Fungal inoculum properties: extracellular enzyme expression and pentachlorophenol removal in highly contaminated field soils

This study was conducted to improve the pentachlorophenol (PCP) bioremediation ability of white-rot fungi in highly contaminated field soils by manipulating bioaugmentation variables. These were the dry weight percentage of fungal inoculum addition (31-175 g kg(-1)), PCP concentration (100-2137 mg kg(-1) PCP), fungal inoculum formulation, and time (1-7 wk). Five fungal isolates were used: the New Zealand isolates Trametes versicolor (L.: Fr.) HR131 and Trametes sp. HR577; the North American isolates Phanerochaete chrysosporium Burds. (two isolates) and Phanerochaete sordida (Karst.) Erikss. & Ryv. Pentachlorophenol removal, manganese peroxidase, and laccase activity, and the formation of chloroanisoles in the contaminated field soils were measured. The majority of PCP removed by the Trametes isolates was in the first week after bioaugmentation. The maximum PCP removal by the fungi varied from 50 to 65% from a 1065 mg kg(-1) PCP contaminated field soil. Pentachlorophenol was preferentially converted to pentachloroanisole (PCA) by the Phanerochaete isolates (>60%), while 2 to 9% of the PCP removed by two Trametes isolates was converted to PCA. A pH increase was measured following bioaugmentation that was dependent on PCP concentration, fungal inoculum addition, and formulation. This, together with rapid initial PCP removal, possibly changed the bioavailability of the remaining PCP to the fungi and significantly decreased the sequestering of PCP in the contaminated field soils. The research supports the conclusion that New Zealand Trametes spp. can rapidly remove PCP in contaminated field soils. Bioavailability and extractability of PCP in the contaminated field soil may significantly increase after bioaugmentation.     

Growth substrate selection and biodegradation of PCP by New Zealand white-rot fungi

Nine New Zealand native white-rot fungi were studied for their ability to grow and survive on different substrates formulated from bark, wheat straw, sawdust, apple pomace and maize products in order to identify their pentachlorophenol (PCP) biodegradation potential and to select a fungal carrier for bioaugmentation of polluted soils. Isolates were also evaluated to mineralize (14)C-PCP in liquid culture and in soil. The American fungus Phanerochaete chrysosporium outgrew the native fungi on the substrates tested, but the high colonisation did not result in superior PCP dechlorination as measured by chloride release. Whilst Trametes versicolor inocula produced on wheat straw and SCS (sawdust-corn meal-starch-mix) gave the highest chloride release, colonization of these two substrates as measured by biological potential was lower compared to the pomace and pomace-sawdust-mix. Neither lignin peroxidase nor manganese peroxidase production were measured for New Zealand white-rot fungi during the experiments. Laccase was the only enzyme detected. In liquid culture, the mineralisation rate was higher for T. versicolor isolates compared to P. chyrysoporium. Very little to no pentachloroanisole (PCA) was captured in the volatile fraction of T. versicolor isolates, whereas 75% of the volatile fraction of P. chrysosporium consisted of PCA. The soil microcosms studies, using contaminated soil from a timber treatment site, clearly showed that the New Zealand T. versicolor isolates mineralized PCP. Degradation of PCP in non-sterile soil was higher in the presence of white-rot fungi than in soil without white-rot fungus. This demonstrates that viable white-rot fungus is necessary for significant PCP degradation and that T. versicolor isolates showed PCP remediation potential. Wheat straw and SCS could be suitable carriers for New Zealand native T. versicolor isolates for bioremediation of PCP polluted soil sites.     

Effects of pH and cyclodextrins on pentachlorophenol degradation (mineralization) by white-rot fungi

White-rot fungi (WRF) such as Trametes hirsuta completely degrade (mineralize) pentachlorophenol (PCP) and many other organopollutants. This has led to them being used to decontaminate various substrates (e.g. soil) through biorememediation. However, because PCP is a biocide, it can inhibit fungal growth and thereby its own degradation. It was hypothesized that substrate pH might affect PCP degradation, because when the pH is lower than 4.7 (the pKa for PCP) the phenol predominates, while at higher pH the phenate does. These two PCP species differ markedly in physical and biological properties. The effect of cyclodextrins was also investigated since it is known that the inclusion complexes these form with PCP differ in bioavailability and toxicity from non-complexed PCP. Tests were first made in liquid and agar media (where conditions are relatively easy to control), and then in a sawdust, because it is a common target matrix for WRF bioremediation. Results with the liquid and agar media showed that growth in the presence of inhibitory PCP concentrations decreased as the pH decreased, consistent with the phenol being more toxic. Growth in sawdust was less affected by PCP regardless of the pH, presumably because the PCP sorbed to the wood which decreased its bioavailability. Some cyclodextrins markedly decreased the PCP's toxicity in liquid, agar and sawdust media. Rates of PCP mineralization (measured from production of (14)CO(2) from (14)C PCP) in liquid cultures containing 0.5 mgkg(-1) PCP (a sub-inhibitory concentration) were similar from pH 2.5-7.5, indicating that the phenol and the phenate were equally degradable. Degradation of a growth inhibiting concentration on sawdust (1,000 mgkg(-1)) could be increased slightly by lowering the pH below the pKa, this increasing sorption to the wood. Degradation increased more when the pH was raised well above the pKa, presumably due to the phenate being less toxic and more soluble, making it more available to the degradative system. Although some cyclodextrins decreased growth inhibition, they also interfered with degradation. If this interference could be overcome, cyclodextrins could be used to increase the maximal PCP concentration that could be treated by WRF bioremediation.     

