Biodegradation of pentachlorophenol by white rot fungi under ligninolytic and nonligninolytic conditions

The roles of lignin peroxidase, manganese peroxidase, and laccase were investigated in the biodegradation of pentachlorophenol (PCP) by several white rot fungi. The disappearance of pentachlorophenol from cultures of wild type strains,P. chrysosporium, Trametes sp. andPleurotus sp., was observed. The activities of manganese peroxidase and laccase were detected inTiametes sp. andPleurotus sp. cultures. However, the activities of ligninolytic enzymes were not detected inP. chrysosporium cultures. Therefore, our results showed that PCP was degraded under ligninolytic as well as nonligninolytic conditions. Indicating that lignin peroxidase, manganese peroxidase, and laccase are not essential in the biodegradation of PCP by white rot fungi.     

Wet sand cultures to screen filamentous fungi for the biotransformation of polycyclic aromatic hydrocarbons

The biodegradation of phenanthrene and benzo[a]pyrene was assayed in liquid and wet sand cultures in the presence of five filamentous fungi. In the controls, 85% volatilisation of phenanthrene occurred within 28 days in liquid cultures while it was only 50% in wet sand. In the later system, remaining phenanthrene and benzo[a]pyrene amounted to 6–51 and 53–92% of their initial levels, respectively, according to the strains. Then, wet sand used as a screening tool evidenced Trametes versicolor and Cunninghamella elegans as the most efficient polycyclic aromatic hydrocarbons degraders among ten strains. © Rapid Science Ltd. 1998     

一种快速提取丝状真菌染色体DNA的方法

介绍了一种适用于丝状真菌染色体DNA大片段的快速提取方法,该方法以(100mM Tris,100mM NaGl,50mM EDTA-Na2 2%SDS,pH值9.0)为提取液,经石英砂研磨破壁.应用该方法成功地提取了粗糙脉胞菌(Neurospora crassa)、米曲霉(Aspergillus oryzae)、产黄青霉(Penicillium chrysogenum)和头孢霉菌(Cep- halosporium sp.)等4种不同丝状真菌的染色体DNA大片段,且所提DNA片段均大于20kb,可直接用于限制性酶切、PCR等分子生物学研究.     

Use of coimmobilized biological systems to degrade toxic organic compounds

The concept of coimmobilizing cell mass (and/or enzyme) and adsorbent in a hydrogel matrix for biodegradation of toxic organic chemicals was introduced. Under defined experimental conditions, the coimmobilized system using activated carbon and Phanerochaete chrysosporium was compared with nonimmobilized systems for the degradation of pentachlorophenol (PCP). It was demonstrated that the coimmobilized system degraded PCP more effectively than the nonimmobilized system. A solid substrate included in the coimmobilized system could support the biodegradation. Isolation of the degrading agents from a model interrupting microorganism by the coimmobilized capsule membrane reduced the interference on the biodegradation. In simulated contaminated soil extract and sand, the coimmobilized system also exhibited higher degradative ability and stability than the nonimmobilized systems.     

FTA-DNA直接提取法在病原真菌分子鉴定中的应用

目的建立并评价FTA-DNA直接提取法在病原真菌分子鉴定中的应用。方法采用whatman FTA-DNA直接提取法从25个不同种属的45株培养的菌株和6例临床标本中提取病原真菌DNA,用于病原真菌的测序鉴定。配制不同浓度的孢子悬液探索该方法的检测限和安全性。结果 45株菌株扩增后均能得到1条清晰的DNA扩增片段,并成功测序。应用该方法亦成功从腹水、胸水、口腔拭子、宫颈拭子来源的临床标本中直接提取DNA并成功鉴定病原真菌。该DNA提取方法联合降落PCR能检测到1.0×103个cell/mL的孢子悬液,1.0×104个cell/mL及以下浓度的孢子悬液可以被FTA卡完全灭活。结论 FTA-DNA直接提取法可快速有效地从培养的菌株及部分临床标本中提取并保存病原真菌DNA,用于病原真菌的测序鉴定。     

Effect of adsorbents on degradation of toxic organic compounds by coimmobilized systems

