰(1500～2000 kg·hm^-2)会降低土壤碱解氮和有机质含量,抑制三七的生长。因此,500～1000 kg·hm^-2生石灰可显著改善林下土壤的化学性质和微生物群落,从而促进三七生长,降低根腐病的发生。     

不同固体发酵培养基下三种白腐真菌分泌的木质纤维素酶活性

本研究选取众所周知的典型白腐真菌树舌灵芝Ganoderma applanatum、毛栓孔菌Trametes hirsuta和木蹄层孔菌Fomes fomentarius作为研究对象,对其利用木质纤维生物质进行发酵及添加有机营养、无机盐、金属离子、表面活性剂等进行了探索,期间以测定漆酶、滤纸纤维素酶、木聚糖酶活性表征3种菌株对木质纤维生物质的预处理能力,为确定白腐真菌菌株及单环境因子而达到高效预处理木质纤维生物质提高生物转化效率的目的奠定了重要的理论基础。结果显示,3种菌株分泌的木质纤维素酶在10周内基本都呈现先上升后下降的趋势,且酶活都较高,均可作为木质纤维生物质预处理的备选菌株。相比于针叶树（落叶松）基质,阔叶树（白桦）基质更适宜于3种菌株生长及分泌木质纤维素酶。各环境因子中,Cu²⁺的添加可提高漆酶活性,表面活性剂对于3种酶活的诱导作用均十分显著。     

Decolorization of dyes using white-rot fungi and radical-generating reactions

Decolorization of synthetic dyes was performed using cultures of white-rot fungi producing ligninolytic enzymes and radical-generating reactions that could be involved in the mechanism of fungal decolorization. Among the white-rot fungi tested, Pleurotus ostreatus exhibited the highest decolorization rates, and also the highest production of laccase and Mn-peroxidase. P. ostreatus strain f6 gave 69% decolorization of Eosin Yellowish, 96% of Evans Blue, 75% of Phenol Red (all at 1 mM) and 88% of Poly B-411 (20 ppm) during a 14-day treatment. Treatment with Cu/succinic acid/H₂O₂ resulted in 96% decolorization of Evans Blue and Poly B-411 within 24 h. However, only 48% and 2% decolorization was achieved with Phenol Red and Eosin Yellowish, respectively. Similar decolorization rates were also obtained when Cu was replaced with Co. The results show that treatment of dye-containing solutions with both fungal cultures and biomimetic catalytic reactions results in decolorization.     

Decolorization of textile indigo dye by ligninolytic fungi

The indigo dye is extensively used by textile industries and is considered a recalcitrant substance, which causes environmental concern. Chemical products used on textile processing, which affect the environment through effluents, can be voluminous, colored and varied. Vat textile dyes, like indigo, are often used and dye mainly cellulosic fibers of cotton. Decolorization of this dye in liquid medium was tested with ligninolytic basidiomycete fungi from Brazil. Decolorization started in a few hours and after 4 days the removal of dye by Phellinus gilvus culture was in 100%, by Pleurotus sajor-caju 94%, by Pycnoporus sanguineus 91% and by Phanerochaete chrysosporium 75%. No color decrease was observed in a sterile control. Thin layer chromatography of fungi culture extracts revealed only one unknown metabolite of Rf=0.60, as a result of dye degradation.     

Reevaluation of the manganese requirement for the biobleaching of kraft pulp by white rot fungi

Manganese dependent peroxidase (MnP) is the main enzyme implicated in the biobleaching of kraft pulps by white rot fungi. The goal of this study was to evaluate the Mn requirement for biobleaching of eucalyptus oxygen delignified kraft pulp (OKP) by various white rot fungi: Trametes versicolor, Phanerochaete sordida, Phlebia radiata, Stereum hirsutum and Bjerkandera sp. strain BOS55. All of the strains tested produced MnP and provided extensive bleaching of OKP when 33 μM Mn was included in the medium. Bjerkandera sp. strain BOS55 was the only strain that also displayed MnP production and biobleaching activity of EDTA-extracted OKP in the complete absence of Mn. However, MnP and biobleaching activity in the absence of Mn was dependent on the presence of organic acids in the medium. The fact the biobleaching was correlated to MnP activity irrespective of whether Mn was present or absent suggests that there may be roles for MnP in Bjerkandera under Mn-deficient conditions. Although manganese-independent peroxidase (MIP) and lignin peroxidase (LiP) were also detected, the titres were much smaller in comparison with those of MnP, so their relative role in biobleaching can be predicted to have a minor importance in comparison with MnP. Only in the case of Bjerkandera, was the expression of LiP stimulated in the presence of oxalate but final brightness was not substantially affected.     

