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.     

Elimination and detoxification of softwood extractives by white-rot fungi

The ability of several white-rot fungal strains to remove and detoxify acetone extractives (pitch or resin) in Scots pine sapwood was investigated in stationary laboratory batch assays. Fungal pretreatment provided up to 62% total pitch reduction and significant decreases in pitch toxicity. The best strains were Bjerkandera sp. strain BOS55, Stereum hirsutum and Trametes versicolor that eliminated over 93% of the problematic triglyceride fraction and 58–87% of other lipophilic extractive classes in only 2 weeks. Fungal removal of the wood extractives was accompanied by a 7.4–16.9-fold decrease in their inhibitory effect, as determined in the Microtox bioassay. Wood pretreatment by Bjerkandera sp. and T. versicolor caused limited losses of woody mass (less than 4% in 4 weeks); whereas S. hirsutum led to somewhat higher mass losses (7% in 4 weeks). These results indicate the potential of white rot fungi to control pitch deposition problems in pulping and to reduce the aquatic toxicity caused by naturally-occurring lipophilic extractives in forest industry effluents.     

Disruption of the Subtilase Gene, albin1, in Ophiostoma piliferum

Wood sapstaining fungi produce multiple proteases that break down wood protein. Three groups of subtilases have been identified in sapstaining fungi; however, it is not known if these groups have distinct physiological roles (B. Hoffman and C. Breuil, Curr. Genet. 41:168-175, 2002). In this work we examined the role of the subtilase Albin1 from Ophiostoma piliferum. Reamplification of cDNA ends PCR was used to obtain the albin1 gene sequence. The encoded subtilase is probably extracellular and involved in nutrient acquisition. This gene was disrupted with an Agrobacterium tumefaciens-mediated transformation system. Two of the disruptants obtained had significantly lower levels of proteolytic activity, slower growth in bovine serum albumin, and significantly reduced growth on wood. Thus, albin1 plays an important role in O. piliferum's ability to acquire nitrogen from wood proteins.     

Effect of volatiles from bacteria and yeast on the growth and pigmentation of sapstain fungi

Sapstain fungi affect the appearance of wood due to colonisation by pigmented hyphae but without producing significant strength losses. This is due to the production of melanin in the fungal cell walls of the staining fungi. Any biological control strategy targeted against this type of deterioration would therefore be considered successful if it inhibited either fungal growth or pigment production. Previous work has established that specific bacterial and yeast isolates selected on the basis of agar screening studies could significantly reduce levels of staining in wood block tests. This paper presents the results of a study to examine the role of volatile organic compounds (VOCs) produced by four bacterial and three yeast isolates on the growth and pigment production by a range of five sapstain fungi on three media types. VOCs from three of the four bacterial strains tested completely inhibited growth of the five target sapstain fungi but only when the antagonists were grown on tryptone soya media. When antagonists were grown on either malt agar or a low nutrient medium levels of inhibition were either significantly reduced or non-existent. Yeast antagonists generally produced lower levels of growth inhibition than the bacteria but a Williopsis mrakii isolate gave 100% inhibition of three of the five sapstain fungi. Production of inhibitory VOCs was highly dependent on the specific antagonist as well as its growth substrate and all five sapstain fungi showed varying sensitivities to the VOCs produced. Not all fungi were inhibited, growth of O. piliferum and A. pullulans being stimulated by the VOCs from antagonists but only when grown under low nutrient conditions. In some instances, where growth was only slightly reduced, the level of pigmentation of the sapstain colony was significantly reduced compared with corresponding controls. The implications of this work for the biological control of sapstain fungi are discussed.     

