Differences in crystalline cellulose modification due to degradation by brown and white rot fungi

Wood-decaying basidiomycetes are some of the most effective bioconverters of lignocellulose in nature, however the way they alter wood crystalline cellulose on a molecular level is still not well understood. To address this, we examined and compared changes in wood undergoing decay by two species of brown rot fungi, Gloeophyllum trabeum and Meruliporia incrassata, and two species of white rot fungi, Irpex lacteus and Pycnoporus sanguineus, using X-ray diffraction (XRD) and ¹³C solid-state nuclear magnetic resonance (NMR) spectroscopy. The overall percent crystallinity in wood undergoing decay by M. incrassata, G. trabeum, and I. lacteus appeared to decrease according to the stage of decay, while in wood decayed by P. sanguineus the crystallinity was found to increase during some stages of degradation. This result is suggested to be potentially due to the different decay strategies employed by these fungi. The average spacing between the 200 cellulose crystal planes was significantly decreased in wood degraded by brown rot, whereas changes observed in wood degraded by the two white rot fungi examined varied according to the selectivity for lignin. The conclusions were supported by a quantitative analysis of the structural components in the wood before and during decay confirming the distinct differences observed for brown and white rot fungi. The results from this study were consistent with differences in degradation methods previously reported among fungal species, specifically more non-enzymatic degradation in brown rot versus more enzymatic degradation in white rot.     

Investigations of Biodeterioration by Fungi in Historic Wooden Churches of Chiloé, Chile

The use of wood in construction has had a long history and Chile has a rich cultural heritage of using native woods for building churches and other important structures. In 2000, UNESCO designated a number of the historic churches of Chiloé, built entirely of native woods, as World Heritage Sites. These unique churches were built in the late 1700 s and throughout the 1800 s, and because of their age and exposure to the environment, they have been found to have serious deterioration problems. Efforts are underway to better understand these decay processes and to carryout conservation efforts for the long-term preservation of these important structures. This study characterized the types of degradation taking place and identified the wood decay fungi obtained from eight historic churches in Chiloé, seven of them designated as UNESCO World Heritage sites. Micromorphological observations identified white, brown and soft rot in the structural woods and isolations provided pure cultures of fungi that were identified by sequencing of the internal transcribed region of rDNA. Twenty-nine Basidiomycota and 18 Ascomycota were found. These diverse groups of fungi represent several genera and species not previously reported from Chile and demonstrates a varied microflora is causing decay in these historic buildings.     

Degradation of fluorene,anthracene, phenanthrene,fluoranthene, and pyrene lacks connection to the production of extracellular enzymes by Pleurotus ostreatus and Bjerkandera adusta

The degradation of polycyclic aromatic hydrocarbons (PAH) was studied in liquid cultures of Bjerkandera adusta and Pleurotus ostreatus during 7 weeks of cultivation. During only 3 days of incubation, B. adusta removed 56% and 38% of fluorene and anthracene, while P. ostreatus degraded 43% and 60% of these compounds; other PAH were degraded to a lower extent. Except for anthracene in cultures of P. ostreatus, all PAH were removed uniformly during the cultivation time but fluorene and anthracene were degraded faster than other PAH. Supplementation of liquid cultures with milled wood decreased the concentration of PAH in the solution and diminished the degradation of PAH. The fungi produced valuable activity of manganese-dependent peroxidase; laccase was secreted only by P. ostreatus and was strongly induced by the addition of milled wood. The production of the oxidative enzymes did not correlate directly to the metabolisation of PAH.     