西南亚高山针叶林主要树种互作及增温对根区土壤微生物群落的影响

温度与植物种类是生态系统土壤微生物群落组成与结构的重要影响因子。气候变暖背景下, 不同树种及树种互作对土壤微生物群落产生的影响仍不清楚。该文以西南亚高山针叶林主要建群种粗枝云杉(Picea asperata)和岷江冷杉(Abies faxoniana)为研究对象, 采用红外加热器模拟增温, 通过不同种植方式(云杉、冷杉单种和二者混种, 以及裸地对照), 研究不同物种及增温对土壤微生物磷脂脂肪酸(PLFAs)含量与群落结构的影响。结果表明: (1)无论增温与否, 与裸地相比, 云杉与冷杉单种均显著增加了土壤微生物群落主要类群及总PLFAs含量, 而混种仅在非增温条件下增加了微生物群落PLFAs含量; 另一方面, 增温显著促进了裸地真菌(F)和云杉根区革兰氏阴性菌(GN)的生长, 但对冷杉与冷杉-云杉混种小区微生物群落具有显著的抑制作用。(2)主成分分析(PCA)表明, 非增温条件下, 植物种植对土壤微生物群落组成的影响更为明显。非增温情况下云杉、冷杉单种和混种均对微生物群落结构有显著影响, 显著降低了土壤革兰氏阳性菌/阴性菌(GP/GN), 增加了土壤真菌细菌比(F/B)(64.29%-35.71%), 而增温时, 仅冷杉单种对GP/GN和F/B有显著影响。(3) PLFAs含量与土壤碳含量显著正相关, 微生物群落结构(F/B)则与土壤pH及无机氮含量有显著相关关系。以上结果说明, 在非增温情况下, 无论单种还是混种均有利于土壤微生物生长, 但在增温情况下混种对微生物群落PLFAs含量无显著影响, 两个物种对微生物群落结构的影响在增温条件下也有减弱的趋势。     

Effects of plant interspecific interaction and warming on soil microbial community in root zone soil of two dominant tree species in the subalpine coniferous forest in southwestern China

温度与植物种类是生态系统土壤微生物群落组成与结构的重要影响因子。气候变暖背景下, 不同树种及树种互作对土壤微生物群落产生的影响仍不清楚。该文以西南亚高山针叶林主要建群种粗枝云杉(Picea asperata)和岷江冷杉(Abies faxoniana)为研究对象, 采用红外加热器模拟增温, 通过不同种植方式(云杉、冷杉单种和二者混种, 以及裸地对照), 研究不同物种及增温对土壤微生物磷脂脂肪酸(PLFAs)含量与群落结构的影响。结果表明: (1)无论增温与否, 与裸地相比, 云杉与冷杉单种均显著增加了土壤微生物群落主要类群及总PLFAs含量, 而混种仅在非增温条件下增加了微生物群落PLFAs含量; 另一方面, 增温显著促进了裸地真菌(F)和云杉根区革兰氏阴性菌(GN)的生长, 但对冷杉与冷杉-云杉混种小区微生物群落具有显著的抑制作用。(2)主成分分析(PCA)表明, 非增温条件下, 植物种植对土壤微生物群落组成的影响更为明显。非增温情况下云杉、冷杉单种和混种均对微生物群落结构有显著影响, 显著降低了土壤革兰氏阳性菌/阴性菌(GP/GN), 增加了土壤真菌细菌比(F/B)(64.29%-35.71%), 而增温时, 仅冷杉单种对GP/GN和F/B有显著影响。(3) PLFAs含量与土壤碳含量显著正相关, 微生物群落结构(F/B)则与土壤pH及无机氮含量有显著相关关系。以上结果说明, 在非增温情况下, 无论单种还是混种均有利于土壤微生物生长, 但在增温情况下混种对微生物群落PLFAs含量无显著影响, 两个物种对微生物群落结构的影响在增温条件下也有减弱的趋势。     