Decolourization of Municipal Effluent and Sludge by Pleurotus sajor-caju and Pleurotus ostreatus

Summary The Americana Municipal Treatment Station, S?o Paulo, Brazil, manages 400 l of effluent s⁻¹, from domestic and textile origin, which produces an average of 20 t of sludge per day. The decolourization of the effluent and sludge by three strains of Pleurotus (Pleurotus sajor-caju F2, F6 and Pleurotus ostreatus) was evaluated. The strains of P. sajor-caju F2 and F6 were able to decolourize the sludge, while P. ostreatus was less efficient. Detoxification was appraised with three bioassays comprising the cnidarian Hydra attenuata, the alga Selenastrum capricornutum and lettuce seeds. After exposure to fungi, effluent toxicity decreased but not that of its sludge. Strain P. sajor-caju F6 presented signs of toxicity shown by electron microscopy in the presence of the effluent. The three strains produced high amounts of manganese-peroxidase (Mn–P) and laccase in the presence of the sludge. Although P. ostreatus produced large amount of Mn–P and laccase enzymes, these enzymes did not result in decolourization of the sludge, suggesting that other factors are likely to be involved. Carbon content decreased only in the treatment with P. ostreatus.     

Isolation and characterization of bacterial strains that have high ability to degrade 1,4-dioxane as a sole carbon and energy source

Four novel metabolic 1,4-dioxane degrading bacteria possessing high ability to degrade 1,4-dioxane (designated strains D1, D6, D11 and D17) were isolated from soil in the drainage area of a chemical factory. Strains D6, D11 and D17 were allocated to Gram-positive actinomycetes, similar to previously reported metabolic 1,4-dioxane degrading bacteria, whereas strain D1 was allocated to Gram-negative Afipia sp. The isolated strains could utilize a variety of carbon sources, including cyclic ethers, especially those with carbons at position 2 that were modified with methyl- or carbonyl-groups. The cell yields on 1,4-dioxane were relatively low (0.179–0.223 mg-protein (mg-1,4-dioxane)^?1), which was likely due to requiring energy for C–O bond fission. The isolated strains showed 2.6–13 times higher specific 1,4-dioxane degradation rates (0.052–0.263 mg-1,4-dioxane (mg-protein)^?1 h^?1) and 2.3–7.8 fold lower half saturation constants (20.6–69.8 mg L^?1) than the most effective 1,4-dioxane degrading bacterium reported to date, Pseudonocardia dioxanivorans CB1190, suggesting high activity and affinity toward 1,4-dioxane degradation. Strains D1 and D6 possessed inducible 1,4-dioxane degrading enzymes, whereas strains D11 and D17 possessed constitutive ones. 1,4-Dioxane degradation (100 mg L^?1) by Afipia sp. D1 was not affected by the co-existence of up to 3,000 mg L^?1 of ethylene glycol. The effects of initial pH, incubation temperature and NaCl concentration on 1,4-dioxane degradation by the four strains revealed that they could degrade 1,4-dioxane under a relatively wide range of conditions, suggesting that they have a certain adaptability and applicability for industrial wastewater treatment.     

Mycotoxigenic fungi contaminating wheat; toxicity of different Alternaria compacta strains

We studied mycotoxigenic fungi contaminating stored wheat grain, measured the toxins they secreted, and assessed their harmfulness. We focused on one common genus Alternaria, and chose 19 isolates representing A. compacta to study how different strains differed in their mycotoxin secretion and toxicity. Toxicity was assessed in a bioassay with a model bacteria Bacillus subtilis. All 19 A. compacta strains secreted toxins. Both the mycotoxin pattern and the fungal toxicity differed between the A. compacta stains. It seemed that some other toxins than alternariols or altenue acted as the main virulence factors of A. compacta against B. subtilis. We suggest that the most commonly studied mycotoxins do not necessarily indicate the toxicity of the fungi. The high variation in the amounts and toxins that different Alternaria species and strains secrete pose a challenge to the food supply chain.     