An Antarctic Hot Spot for Fungi at Shackleton's Historic Hut on Cape Royds

The historic expedition huts located in the Ross Sea Region of the Antarctic and the thousands of artifacts left behind by the early explorers represent important cultural heritage from the “Heroic Era” of Polar exploration. The hut at Cape Royds built by Ernest Shackleton and members of the 1907–1908 British Antarctic Expedition has survived the extreme Antarctic environment for over 100 years, but recent studies have shown many forms of deterioration are causing serious problems, and microbial degradation is evident in the historic wood. Conservation work to reduce moisture at the hut required removal of fodder, wood, and many different types of organic materials from the stables area on the north side of the structure allowing large numbers of samples to be obtained for these investigations. In addition, wood from historic food storage boxes exposed in a ravine adjacent to the hut were also sampled. Fungi were cultured on several different media, and pure cultures were obtained and identified by sequencing of the internal transcribed spacer region of rDNA. From the 69 cultures of filamentous fungi obtained, the most predominant genera were Cadophora (44%) followed by Thielavia (17%) and Geomyces (15%). Other fungi found included Cladosporium, Chaetomium, and isolates identified as being in Pezizomycotina, Onygenales, Nectriaceae, and others. No filamentous basidiomycetes were found. Phylogenetic analyses of the Cadophora species showed great species diversity present revealing Cadophora malorum, Cadophora luteo-olivacea, Cadophora fastigiata, as well as Cadophora sp. 4E71-1, a C. malorum-like species, and Cadophora sp. 7R16-1, a C. fastigiata-like species. Scanning electron microscopy showed extensive decay was present in the wood samples with type 1 and type 2 forms of soft rot evident in pine and birch wood, respectively. Fungi causing decay in the historic wooden structures and artifacts are of great concern, and this investigation provides insight into the identity and species diversity of fungi found at the site. The historic woods and other organic materials at this site represent a large input of carbon into the Antarctic environment. This as well as nutrient additions from the nearby Adélie penguin (Pygoscelis adeliae) colony and favorable conditions for fungal growth at Cape Royds appear responsible for the significant fungal diversity, and where extensive decay is taking place in wood in contact with the ground.     

Temporal changes in wood crystalline cellulose during degradation by brown rot fungi

The degradation of wood by brown rot fungi has been studied intensely for many years in order to facilitate the preservation of in-service wood. In this work we used X-ray diffraction to examine changes in wood cellulose crystallinity caused by the brown rot fungi Gloeophyllum trabeum, Coniophora puteana, and two isolates of Serpula lacrymans. All fungi increased apparent percent crystallinity early in the decay process while decreasing total amounts of both crystalline and amorphous material. Data also showed an apparent decrease of approximately 0.05 Å in the average spacing of the crystal planes in all degraded samples after roughly 20% weight loss, as well as a decrease in the average observed relative peak width at 2θ = 22.2°. These results may indicate a disruption of the outer most semi-crystalline cellulose chains comprising the wood microfibril. X-ray diffraction analysis of wood subjected to biological attack by fungi may provide insight into degradative processes and wood cellulose structure.     

Assessment of fungal diversity and deterioration in a wooden structure at New Harbor, Antarctica

Scientists working at New Harbor, Antarctica in November 1959 used a wooden crate as a makeshift workspace and kitchen. The structure has been used intermittently over the subsequent decades and still remains at the site with various materials left in and around it. The wooden structure was assessed for deterioration and samples collected to determine the diversity of fungi at the site after 43 years in the Antarctic environment. Results from these investigations are compared to the results from research on the historic huts of Ross Island, approximately 70 km east of New Harbor that were built 48–58 years earlier. Our analysis shows the wood of the New Harbor structure is extremely weathered and soft rot decay was detected in the wood in contact with the ground. Fungal cultures isolated from wood of the structure were identified using sequences of the internal transcribed spacer region of the rDNA. Several species of Cadophora were identified including C. malorum, C. luteo-olivacea, C. fastigiata and a previously undescribed species designated C. sp. NH. Laboratory decay experiments using two Cadophora species isolated from New Harbor demonstrated extensive decay and loss of biomass in hardwood wafers after 16 weeks. Other fungi isolated from the wood included species of Cladosporium, Hormonema, Penicillium and Lecythophora. Wind erosion has also severely affected the structure’s exterior wood causing deep furrowing between earlywood and latewood cells. In general, the deterioration and fungi found at the site were similar to those found at the historic expedition huts on Ross Island, however, one species obtained is unique to the New Harbor site. This research expands our knowledge of the microbes colonizing wood brought into the polar environment and provides additional information on deterioration and decomposition processes occurring in Antarctica.     