Effect of nonylphenol surfactants on fungi following the application of sewage sludge on agricultural soils

The effect of nonylphenol on fungi following the application of contaminated sewage sludge on agricultural soil was studied in laboratory experiments. Nonylphenol bioavailability and adsorption were determined in the soil alone and soil-sludge mixtures. Mixing the soil with sludge made it possible to measure the nonylphenol concentration in the soil solution, which comprised between 6.6 x 10(-6) and 3.8 x 10(-7) M, according to the sludge. We then examined the dose-response relationship between nonylphenol concentration in the culture medium and both biomass production and germination rate of the spores from several strains of filamentous fungi. When applied in this range of concentration, nonylphenol was without noticeable short-term effect on these endpoints. Long-term exposure of fungi to nonylphenol was also assessed. The most intensive effect was a strong stimulation of spore production and germination in Fusarium oxysporum Schlechtendahl. Biomass production by the Fusarium strains also increased. Finally, nonylphenol was shown to induce laccase production in Trametes versicolor. We conclude that the potential of nonylphenol to adversely affect several soil fungi remains low.     

Cost analysis in laccase production

In this paper the cost of producing the enzyme laccase by the white-rot fungus Trametes pubescens under both submerged (SmF) and solid-state fermentation (SSF) conditions was studied. The fungus was cultured using more than 45 culture medium compositions. The cost of production was estimated by analyzing the cost of the culture medium, the cost of equipment and the operating costs. The cost of the culture medium represented, in all cases, the highest contribution to the total cost, while, the cost of equipment was significantly low, representing less than 2% of the total costs. The cultivation under SSF conditions presented a final cost 50-fold lower than the one obtained when culturing under SmF conditions at flask scale. In addition, the laccase production under SSF conditions in tray bioreactors reduced the final cost 4-fold compared to the one obtained under SSF conditions at flask scale, obtaining a final price of 0.04 cent €/U.     

Enzymatic degradation of low-rank coals by a cell-free enzymatic system from Coriolus versicolor

An extracellular fraction from the lignin degrading white-rot fungus, Coriolus versicolor, can biosolubilize leonardite and pretreated lignites in vitro. Leonardite is often found as an overburden to lignite deposits, and is related to humic acid, and is more oxidized than lignites. Extracts from certain fungi such as C. versicolor solubilize leonardite. The biosolubilization activity is at least in part enzymatic in nature. A protein which is at least partly responsible for the solubilization effect has been substantially purified and concentrated by ultrafiltration and ion-exchange chromatography from the broth of an agitated submerged culture of C. versicolor grown in a defined medium. An in vitro coal biosolubilization assay was used to establish the effects of various physical and chemical factors affecting enzyme activity, including temperature and the salt and buffer composition of the reaction mixture. Some initial studies on the mechanism of the enzymatic leonardite biosolubilization reaction are described.     

Effects of tree root-derived substrates and inorganic nutrients on pyrene mineralization in rhizosphere and bulk soil

This study investigated the effects of organic and inorganic nutrients on the microbial degradation of the common soil contaminant pyrene. The material used in this investigation was collected from potted trees that had been growing for over a year in a soil artificially contaminated with polycyclic aromatic hydrocarbons. Soil was removed from the nonroot (bulk) and root (rhizosphere) zones of these pots and used in mineralization studies that tracked microbial degradation of 14C-pyrene. The factors influencing degradation in these zones were then tested by amendment with essential inorganic nutrients or with root-derived materials. As expected, pyrene mineralization was greater in soil removed from the rhizosphere than in bulk soil. The rate of mineralization in rhizosphere soil was inhibited by inorganic nutrient amendment, whereas nutrients stimulated mineralization in the bulk soil. Pyrene mineralization in bulk soil was also increased by the addition of root extracts intended to mimic exudation by living roots. However, amendment with excised fine roots that were allowed to decay over time in soil initially inhibited mineralization. With time, the rate of mineralization increased, eventually exceeding that of unamended bulk soil. Combined, the initial inhibition and subsequent stimulation produced a zero net impact of decaying fine roots on bulk soil mineralization. Our results, in conjunction with known temporal patterns of fine root dynamics in natural systems, support the idea that seasonal variations in nutrient and substrate availability may influence the long-term effect of plants on organic degradation in soil, possibly reducing or negating the beneficial effects of vegetation that are often observed in short-term studies.     