Novel benzimidazol-2-ylidene carbene precursors and their silver(I) complexes: Potential antimicrobial agents

Novel benzimidazolium salts were synthesized as N-heterocyclic carbene (NHC) precursors, these NHC precursors were metallated with Ag₂O in dichloromethane at room temperature to give novel silver(I)–NHC complexes. Structures of these benzimidazolium salts and silver(I)–NHC complexes were characterized on the basis of elemental analysis, ¹H NMR, ¹³C NMR, IR and LC–MS spectroscopic techniques. A series of benzimidazolium salts and silver(I)–NHC complexes were tested against standard bacterial strains: Enterococcus faecalis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and the fungal strains: Candida albicans and Candida tropicalis. The results showed that benzimidazolium salts inhibited the growth of all bacteria and fungi strains and all silver(I)–NHC complexes performed good activities against different microorganisms.     

Investigations on ultraviolet light and nitrous acid induced mutations of halotolerant bacterial strains for the treatment of tannery soak liquor

The objective of this research work is to study the effect of physical and chemical mutagenesis on biological treatment of tannery saline wastewater (soak liquor) employing halotolerant bacterial strains. Four halotolerant bacterial strains isolated from saline sources were used. The strains were identified as Pseudomonas aeruginosa, Bacillus flexus, Exiguobacterium homiense and Staphylococcus aureus, respectively. The isolates were found to grow well in medium containing 0–10% NaCl. At high saline concentration (>5%), the identified strains and their mixed consortia showed a low degrading efficiency of soak liquor (35–45%). UV light and nitrous acid mutagenesis were performed over the strains and the mutated strains were employed for degradation of soak liquor at high salinity level (6% by wt). Comparison of Chemical Oxygen Demand (COD) removal rates for both pure mutant isolates and mixed mutated consortia showed that nitrous acid mutagenesis resulted in degradation of 71% COD removal. Ultraviolet (UV) mutagenesis has no effect on degradation effectiveness. Biomass sludge (Mixed Liquor Volatile Suspended Solids) growth was also found to be high in nitrous acid treated strains.     

Variation and distribution of esterase isozymes inHeteranthelium andTaeniatherum of the tribe triticeae,gramineae

Esterase isozyme variations ofHeteranthelium piliferum, Taeniatherum asperum andT. crinitum collected in Iraq, Turkey and Iran were analysed by gel isoelectric focusing. InH. piliferum two types of esterase zymogram, H1 and H2, were found. It was demonstrated that a pair of allelic genes, which is shown in the heterozygotes, controls the difference between type H1 and H2. Two types of esterase zymogram, T1 and T2, were observed in two species ofTaeniatherum. The majority of strains having type H1 ofH. piliferum and type T1 ofT. crinitum was distributed in the highly elevated plateau of the Anatolian and Iranian highlands, while strains with H2 and T2 of these two species were found in the western foot-hills of the Zagros Mountains, the Tigris basin of Mesopotamia and the central Anatolian plateau.     

Identification, phylogenetic analysis and characterization of obligate halophilic fungi isolated from a man-made solar saltern in Phetchaburi province, Thailand

A total of 17 species from 43 isolates were obtained through serial dilutions of soil samples isolated from one of the man-made solar salterns located in Ban Laem district of Phetchaburi province, Thailand. Soil analysis of the sample revealed high salinity and moisture content, slight alkalinity and low amounts of nitrogen, total organic carbon and organic matter in the habitat. Morphological analysis was performed on all isolates, and molecular identification and phylogenetic analysis were carried out only on the halophilic fungi isolated. Six halophilic fungi, belonging to four species, were identified among the isolates, including five strains of Aspergillus genus [Aspergillus flavus, A. gracilis, A. penicillioides (2 strains) and A. restrictus]. One species was found to be a yeast, namely, Sterigmatomyces halophilus, which was the most frequent isolate found among the halophilic fungi. All other isolates were halotolerant fungi. Characterization of the halophilic fungal isolates showed that they were best adapted to conditions of 10–15 % NaCl (w/v), slight alkalinity (pH 7.0–7.5) and a temperature range of 30–35 °C.     

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.     