Azole-based antimycotic agents inhibit mold on unseasoned pine

Inhibiting the growth of mold fungi on cellulose-based building materials may be achievable through the use of azole-based antimycotics. Azoles were variably effective against mold fungi that are frequently found on wood and wood products. Unseasoned southern yellow pine specimens that were dip-treated with varying concentrations of eight azoles were evaluated for their ability to resist mold infestation when challenged with Aspergillus niger, Penicillium chrysogenum, and Trichoderma viride spores. Minimal fungicidal concentrations (MFC₉₀) were determined to be 0.016% for thiabendazole and 0.043% for voriconazole, the most efficacious azoles against the challenge fungi. We conclude that thiabendazole or voriconazole may be used alone or in combination to inhibit mold fungi on unseasoned pine.     

Selection of white-rot fungi for biopulping

Different rates of wood decay and ligninolytic activity were found in wood decayed by various white-rot fungi. Chemical and ultrastructural analyses showed wood decayed by Coriolus versicolor consisted of a nonselective attack on all cell wall components. Lignin degradation was restricted to the cell wall adjacent to hyphae or around the circumference of cell lumina. Decay by Phellinus pini, Phlebia tremellosus, Poria medullapanis and Scytinostroma galactinum was selective for lignin degradation. Secondary walls were void of lignin and middle lamellae were extensively degraded. A diffuse attack on lignin occurred throughout all cell wall layers. Variation in ligninolytic activity was found among strains of Phanerochaete chrysosporium. Differences in weight loss as well as lignin and polysaccharide degradation were also found when wood of different coniferous and deciduous tree species was decayed by various white-rot fungi.     

Biodegradation of phenol, <Emphasis Type="Italic">o-</Emphasis>cresol, <Emphasis Type="Italic">m-</Emphasis>cresol and <Emphasis Type="Italic">p-</Emphasis>cresol by indigenous soil fungi in soil contaminated with creosote

Seven non-basidiomycetic fungi, Aspergillus, Candida, Cladosporium, Fusarium, Monicillium, Trichoderma and Penicillium, and two basidiomycetic fungi, Pleurotus and Phanerochaete were isolated from a creosote-contaminated soil by using mineral salts medium and soil extract broth containing antibiotics. Soil contaminated with phenol, o-cresol, m-cresol and p-cresol was collected from the yard of a wood treatment plant in South Africa and inoculated with the strains of Aspergillus, Cladosporium, Fusarium, Monicillium, Penicillium and Phanerochaete, selected from the isolate. The soil in some of the treatment reactors was amended with nutrient supplements to give a C:N:P ratio of 25:5:1. A total of 18 duplicate treatments were established and incubated in the dark at 25°C for 70days. The soil in all the reactors was tilled weekly and moisture was maintained at 70% field capacity. Soil samples were collected every 2weeks for analysis of residual concentrations of the phenols tested, pH measurement and moisture content determination. The nutrient-supplemented treatments were more effective in degrading the phenols (between 84 and 100%) than those that were not supplemented. Barley, which was used as bulking agent enhanced the growth of the fungi and subsequently the degradation of the phenols. Inoculation with a mixture of the six fungal isolates promoted more phenol degradation than with single isolates.     