The use of coimmobilized systems for treatment of toxic organic compounds has been proposed. The proposed approach combines the use of adsorbents and laboratory identified microorganisms immobilized in a protective permeable barrier to achieve a greater degree of control over the remediation process. This study was launched to understand the effect of adsorbents and changes in adsorption on the degradation of toxic compounds by coimmobilized systems. The specific case studied involved the degradation of pentachlorophenol (PCP) by Arthrobacter (ATCC 33790) coimmobilized with powdered activated carbon within calcium alginate capsules.The design parameters studied included adsorbent content and type as well as the effect of solution pH and surfactant concentration on adsorption and biodegradation. It was found that the equilibrium adsorption behavior of PCP was strongly influenced by solution pH and surfactant concentration. A mathematical model was developed that combined the physical processes of mass transfer and adsorption with biological degradation of PCP. The model was used to predict the effect of various parameters on the degradation of PCP. Based on model predictions, the degradation of PCP. Based on model predictions, the degradation of PCP was strongly dependent on variations in adsorbent capacity and affinity for this contaminant.     

A high-throughput chemical screen for resistance to Pseudomonas syringae in Arabidopsis

The study of plant pathogenesis and the development of effective treatments to protect plants from diseases could be greatly facilitated by a high-throughput pathosystem to evaluate small-molecule libraries for inhibitors of pathogen virulence. The interaction between the Gram-negative bacterium Pseudomonas syringae and Arabidopsis thaliana is a model for plant pathogenesis. However, a robust high-throughput assay to score the outcome of this interaction is currently lacking. We demonstrate that Arabidopsis seedlings incubated with P. syringae in liquid culture display a macroscopically visible 'bleaching' symptom within 5 days of infection. Bleaching is associated with a loss of chlorophyll from cotyledonary tissues, and is correlated with bacterial virulence. Gene-for-gene resistance is absent in the liquid environment, possibly because of the suppression of the hypersensitive response under these conditions. Importantly, bleaching can be prevented by treating seedlings with known inducers of plant defence, such as salicylic acid (SA) or a basal defence-inducing peptide of bacterial flagellin (flg22) prior to inoculation. Based on these observations, we have devised a high-throughput liquid assay using standard 96-well plates to investigate the P. syringae-Arabidopsis interaction. An initial screen of small molecules active on Arabidopsis revealed a family of sulfanilamide compounds that afford protection against the bleaching symptom. The most active compound, sulfamethoxazole, also reduced in planta bacterial growth when applied to mature soil-grown plants. The whole-organism liquid assay provides a novel approach to probe chemical libraries in a high-throughput manner for compounds that reduce bacterial virulence in plants.     

Distribution of acetyl esterase in wood-rotting fungi

The distribution of acetyl esterase was studied in 30 strains of wood-rotting fungi. A screening test on agar plates using glucose β-d-pentaacetate as a substrate indicated that all tested fungi produced acetyl esterase to form a clear zone on the culture. All fungi also showed positive responses in an agar test using carboxymethyl cellulose acetate. Enzyme assay showed that extracellular acetylxylan esterase activity was present in the filtrates of wood-meal culture of all these fungi. The ratio of fungal acetylxylan esterase activity to 4-nitrophenyl acetyl esterase activity were higher than that of porcine liver esterase, indicating that fungal esterases have high affinity for acetylated carbohydrates. Acetyl esterase is suggested to be distributed widely in wood-rotting fungi for degradation of native acetylated hemicelluloses.     

A screening method for detecting iron reducing wood-rot fungi

A plate assay using the Fe(II) selective dye, ferrozine, for detecting wood-rot fungi with Fe(III) reductive abilities, was developed. The assay is fast, simple and, in most cases, more sensitive than the corresponding liquid medium test. The brown rot fungi, Gloeophyllum trabeum and Laetiporeus sulphureus, displayed higher iron reductive capabilities than white rot fungi, Trametes versicolor, Ganoderma australe and Ceriporiopsis subvermispora.     

A PCR-based method for detecting the mycelia of stipitate hydnoid fungi in soil

To reduce the reliance on sporocarp records for conservation efforts, information on the below-ground distribution of specific fungal species, such as stipitate hydnoid fungi, is required. Species-specific primers were developed within the internal transcribed spacer (ITS1 and ITS2) regions for 12 hydnoid fungal species including Bankera fuligineoalba, Hydnellum aurantiacum, H. caeruleum, H. concrescens, H. ferrugineum, H. peckii, Phellodon confluens, P. melaleucus, P. niger, P. tomentosus, Sarcodon glaucopus and S. squamosus. The specificity of the primer pairs was tested using BLAST searches and PCR amplifications. All primers amplified DNA only of the target species with the exception of those designed for P. melaleucus. In order to assess the ability of the primers to detect DNA from mycelium in soil, DNA extracted from soil samples taken from around solitary H. peckii sporocarps was amplified with the H. peckii primer 1peck and ITS2. H. peckii DNA was detected in 70% of all soil samples and up to 40 cm away from the base of individual sporocarps. The development of these species-specific primers provides a below-ground alternative for monitoring the distribution of these rare fungi.     