Fungal bio-treatment of spruce wood with Trametes versicolor for pitch control: influence on extractive contents, pulping process parameters, paper quality and effluent toxicity

Lipophilic low molar-mass constituents in wood chips for the paper industry result in low quality pulp, pitch deposition, and effluent toxicity. New biotechnological solutions such as fungal pre-treatment of wood chips can reduce pitch problems. This laboratory-scale study focuses on the potential and limitations of a fungal bio-treatment of Norway spruce chips with the white-rot fungus Trametes versicolor. Different fungal treatment conditions were compared. A 4-week fungal treatment reduced the concentration of resin acids and triglycerides by 40% and 100%, respectively, but neither lowered the energy requirements of the TMP process nor significantly affected the morphological fiber characteristics and the physical pulp properties. The pre-treatment led to slightly poorer optical properties. The Trametes versicolor fungal treatment contributed to a less toxic effluent and improved the biodegradability. A treatment of 2-3 weeks appears optimal.     

Chaetoglobosins and azaphilones produced by Canadian strains of Chaetomium globosum isolated from the indoor environment

Chaetomium globosum is one of the most common species of fungi found growing on damp building materials in North America and Europe. At doses that could be experienced in a building with some mould damage, exposure to metabolites from other fungi results in inflammatory changes in vivo and in vitro. This research requires knowledge of the dominant toxins produced by fungal strains from the built environment and characterization of pure compounds for toxicity testing. We examined 25 strains of C. globosum isolated from the built environment in Canada. In varying amounts, these strains primarily produced chaetoglobosin A, C and F, chaetomugilin D, and chaetoviridin A. Spectroscopic data of the major isolated compounds are provided. Previous studies reported a number of metabolites from this species that we did not find. However, this appears to be due to misidentifications of the fungi they examined as well as problems with the analytical methods used. In addition, our data support the use of metabolite profiles for resolving the taxonomy of some economically important Chaetomium species.     

Development of a microbial consortium for dairy wastewater treatment

The wastewater from the dairy industries usually contains high concentrations of contaminants and, since the volume generated is also high, the total contaminant load is very significant. Among the available options for treatment, biological degradation looks like the most promising one. Furthermore, the supplementation of the native microbial populations with external microorganisms with high specific degradation rates (bio-augmentation) has demonstrated to improve the performance of treatment. The main objective of this research was to select a combination of bacteria to improve the aerobic treatment of dairy processing wastewater. For this purpose, eleven fat/protein-degrading microorganisms belonging to the genera Bacillus, Serratia, Lactococcus, Enterococcus, Stenotrophomonas, Klebsiella and Escherichia, were evaluated as potential degrading bacteria using a Plackett-Burman design. Assays were carried out to select the strains that most significantly influenced the degradation of wastewater and biomass yield, in terms of COD removal. A simulated dairy industry effluent was used as culture medium. Four strains were selected as potential members of the microbial consortium: Lactococcus garvieae, Bacillus thuringiensis, Escherichia coli and Stenotrophomonas sp. The optimal operation temperature and pH range of the selected consortium were 32°C and 6 ～ 8, respectively. The degradation percentages reached with the selected consortium were 80.67 and 83.44% at 24 and 48 h, respectively. The selected consortium significantly improved the degradation of the dairy wastewater, and the degradation degree achieved by this consortium was higher than by using the strains individually.     

Polysaccharide-Degrading Activity in Marine and Terrestrial Strains of Mycelial Fungi