Fungal biodiversity in indoor environments in Havana, Cuba

A study was made of the indoor mycobiota in a range of public and private buildings (libraries, museums, laboratories and offices, warehouses, homes and a school) in the city of Havana (Cuba). Culturable airborne fungi were sampled using a slit-to-agar impactor (Chirana aeroscope). High levels of contamination were recorded, with more than 700 colony-forming units per cubic metre of air in 85% of the buildings sampled, and more than 1,000 in 55% of buildings. Of the 28 genera and 31 species identified, the most common were Aspergillus flavus, Aspergillus niger, Penicillium citrinum, Cladosporium cladosporioides and Cladosporium sphaerospermum. Genus and species diversity was analysed, and new findings were obtained regarding Cuba’s atmospheric ecosystem.     

Can co-culturing of two white-rot fungi increase lignin degradation and the production of lignin-degrading enzymes?

The aim of this work was to investigate the poorly understood effects of co-culturing of two white rot fungi on the production of lignin-degrading enzyme activities. Four species, Ceriporiopsis subvermispora, Physisporinus rivulosus, Phanerochaete chrysosporium and Pleurotus ostreatus were cultured in pairs to study the degradation of aspen wood and the production of lignin-degrading enzymes. Potential of co-culturing for biopulping was evaluated. Chemical analysis of decayed aspen wood blocks showed that co-culturing of C. subvermispora with P. ostreatus could significantly stimulate wood decay, when compared to monocultures. Based on the fungi tested here, however, this effect is species-specific. Other combinations of fungi were slightly stimulating or not stimulatory. The pattern of lignin degradation was altered towards the acid insoluble part of lignin especially in co-cultures where P. ostreatus was included as a partner. The use of agar plates containing the polymeric dye Poly R-478 showed elevated dye decolourization at the confrontation zone between mycelia. Laccase was significantly stimulated only in the co-culture of P. ostreatus with C. subvermispora. Manganese peroxidase activity was stimulated in co-cultures of P. ostreatus with C. subvermispora or with P. rivulosus. Immunoblotting indicated changes in lignin-degrading enzymes and/or their isoform composition in response to co-culturing. This is the first report on the effects of co-culturing of potential biopulping fungi on wood degradation, and gives basic knowledge on fungal interactions during wood decay that can be utilized in practical applications.     

Evaluation of the selectivity of white rot isolates using near infrared spectroscopic techniques

Seventeen isolates from white rotted beech wood and six strains from a local culture collection were evaluated for their capability to delignify beech and spruce wood selectively. Six peroxidase-positive isolates were found using a colorimetric agar plate test (Poly R-478), and genetically identified by their internal transcribed spacer (ITS1) or 28S rDNA sequences. Colonised on beech and spruce wood veneers, some of the peroxidase-positive isolates caused selective white rot on both wood species. Weight loss and lignin content of the degraded veneers were estimated from FT-NIR spectra with established linear regression models and multivariate models based on partial least squares regression (PLSR). Weight loss of the samples was also determined gravimetrically. A measure for the relative selectivity of the strains for lignin degradation was formulated and the values were calculated. Two strains that were identified as Oxyporus latemarginatus and Trametes cervina exhibited high selectivity on spruce wood, but the lignin content of the decayed wood was higher than that degraded by the reference strain Ceriporiopsis subvermispora. One strain – identified as Phlebia tremellosa – led to a lower lignin content of beech wood but caused also comparably high weight loss and thus exhibited an overall lower selectivity. The NIR spectroscopic method proved to be convenient for the quick screening of selective white rot fungi. Furthermore, the results revealed that high selectivity for lignin degradation is much more pronounced in early degradation stages.     