褐环乳牛肝菌对樟子松和油松根际土壤真菌多样性的影响

为了解褐环乳牛肝菌Suillus luteus对樟子松Pinus sylvestris var. mongolica和油松Pinus tabulaeformis根际土壤真菌群落结构的影响,采用Illumina MiSeq高通量测序,对两种松树接种S. luteus与对照组的根际土壤真菌群落结构进行研究。结果显示,4个样品共获得原始序列347 681条,归为5个真菌门。综合各土壤样品真菌Alpha多样性指数及OTUs-Venn图,发现接种S. luteus后樟子松和油松根际土壤真菌相对丰度与对照存在一定差异。群落结构分析表明,接种S. luteus提高了樟子松和油松根际土壤担子菌和壶菌的相对丰度,抑制了子囊菌门真菌。优势属由原来的Geopora转变为Suillus。通过PCA分析与NMDS分析发现接种S. luteus后樟子松和油松根际土壤真菌群落差异性增加,且接种S. luteus后樟子松根际土壤真菌群落结构相似性显著高于油松根际土壤真菌群落结构相似性。接种S. luteus的樟子松和油松土壤真菌丰富度降低、多样性增加,接种S. luteus改变了樟子松和油松根际土壤真菌群落结构和优势真菌种群。试验结果为樟子松和油松促生真菌菌种筛选和育苗工作提供了依据。     

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 .     

呼伦贝尔沙地樟子松外生菌根真菌多样性

沙地樟子松是我国北方重要的防风固沙造林树种,也是一种典型的外生菌根依赖型树种。为揭示呼伦贝尔沙地樟子松外生菌根真菌多样性,以中龄、近熟、成熟3个龄组沙地樟子松人工林和沙地樟子松天然林为研究对象,采用野外调查和分子生物学相结合的研究方法,鉴定分析沙地樟子松外生菌根真菌种群特征。研究结果表明：（1）呼伦贝尔沙地樟子松外生菌根真菌共有10个OTU属于子囊菌,48个OTU属于担子菌,隶属于21科25属。（2）天然林优势菌为糙缘腺革菌属Amphinema、丝膜菌属Cortinarius和乳牛肝菌属Suillus,人工林优势菌为乳牛肝菌属,其余菌种相对丰度随着林龄变化波动较大。（3）天然林与人工林外生菌根真菌种群Shannon、Simpson和Pielou指数存在显著差异（P<0.05）,人工林间alpha多样性指数差异不显著（P>0.05）。（4）呼伦贝尔沙地樟子松天然林和人工林外生菌根真菌种群组成存在较大差异,其中近熟林的外生菌根真菌群落组成与天然林的最为接近。     

Wood decay under the microscope

Many aspects of the interactions between host wood structure and fungal activity can be revealed by high resolution light microscopy, and this technique has provided much of the information discussed here. A wide range of different types of decay can result from permutations of host species, fungal species and conditions within wood. Within this spectrum, three main types are commonly recognised: brown rot, white rot and soft rot. The present review explores parts of the range of variation that each of these encompasses and emphasizes that degradation modes appear to reflect a co-evolutionary adaptation of decay fungi to different wood species or the lignin composition within more primitive and advanced wood cell types. One objective of this review is to provide evidence that the terms brown rot, white rot and soft rot may not be obsolete, but rigid definitions for fungi that are placed into these categories may be less appropriate than thought previously. Detailed knowledge of decomposition processes does not only aid prognosis of decay development in living trees for hazard assessment but also allows the identification of wood decay fungi that can be used for biotechnology processes in the wood industry. In contrast to bacteria or commercial enzymes, hyphae can completely ramify through solid wood. In this review evidence is provided that wood decay fungi can effectively induce permeability changes in gymnospermous heartwood or can be applied to facilitate the identification of tree rings in diffuse porous wood of angiosperms. The specificity of their enzymes and the mild conditions under which degradation proceeds is partly detrimental for trees, but also make wood decay fungi potentially efficient biotechnological tools.     

The effect of phenolic acids and molasses spent wash concentration on distillery wastewater remediation by fungi

The effect of gallic acid, vanillic acid, and molasses spent wash (MSW) concentration on growth and decolourizing capability of four fungi (Geotrichum candidum, Coriolus versicolor, Phanerochaete chrysosporium and Mycelia sterilia) was studied. Fungal growth was inhibited to a varying extent in the presence of gallic and vanillic acid, except for G. candidum, which was unaffected by gallic acid. G. candidum and P. chrysosporium growth rates increased in the presence of increasing concentrations of MSW (up to 50% v/v), however, growth of M. sterilia and C. versicolor was inhibited at spent wash concentrations above 5% (v/v). Increasing the concentration of MSW from 6·25% (v/v) to 12·5% (v/v) increased the decolourizing ability of each fungus, except for M. sterilia. C. versicolor exhibited greatest colour removal with a reduction of 0·43 units at A₄₇₅ (equivalent to 53% colour reduction) after 10 days growth in 12·5% (v/v) MSW.