Microbial biomass governs enzyme activity decay during aging of worm-worked substrates through vermicomposting

Vermicomposting is the biooxidation and stabilization of organic matter involving the joint action of earthworms and microorganisms, thereby turning wastes into a valuable soil amendment called vermicompost. Studies have focused on the changes in the type of substrates available before and after vermicomposting, but little is known on how these changes take place, especially those changes related with maturation of vermicompost. This study investigated the effects of aging on the microbiological properties of fresh vermicompost produced from pig slurry by analyzing the substrate after the earthworms had left it. We incubated 16-wk-old vermicompost and sampled it after 15, 30, 45, and 60 d analyzing microbial biomass and activity (assessed as microbial biomass N and basal respiration respectively) and four enzymatic activities (beta-glucosidase, cellulase, protease, and alkaline phosphatase). Aging of vermicompost resulted in decreases of microbial biomass and activity. Three of the four enzymes analyzed also showed decrease. An initial increase followed by a rapid decrease in alkaline phosphatase was also recorded. High and significant correlations between microbial biomass and beta-glucosidase (r = 0.62, P < 0.001), cellulase (r = 0.56, P < 0.01), and protease (r = 0.82, P < 0.001) were found. Results suggest that there may be two steps involved in the aging dynamics of vermicompost with regards to extracellular enzyme activity; the first step was characterized by a decrease in microbial populations, which resulted in a reduction in the synthesis of new enzymes. The second step was the degradation of the pool of remaining enzymes. This dynamic does not seem to be affected by earthworms because similar decaying patterns of microbial biomass and activity were found in substrate where earthworms were present.     

Impact of ectomycorrhizal colonization of hybrid poplar on the remediation of diesel-contaminated soil

Infection by ectomycorrhizal (ECM) fungi may benefit hybrid poplar growing in contaminated soils by providing greater access to water and nutrients and possibly protecting the trees from direct contact with toxic contaminants. The objective of this research was to determine the effect of colonization of the ECM fungus Pisolithus tinctorius (Pers.) Coker & Couch on hybrid poplar fine root production, biomass and N and P uptake when grown in diesel-contaminated soil (5000 mg diesel fuel kg soil(-1)). Commercially available Mycogrow Tree Tabs were the source of inoculum. A minirhizotron camera was used to provide the data necessary for estimating fine root production. Colonization of hybrid poplar roots (P. deltoides x [P. laurifolia x P. nigra] cv. Walker) by P. tinctorius increased total fine root production in diesel-contaminated soil to 56.58 g m(-2) compared to 22.59 g m(-2) in the uncolonized, diesel-contaminated treatment. Hybrid poplar leaf N and P concentrations were significantly greater in the diesel-contaminated/ECM-colonized treatment compared to the diesel-contaminated/uncolonized treatment after 12 wk, while significantly less diesel fuel was recovered from the soil of the uncolonized treatment compared to the colonized treatment. Both planted treatments removed more contaminants from the soil than an unplanted control. Significantly greater concentrations of total petroleum hydrocarbons (TPH) were found sequestered in hybrid poplar root/fungal-sheath complexes from the colonized treatment compared to the roots of the uncolonized treatment. The results of this study indicate that over a 12-wk growth period, ECM colonization of hybrid poplar in diesel-contaminated soils increased fine root production and whole-plant biomass, but inhibited removal of TPH from the soil.     

Depolymerization of polyaromatic hydrocarbons by Penicillium spp. inhabit the phyllosphere of urban ornamental plants