White rot Basidiomycetes isolated from Chiloé National Park in Los Lagos region, Chile

Wood decomposition is an important component in forest ecosystems but information about the diversity of fungi causing decay is lacking. This is especially true for the temperate rain forests in Chile. These investigations show results of a biodiversity study of white-rot fungi in wood obtained from Chiloé National Park in Los Lagos region, Chile. Culturing from white-rotted wood followed by sequencing of the complete internal transcribed spacer region of the ribosomal DNA (rDNA) or partial large subunit region of the rDNA, identified 12 different species in the Basidiomycota. All of these fungi were characterized as white rot fungi and were identified with a BLAST match of 97 % or greater to sequences in the GenBank database. Fungi obtained were species of Phlebia, Mycoacia, Hyphodontia, Bjerkandera, Phanerochaete, Stereum, Trametes, and Ceriporiopsis. This report identifies for the first time in Chile the species Ceriporiopsis subvermispora, Hyphodontia radula, Phlebia radiata, Phanerochaete affinis, Peniophora cinerea, Stereum gausapatum, Phlebia setulosa and Phanerochaete sordida. Scanning electron microscopy was used to characterize the type of decay caused by the fungi that were isolated and a combination of selective lignin degraders and simultaneous white rot fungi were found. Fungi that cause a selective degradation of lignin are of interest for bioprocessing technologies that require modification or degradation of lignin without cellulose removal.     

Biodegradation of Lignocellulose by White-Rot Fungi: Structural Characterization of Water-Soluble Hemicelluloses

In the biological pretreatment process, white-rot fungi are mostly used to degrade lignin and carbohydrates in lignocellulosic biomass. In this study, water-soluble hemicelluloses were recovered from birch wood (Betula alnoides) decayed by white-rot fungi (Ganoderma lucidum C7016) for different durations up to 16 weeks. Accordingly, the dimethyl sulfoxide (DMSO)-soluble hemicelluloses were isolated from the untreated birch wood as a comparison. Results showed that the fungal-degraded polysaccharides were acidic hemicelluloses having a high content of uronic acids ranging from 20.6 to 22.5 %. Gel permeation chromatography analysis demonstrated that the recovered water-soluble hemicelluloses had a lower average molecular weight (M _w, 15,990–27,560 g?mol^?1) than that of the DMSO-soluble hemicelluloses (M _w , 33,960 g?mol^?1). Fourier transform infrared spectroscopy, scanning electron microscopy, one- and two-dimensional nuclear magnetic resonance spectroscopy also revealed significantly changes between those of fungal degraded and DMSO-soluble hemicelluloses. It was proposed that the hemicelluloses with low molecular weights were easily removed from wood by fungal degradation. This research revealed the changes of hemicelluloses in fungal degradation in the natural environment, which may enable the exploration of novel methods in bioconversion of lignocellulosic biomass for the production of biofuels and biopolymers, in addition to the development of new and better ways to protect wood from biodegradation by microorganisms.     

Separation of Fungal Propagules by Partition in Aqueous Polymer Two-Phase Systems

Conidia of Penicillium chrysogenum and Penicillium frequentans and sporangiospores of Rhizopus rhizopodiformis, Rhizomucor pusillus, and Mucor racemosus were subjected to partition in aqueous polymer two-phase systems. The partition behavior differed drastically between the conidia of the two Penicillium species and the sporangiospores of the three species of phycomycetes. This difference in partition behavior can be used for purification of fungi belonging to different taxonomical groups. P. frequentans was completely separated from M. racemosus by two extractions, whereas four extractions were needed to purify M. racemosus. This method was used on an air sample from a locality where wood fuel chips are handled. The conidia of the fungi Trichoderma viride and Rhizopus rhizopodiformis were removed completely by only two extractions.     

