Frantisek Zadrazil

Influence of cadmium and mercury on activities of ligninolytic enzymes and degradation of polycyclic aromatic hydrocarbons by Pleurotus ostreatus in soil

ABSTRACT The white-rot fungus Pleurotus ostreatus was able to degrade the polycyclic aromatic hydrocarbons (PAHs) benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenzo[a,h]anthracene, and benzo[ghi]perylene in nonsterile soil both in the presence and in the absence of cadmium and mercury. During 15 weeks of incubation, recovery of individual compounds was 16 to 69% in soil without additional metal. While soil microflora contributed mostly to degradation of pyrene (82%) and benzo[a]anthracene (41%), the fungus enhanced the disappearance of less-soluble polycyclic aromatic compounds containing five or six aromatic rings. Although the heavy metals in the soil affected the activity of ligninolytic enzymes produced by the fungus (laccase and Mn-dependent peroxidase), no decrease in PAH degradation was found in soil containing Cd or Hg at 10 to 100 ppm. In the presence of cadmium at 500 ppm in soil, degradation of PAHs by soil microflora was not affected whereas the contribution of fungus was negligible, probably due to the absence of Mn-dependent peroxidase activity. In the presence of Hg at 50 to 100 ppm or Cd at 100 to 500 ppm, the extent of soil colonization by the fungus was limited.

Screening of white-rot fungi for their ability to mineralize polycyclic aromatic hydrocarbons in soil.

Soil samples from an agricultural field contaminated with 10 ppm14C-benz(a)anthracene in glass tubes were brought into contact with cultures of wood-rotting fungi, precultivated on wheat straw substrate. Forty-five strains of white-rot fungi and four brown-rot fungi were tested for their ability to colonize the soil and to mineralize14C-benz(a)anthracene to14CO2 within a 20-week incubation time. Twenty-two white-rot fungi and all brown-rot fungi were unable to colonize the soil. Twenty-three strains of white-rot fungi, all belonging to the genusPleurotus, colonized the soil. During the experiment the noncolonizing fungi and their substrate disintegrated more and more to a nonstructured pulp from which water diffused into the soil. The same phenomenon was observed in the control which contained only straw without fungus and contaminated soil. In samples with colonizing fungi the substrate as well as the mycelia in the soil remained visibly unchanged during the entire experiment. Surprisingly, most samples with fungi not colonizing the soil and the control without fungus liberated between 40 and 58 % of the applied radioactivity as14CO2 whereas the samples with the colonizing fungi respired only 15–25 % as14CO2. This was 3–5 times more14CO2 than that liberated from the control (4.9 %) which contained only contaminated soil without straw and fungus. A similar result was obtained with selected colonizing and noncolonizing fungi and soil contaminated with 10 ppm14C-pyrene. However, in pure culture studies in which14C-pyrene was added to the straw substrate,Pleurotus sp. (P2), as a representative of the colonizing fungi, mineralized 40.3 % of the added radioactivity to14CO2. The noncolonizing fungiDichomitus squalens andFlammulina velutipes liberated only 17.2 or 1.7 %, respectively, as14CO2. These results lead to the hypothesis that the native soil microflora stimulated by the formed products of straw lysis is responsible for high degradation rates found with noncolonizing fungi.

Two-step degradation of pyrene by white-rot fungi and soil microorganisms.

Abstract  The effect of soil microorganisms on mineralization of 14C-labelled pyrene by white-rot fungi in solid-state fermentation was investigated. Two strains of white-rot fungi, Dichomitus squalens and a Pleurotus sp., were tested. The fungi were incubated on milled wheat straw contaminated with [14C]pyrene for 15 weeks. CO2 and 14CO2 liberated from the cultures were determined weekly. To study the effect of soil microorganisms on respiration and [14C]pyrene mineralization in different periods of fungal development, the fungal substrate was covered with soil at different times of incubation (after 0, 1, 3, 5, 7, 9 or 11 weeks). The two fungi showed contrasting ecological behaviour in competition with the soil microflora. Pleurotus sp. was highly resistant to microbial attack and had the ability to penetrate the soil. D. squalens was less competitive and did not colonize the soil. The resistance of the fungus was dependent on the duration of fungal preincubation. Mineralization of [14C]pyrene by mixed cultures of D. squalens and soil microorganisms was higher than by the fungus or the soil microflora alone when soil was added after 3 weeks of incubation or later. With Pleurotus sp., the mineralization of [14C]pyrene was enhanced by the soil microflora irrespective of the time of soil application. With D. squalens, which in pure culture mineralized less [14C]pyrene than did Pleurotus sp., the increase of [14C]pyrene mineralization caused by soil application was higher than with Pleurotus sp.