A variety of anthropogenic sources release hazardous polyaromatic hydrocarbons (PAHs) into the phyllosphere which is an excellent niche for diverse fungi, and some of them have PAHs degradation capabilities. Therefore, this research attempted to determine the PAHs (phenanthrene, anthracene, naphthalene, and pyrene) degradation capability of phyllosphere inhabited Penicillium species. The leaf samples were collected from highly polluted urban areas (Panchikawatta, Pettah, Orugodawatta, Maradana, Sapugaskanda, and Colombo Fort) in Sri Lanka to isolate fungal species inhabiting the phyllosphere. Furthermore, their distribution patterns among the leaf tissue layers were studied using bright-field microscopic observations. Moreover, the best PAH degraders were screened out using plate assays and confirmed through High Performance Liquid Chromatography (HPLC) analysis. Further, their enzymatic activities during the PAHs degradation were analyzed. As per the microscopic observations, the highest fungal distribution was in the upper epidermis of the leaves followed by the fungal distribution in the interspaces of palisade mesophyll layers. Out of isolated fungal species, two Penicillium spp. (Penicillium citrinum P₂₃B-91 and Penicillium griseofulvum P₉B - 30) showed the highest PAHs (phenanthrene, anthracene, naphthalene, and pyrene) degradation capabilities. Manganese peroxidase (MnP) enzyme dominated phenanthrene degradation in P. griseofulvum P₉B - 30, which showed the highest phenanthrene degradation ability (61%). In addition, P. citrinum P23B-91 was good at degrading anthracene (88%) and also displayed a higher MnP activity during the anthracene degradation than laccase and lignin peroxidase activities. The discoveries from the toxicity assay during the PAHs degradation processes revealed that the produced byproducts had no toxic effects on the fungal growth cycle and the phyllosphere. Therefore this phyllosphere Penicillium spp. are ideal for the bioremediation of polluted air in urbanized areas.     

Cadmium and zinc in vegetation and litter of a voluntary woodland that has developed on contaminated sediment-derived soil

Vegetation that develops spontaneously on metal-contaminated soils presents an opportunity to evaluate both metal bioavailability and the risks posed to biota. The behavior of Cd and Zn in the species of a spontaneously developed woodland, colonizing a canal embankment, has been investigated. Nitric-acid-extractable metal concentrations in the sediment-derived substrate ranged between 5.0 to 376 mg kg(-1)dry wt. Cd and 83.0 to 784 mg kg(-1)dry wt. Zn. The woodland is dominated by Willow (Salix) species. Salix caprea selectively accumulated Cd in all stem tissues, in contrast to S. viminalis, which regulated tissue Cd content. Both species showed an effective regulation of tissue Zn. Cadmium uptake by S. caprea was correlated with differences in soil pH, while Zn uptake was not. There was no relationship between tissue metal concentrations and soil metal nitric acid-extractable concentrations. Other aspects of ecosystem function appeared unaffected by the elevated Cd flux in S. caprea; leaf litter organisms present represented all major groups and there was no accumulation of organic matter. The woodland represents a potentially sustainable option for remediating a low value site with difficult access that does not involve removal of the contaminated material to a landfill or making a permanent inert cover.     

Bioremediation of cutting fluids contaminated soil by Pleurotus tuber-regium Singer

This study investigated the effect of inoculation of Pleurotus tuber-regium, a Nigerian white rot fungus, period of incubation, different levels of contamination on cutting fluids degradation in contaminated soil over 30, 60, and 90 days. Control for different levels of cutting fluids was also used to compare rates of bioremediation of the contaminant in the soil. At the end of each incubation period, the mycelia-ramified substrate was separated from the soil layer and dried. The soil samples were analyzed for physico-chemical parameters; total petroleum hydrocarbon, lignin content by determining the acid detergent fraction (ADF), heavy metals content of the soil using flame atomic absorption spectrophotometer, and changes in the polyphenol oxidase and peroxidase activities were also determined after 1, 2, and 3 months. P. tuber-regium improved the nutrient status of the soil and increased enzyme activity was recorded. A reduction in the pH and heavy metal contents of the soil at all levels of cutting fluids concentrations was detected. The lignin in the rice straw decreased from 34.50% in the control to 8.06% at 30% cutting fluids concentration after 3 months of incubation. The highest TPH loss of 30.84% was recorded at 20% cutting fluids contamination after 3 months compared to 13.75% at the onset of the experiment. The improvement of the nutrient contents of the soil, bioaccumulation of heavy metals, degradation of TPH, lignin, and increased activity of polyphenol oxidase and peroxidase was due to biodegradation of the cutting fluids.     

S-TE剩余污泥溶解过程中VSS的变化研究

从土壤中筛选出1株能分泌胞外酶、促进污泥溶解的嗜热脂肪芽孢杆菌Bacillus stearothermophilusAT07-1,将AT07-1培养液接种到不同浓度（TSS约为7.5、14.5、20.5 g/L）的剩余污泥中,于65℃进行嗜热菌溶解试验,并与未接种试验进行对比。结果表明,接种嗜热菌AT07-1促进了污泥中悬浮固体的溶解。60h时接种试验VSS的溶解率分别为61.41%、53.79%、41.86%,比不接种试验同期分别提高了27.57%、25.24%、16.26%。试验条件下VSS溶解过程在初始阶段（t=3d）符合一级反应动力学模型,接种后溶解速率常数Kd显著提高,从0.12 d-1、0.08 d-1、0.09 d^-1分别增加到0.27 d^-1、0.24 d^-1、0.13 d^-1。