Fungal Degradation of Lipophilic Extractives in Eucalyptus globulus Wood

Solid-state fermentation of eucalypt wood with several fungal strains was investigated as a possible biological pretreatment for decreasing the content of compounds responsible for pitch deposition during Cl₂-free manufacture of paper pulp. First, different pitch deposits were characterized by gas chromatography (GC) and GC-mass spectrometry (MS). The chemical species identified arose from lipophilic wood extractives that survived the pulping and bleaching processes. Second, a detailed GC-MS analysis of the lipophilic fraction after fungal treatment of wood was carried out, and different degradation patterns were observed. The results showed that some basidiomycetes that decreased the lipophilic fraction also released significant amounts of polar extractives, which were identified by thermochemolysis as originating from lignin depolymerization. Therefore, the abilities of fungi to control pitch should be evaluated after analysis of compounds involved in deposit formation and not simply by estimating the decrease in the total extractive content. In this way, Phlebia radiata, Funalia trogii, Bjerkandera adusta, and Poria subvermispora strains were identified as the most promising organisms for pitch biocontrol, since they degraded 75 to 100% of both free and esterified sterols, as well as other lipophilic components of the eucalypt wood extractives. Ophiostoma piliferum, a fungus used commercially for pitch control, hydrolyzed the sterol esters and triglycerides, but it did not appear to be suitable for eucalypt wood treatment because it increased the content of free sitosterol, a major compound in pitch deposits.     

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

Summary Of 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.These results were reported in part at the Journées Internationales d'Etudes du Groupe Polyphenols, 29. 9.–1. 10. 1982, Université Paul Sabatier, Toulouse, France     

Fungal accumulation of metals from building materials during brown rot wood decay

This study analyzes the accumulation and translocation of metal ions in wood during the degradation performed by one strain of each of the three brown rot fungi; Serpula lacrymans, Meruliporia incrassata and Coniophora puteana. These fungi species are inhabitants of the built environment where the prevention and understanding of fungal decay is of high priority. This study focuses on the influence of various building materials in relation to fungal growth and metal uptake. Changes in the concentration of iron, manganese, calcium and copper ions in the decayed wood were analyzed by induced coupled plasma spectroscopy and related to wood weight loss and oxalic acid accumulation. Metal transport into the fungal inoculated wood was found to be dependent on the individual strain/species. The S. lacrymans strain caused a significant increase in total iron whereas the concentration of copper ions in the wood appeared decreased after 10 weeks of decay. Wood inoculated with the M. incrassata isolate showed the contrary tendency with high copper accumulation and low iron increase despite similar weight losses for the two strains. However, significantly lower oxalic acid accumulation was recorded in M. incrassata degraded wood. The addition of a building material resulted in increased weight loss in wood degraded by C. puteana in the soil-block test; however, this could not be directly linked specifically to the accumulation of any of the four metals recorded. The accumulation of oxalic acid seemed to influence the iron uptake. The study assessing the influence of the presence of soil and glass in the soil-block test revealed that soil contributed the majority of the metals for uptake by the fungi and contributed to increased weight loss. The varying uptake observed among the three brown rot fungi strains toward the four metals analyzed may be related to the specific non-enzymatic and enzymatic properties including bio-chelators employed by each of the species during wood decay.     

Endoglucanase-producing fungi isolated from Cape Evans historic expedition hut on Ross Island, Antarctica

Early explorers of Antarctica's Heroic Era erected wooden buildings and brought large quantities of supplies to survive in Antarctica. The introduction of wood and other organic materials provided nutrient sources for fungi that were indigenous to Antarctica or were brought in with the materials and adapted to the harsh conditions. Seventy-two isolates of filamentous fungi were cultured on selective media from interior structural wood of the Cape Evans historic hut and 27 of these screened positive for the ability to degrade carboxymethyl cellulose (CMC). Four non-CMC-degrading isolates were added to a group of 14 CMC-degrading isolates for further study, and endo-1, 4-beta-glucanase activity was demonstrated in the extracellular supernatant from all of these 18 isolates when grown at 4 degrees C, and also when they were grown at 15 degrees C. Isolates of Penicillium roquefortii and Cadophora malorum showed preference for growth at 15 degrees C rather than 25 degrees C or 4 degrees C indicating psychrotrophic characteristics. These results demonstrate that cellulolytic filamentous fungi found in Antarctica are capable of growth at cold temperatures and possess the ability to produce extracellular endo-1, 4-beta-glucanase when cultured at cold and temperate temperatures.