A rapid and simple method for DNA extraction from yeasts and fungi isolated from Agave fourcroydes

A simple and easy protocol for extracting high-quality DNA from different yeast and filamentous fungal species is described. This method involves two important steps: first, the disruption of cell walls by mechanical means and freezing; and second, the extraction, isolation, and precipitation of genomic DNA. The absorbance ratios (A₂₆₀/A₂₈₀) obtained ranged from 1.6 to 2.0. The main objective of this procedure is to extract pure DNA from yeast and filamentous fungi, including those with high contents of proteins, polysaccharides, and other complex compounds in their cell walls. The yield and quality of the DNAs obtained were suitable for micro/minisatellite primer-polymerase chain reaction (MSP-PCR) fingerprinting as well as for the sequence of the D1/D2 domain of the 26S rDNA.     

Two-liquid-phase bioreactors for enhanced degradation of hydrophobic/toxic compounds

Two-liquid-phase culture systems involve the addition of a water-immiscible, biocompatible and non-biodegradable solvent to enhance a biocatalytic process. Two-liquid-phase bioreactors have been used since the mid-seventies for the microbial and enzymatic bioconversion of hydrophobic/toxic substrates into products of commercial interest. The increasing popularity of bioremediation technologies suggests a new area of application for this type of bioreactor. The toxicity and the limited bioavailability of many pollutants are important obstacles that must first be overcome in order to improve biodegradation processes. Two-liquid-phase bioreactors have the potential to resolve both limitations of biotreatment technologies by the enhancement of the mass-transfer rate of compounds with low bioavailability, and by the controlled delivery of apolar toxic compounds. This technology can also be useful in accelerating the enrichment of microorganisms degrading problematic pollutants. In this paper, we discuss the application of two-liquid-phase bioreactors to enhance the biodegradation of toxic/poorly bioavailable contaminants. Important microbial mechanisms involved in this type of system are described. Uptake of the substrates can be achieved by microorganisms freely dispersed in the aqueous phase and/or bound at the interface between the aqueous and the immiscible phases. Production of surface-active compounds and adhesion abilities are microbial features involved in the process. General guidelines for the design of two-liquid-phase bioreactors for biodegradation purposes are presented. Solvent selection should be established on specific criteria, which depend on the characteristics of target compound(s) and the microorganism(s) implicated in the biodegradation process. The central importance of maximizing the interfacial surface area is highlighted. The potential of this approach as an alternative to current biotreatment technologies is also discussed.     

Application of microscopic fungi isolated from polluted industrial areas for polycyclic aromatic hydrocarbons and pentachlorophenol reduction

The growth abilities of fifteen fungal strains isolated fromcontaminated areas, in the presence of xenobiotics compounds mixture (overworked cuttingfluid, crude and waste oil) were examined. Strains with the richest growth were chosen for anthracene, phenanthrene and pentachlorophenol biodegradation in Sabouraudmedium (with initial xenobiotic concentration 250 mg/l in cultures with polycyclicaromatic hydrocarbons and 10 mg/l for the chlorinated substrate). Strains IM 1063and IM 6325 were able to attack phenanthrene forming its derivative 9-phenanthrenolwith the yields 5.22 mg/l and 2.82 mg/l, respectively. Strain IM 1063 and IM 6325 transformed pentachlorophenol to an intermediatecompound – pentachloromethoxybenzene. Final content of pentachloromethoxybenzene reached 3.46 mg/l and3.2 mg/l, respectively. Strain IM 6203 (contrary to other strains) released an intermediateproduct of pentachlorophenol metabolism – 2,3,5,6-tetrachlorohydroquinone(8.73 mg/l substrate remaining and 1.2 mg/l 2,3,5,6-tetrachlorohydroquinone forming).The IM 6203 strain was identified as Mucor ramosissimus. The chlorinatedpesticide degradation by M. ramossimus was improved significantly on a medium with overworked oil. Only 8.3% of pentachlorophenol and 4.3% of 2,3,5,6-tetrachlorohydroquinone in relation to the introduced substrate (10 mg/l) were found, after7 days of incubation. The growth of M. ramosissimus on medium with overworked oil in pentachlorophenol presence was associated with oil emulgation,which enhanced fungal growth and the pesticide degradation.     