Degradation of eight highly condensed polycyclic aromatic hydrocarbons by Pleurotus sp. Florida in solid wheat straw substrate

Abstract The degradation of eight unlabeled highly condensed polycyclic aromatic hydrocarbons (PAH) and the mineralization of three 14C-labeled PAH by the white-rot fungus Pleurotus sp. Florida was investigated. Three concentrations containing 50, 250 or 1250 μg each unlabeled PAH/5 g straw were added to sterile sea sand. Selected treatments were added subsequently with 14C-labeled pyrene, benzo[a]anthracene or benzo[a]pyrene. The PAH-loaded sea sand was then mixed into straw substrate and incubated. The disappearance of the unlabeled four-to six-ring PAH: pyrene, benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenz[a,h]anthracene and benzo[ghi]perylene, was determined by high-performance liquid chromatography. After 15 weeks of incubation, the recoveries were less than 25% for initial amounts of 50 μg (controls above 85%). The recoveries of unlabeled PAH increased in the inoculated samples with increasing concentrations applied. No correlation could be determined between the number of condensed rings of the PAH and the recoveries of added PAH. Pleurotus sp. Florida mineralized 53% [14C]pyrene, 25% [14C]benzo[a]anthracene and 39% [14C]benzo[a]pyrene to 14CO2 in the presence of eight unlabeled PAH (50 μg applied) within 15 weeks. During the course of cultivation, Pleurotus sp. Florida degraded more than 40% of the wheat straw substrate. Variation of the initial concentration of PAH did not influence the extent of degradation of the organic matter.

The ability of different plant species to remove polycyclic aromatic hydrocarbons and polychlorinated biphenyls from incubation media

The ability of in vitro cultured cells of black nightshade, wheat, barley, soybean, tomato, mulberry and birch to grow in the presence of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) and to metabolise them was compared. No correlation was found between the resistance of the plants and removal of xenobiotics. Up to 20% of PCBs and over 90% of PAHs were removed by wheat cells from nutrient media in two weeks.

The Effect of Interaction Between White-rot Fungi and Indigenous Microorganisms on Degradation of Polycyclic Aromatic Hydrocarbons in Soil

White-rot fungi applied for soil bioremediation have to compete with indigenous soil microorganisms. The effect of competition on both indigenous soil microflora and white-rot fungi was evaluated with regard to degradation of polycyclic aromatic hydrocarbons (PAH) with different persistence in soil. Sterile and non-sterile soil was artificially contaminated with 14C-labeled PAH consisting of three (anthracene), four (pyrene, benz[a]anthracene) and five fused aromatic rings (benzo[a]pyrene, dibenz[a,h]anthracene). The two fungi tested,Dichomitus squalens and Pleurotus ostreatus, produced similar amounts of ligninolytic enzymes in soil, but PAH mineralization by P. ostreatus was significantly higher. Compared to the indigenous soil microflora, P.ostreatus mineralized 5-ring PAH to a larger extent, while the indigenous microflora was superior in mineralizing 3-ring and 4-ring PAH. In coculture the special capabilities of both soil microflora and P. ostreatus were partly restricted due to antagonistic interactions, but essentially preserved. Thus, soil inoculation with P. ostreatus significantly increased the mineralization of high-molecular-weight PAH, and at the same time reduced the mineralization of anthracene and pyrene. Regarding the mineralization of low-molecular-weight PAH, the stimulation of indigenous soil microorganisms by straw amendment was more efficient than application of white-rot fungi.