The activity of extracellular polysaccharide-degrading enzymes and glycosidases from mycelial fungi towards various carbohydrates and carbohydrate derivatives from plant and algal cell walls has been screened. Twenty-three strains of mycelial fungi isolated from the marine sediment and dung were grown by submerged cultivation on a plant-based substrate (a by-product of the grain processing industry) for previous screening for their biomass and protein productivity. Molecular identification allowed for the assignment of marine fungal strains to the following species: Sirastachys phyllophila, Ochroconis mirabilis, Pseudallescheria boydii, Pseudallescheria ellipsoidea, Beauveria felina, Scopulariopsis brevicaulis, Cladosporium sp., and Trichoderma sp. The terrestrial strains belonged to the species Thermomyces thermophilus, Thermomyces dupontii, Thermomyces lanuginosus, Fusarium avenaceum, Mycothermus thermophilum, and Thermothelomyces thermophila. Seven strains of thermophilic terrestrial fungal species T. thermophila, T. thermophilus, T. dupontii and M. thermophilus and two marine fungal strains of S. brevicaulis and Beauveria felina exhibited the highest protein yields and a wide range of polysaccharide-degrading activity when the cultures were cultivated at 22–25°C. The cellulolytic thermophilic strain M. thermophilus 55 isolated from dung demonstrated unusual specificity, most intensive increase of mycelial biomass, and high activity towards algal polysaccharides after seven days of cultivation. The specific activity of laminarinase was one order of magnitude higher than in the marine strains and amounted to 1180 U/mg, and the alginate lyase, carrageenase, polymannuronate lyase, agarase, and fucoidanase activity levels (from 208 to 500 U/mg) were also higher than in all marine strains. All active polysaccharide-degrading strains of thermophilic terrestrial and marine fungi identified in the present study are of considerable interest, as the potential of these fungi for polysaccharide degradation can be applied in the transformation of various agricultural and maricultural waste of plant origin and in the modification of carbohydrate-containing substances in structural research and biotechnology.     

Optimization of triazo Acid Black 210 dye degradation by Providencia sp. SRS82 and elucidation of degradation pathway

The current work is aimed to evaluate the degradation of triazo textile dye Acid Black 210 (AB210) by Providencia sp. SRS82 that degrade 100 mg/L dye within 90 min under optimum conditions and was also found tolerant to as high as 2000 ppm of dye AB210. Optimum conditions for decolourization and degradation of AB210 with the isolate were viz. temperature 30 °C, pH 8, NaCl concentration 2.5% (w/v) and initial cell load of 8 × 10⁸ cells/mL under static condition. Induction of intracellular and extracellular lignin peroxidase, intracellular laccase and tyrosinase, azoreductase, and DCIP reductase indicated their contribution in the biodegradation of AB210. The products obtained from Providencia sp. SRS82 degradation was monitored through UV–Vis spectrophotometer and were characterized by FTIR, HPTLC, HPLC, GC/MS and LCMS. The proposed metabolic pathway for the biodegradation of AB210 is elucidated for the first time, which showed production of 4 molecules of benzene, one of naphthalene and 4-aminophenyl-N-(4-amino phenyl) benzene sulphonamide. Microbial toxicity and cytotoxicity studies revealed the comparatively less toxic nature of metabolites generated after degradation of AB210. Providencia sp. SRS82 was found competent to degrade actual effluent and diverse dyes that could be present in textile industry effluent showing usefulness of the organism for possible commercial application.     

Characterization of new <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains from Iran,based on cytocidal and insecticidal activity,proteomic analysis and gene content

Characterization of new Bacillus thuringiensis strains is a valuable tool to discover novel insecticidal toxins and to manage resistance problems. In this study, seven Iranian Bt strains were selected according to their toxicity against Plodia interpunctella, to be thoroughly characterized based on their toxicity, protein profiling, proteomic analysis, gene content and β-exotoxin production. The toxicity was assessed by insect bioassays and cell viability assays (a less cost, time and material consuming technique), using four lepidopteran pests and four lepidopteran cell lines from Trichoplusia ni (Hi5), Helicoverpa zea (HzGUT), Spodoptera exigua (UCR-SE) and Spodoptera frugiperda (Sf21). The selected Bt strains showed similar protein electrophoretic profiles, but differed in toxicity. LC–MS/MS analysis of solubilized crystal proteins and gene content analyses (PCR screening) were compared and correlated with the toxicity results. Based on our data, three Bt strains could be considered as candidates for development of future bioinsecticides.     

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.