A simple and rapid method to screen for mutant mice lacking D-amino-acid oxidase activity

A simple and rapid method to screen for mutant mice (Mus musculus) lacking D-amino-acid oxidase activity has been devised. Mice were given water containing small amounts (0.02%) of either D-methionine or D-phenylalanine. Urinary levels of the D-amino acid were examined using thin-layer chromatography. Some mice excreted substantial amounts of the D-amino acid through the urine. None of them had detectable D-amino-acid oxidase activity.     

A rapid method for harvesting and immobilization of oleaginous microalgae using pellet-forming filamentous fungi and the application in phytoremediation of secondary effluent

A rapid method for harvesting and immobilization of oleaginous microalgae using pellet-forming filamentous fungi was developed. The suitable conditions for pellet formation by filamentous fungi were determined. Among the strains tested, Trichoderma reesei QM 9414 showed superior pellet forming ability. Its pellets were used to harvest oleaginous microalga Scenedesmus sp. With increasing volume ratio of fungal pellets to microalgae culture up to 1:2, >94% of microalgal cells were rapidly harvested within 10 min. The ratio of fungal pellets could manipulate both harvesting time and initial concentration of microalgal cells in the pellets. The microalgae–fungal pellets were successfully used as immobilized cells for effective phytoremediation of secondary effluent from seafood processing plants under nonsterile condition. The chemical oxygen demand, total nitrogen, and total phosphorus removal were >74%, >44%, and >93%, respectively. The scanning electron microscopy showed that the microalgal cells were not only entrapped in the pellets but also got attached to the fungal hyphae with sticky exopolysaccharides, possibly secreted by the fungi. The extracted lipids from the pellets were mainly composed of C16–C18 (>83%) with their suitability as biodiesel feedstocks. This study has shown the promising strategy to rapidly harvest and immobilize microalgal cells and the possible application in phytoremediation of industrial effluent.     

A rapid method for assessing the resistance of polyurethanes to biodeterioration

A rapid soil burial method for assessing the susceptibility of polyurethanes to biodeterioration was developed. The time of the test was reduced by prestressing the polyurethanes. The degree of deterioration was measured by following changes in the appearance of the polyurethanes and in selected physical properties. It was found that pre-stressing produced significant reductions in the tensile strength of a known susceptible polyurethane after burial in soil for 2 weeks. The reduction was greater than that found with unstressed polyurethanes buried for 26 weeks in active soil. Changes in tensile strength were less after burial for 4 weeks in sterile soil than after burial in active soil for the same period. The results suggest that deterioration of polyurethane during soil burial is a result of both chemical and microbial action.     

A rapid method to screen degradation ability in chlorophenoxyalkanoic acid herbicide-degrading bacteria

AIMS: An agar medium containing a range of related chlorophenoxyalkanoic acid herbicides, 2,4-dichlorophenoxyacetic acid (2,4-D), 2-methyl-4-chlorophenoxyacetic acid (MCPA), racemic mecoprop, (R)-mecoprop and racemic 2,4-DP (2-(2,4-dichlorophenoxy) propionic acid) was developed to assess the catabolic activity of a range of degradative strains. METHODS AND RESULTS: The medium was previously developed containing 2,4-D as a carbon source to visualise degradation by the production of dark violet bacterial colonies. Strains isolated on mecoprop were able to degrade 2,4-D, MCPA, racemic mecoprop, (R)-mecoprop and racemic 2,4-DP, whereas the 2,4-D-enriched strains were limited to 2,4-D and MCPA as carbon sources. Sphingomonas sp. TFD44 solely degraded the dichlorinated compounds, 2,4-D, racemic 2,4-DP and 2,4-DB (2,4-dichlorophenoxybutyric acid). However, Sphingomonas sp. AW5, originally isolated on 2,4,5-T, was the only strain to degrade the phenoxybutyric compound MCPB (4-chloro-2-methylphenoxybutyric acid). CONCLUSION: This medium has proved to be a very effective and rapid method for screening herbicide degradation by bacterial strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This method reduces the problem of assessing the biodegradability of this family of compounds to an achievable level.