Production of ligninolytic exoenzymes and14C-Pyrene mineralization byPleurotus sp. in lignocellulose substrate

Pleurotus sp. was grown in liquid medium and on a solid straw substrate, and activities of laccase and manganese-dependent peroxidase (MnP) were recorded. The activities were the highest in a rich, glucose corn-steep liquid medium. In straw cultures, laccase activity was about ten times lower. Under solid state conditions, MnP production was the highest during days 20–40, when laccase activity already had declined. In straw cultures, mineralization of14C-pyrene was measured as release of14CO2. The highest rates of pyrene mineralization occurred during days 20–45,i.e. the period of high MnP activities, suggesting a role of this enzyme in PAH degradation. Within 60d, 24% of pyrene was mineralized.

BIODEGRADATION OF PAHS IN LONG-TERM CONTAMINATED SOIL CULTIVATED WITH EUROPEAN WHITE BIRCH (BETULA PENDULA) AND RED MULBERRY (MORUS RUBRA) TREE

The ability of birch (Betula pendula) and mulberry (Morus rubra), cultivated either separately or together with perennial ryegrass (Lolium perenne), to enhance the biodegradation of 15 selected polycyclic aromatic hydrocarbons (PAHs) in long-term contaminated soil was studied in a greenhouse experiment. The microecosystems (MESs) were cultivated for either 12 or 18 months at a natural photoperiod. The fact that the soil chosen for cultivation had been contaminated for over 50 years was expected to be the main factor limiting biodegradation. Extracts of both planted and unplanted soil were analyzed using HPLC. After 1 year of cultivation, the overall content of the investigated compounds had declined to 50%. The concentrations of fluoranthene and pyrene, the PAHs originally present in the soil in the highest concentrations (103.5 and 83.3 mg/kg, respectively), had decreased to 28.0 and 18.0 mg/kg, respectively. In addition, other compounds were successfully degraded, including even benzo[a]pyrene. Benzo[ghi]perylene and indeno[1,2,3-cd]pyrene were the only PAHs remaining and were almost entirely undegraded. Because few significant differences were found between the various types of MESs studied, degradation can primarily be attributed to the presence of degrading microorganisms in the soil.

Enhancing the mineralization of [U-14C]dibenzo-p-dioxin in three different soils by addition of organic substrate or inoculation with white-rot fungi

Abstract The potential for aerobic mineralization of [U-14C]dibenzo-p-dioxin (DD) was investigated in samples of three different agricultural soils already contaminated with polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/F) by industrial activities. The influence of amendments, i.e. wheat straw and compost, and of soil treatment by inoculation with lignolytic fungi, grown on wheat straw substrate, was tested. All the soils tested contained an indigenous DD-mineralizing microflora. The soil characterized by the highest organic matter content and the highest content of soil microbial biomass displayed the best DD mineralization of 36.6% within 70 days, compared with the two organic-matter-poor soils with an endogenous DD mineralization of 19.5% and 23.3% respectively. Amendments with compost increased DD mineralization up to 28% in both soils with low organic matter and microbial biomass content, but did not affect mineralization in the organic-matter-rich soil. Addition of wheat straw had no constant influence on DD mineralization in the soils tested. The best DD mineralization resulted from inoculation with lignolytic white-rot fungi (Phanerochaete chrysosporium, Pleurotus sp. Florida, Dichomitus squalens) and with an unidentified lignolytic fungus, which was isolated originally from a long-term PCDD/F-contaminated soil. A mineralization of up to 50% within 70 days was reached by this treatment. The influence of inoculated fungi on mineralization differed between the soils investigated.

Polycyclic aromatic hydrocarbon degradation by white rot fungi

Polycyclic aromatic hydrocarbons (PAH) are ubiquitous environmental pollutants, usually formed by incomplete combustion processes. Many PAH are known to be mutagenic and carcinogenic. In addition, PAH with more than three condensed rings are characterised by microbial recalcitrance.