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

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.

Influence of incubation temperature on activity of ligninolytic enzymes in sterile soil by Pleurotus sp. and Dichomitus squalens.

Solid state straw cultures of the white rot fungi Pleurotus sp. and Dichomitus squalens covered with a layer of sterile soil were incubated at 20 degrees C, 25 degrees C, 30 degrees C, and 34 degrees C. The activities of the extracellular enzymes laccase and manganese peroxidase (MnP) in the soil layers were measured over eight weeks. Pleurotus sp. produced high enzyme activity at low temperatures. Laccase was maximum at 25-30 degrees C and manganese peroxidase at 20 degrees C. D. squalens showed uniformly high levels of manganese peroxidase at 20-30 degrees C whereas at 34 degrees C, MnP was low and laccase showed an atypical time pattern. The discrepancy between low activity of the main enzyme, MnP, and high straw decomposition at 34 degrees C by D. squalens warrants further investigation of the enzyme inventory produced at that temperature.

Screening of Pleurotus ostreatus isolates for their ligninolytic properties during cultivation on natural substrates.

Thirteen basidiospore-derived isolates of Pleurotus ostreatus f6 strain differing in the level of ligninolytic enzyme production and other characteristics (mycelium extension rate, colony morphology) from the parental strain were cultivated on natural substrates. Under these conditions ligninolytic enzyme activity, loss of organic mass, polycyclic aromatic hydrocarbons (PAHs) degradation and colonization of sterile and nonsterile soil were studied. The activity of ligninolytic enzymes was substantially higher in straw than in liquid culture, although the differences between the isolates were less pronounced on this substrate. Some of the isolates showed a very good ability to decompose the lignocellulosic substrate (straw) and a relatively high loss of organic mass was found after 50 days of cultivation in these strains. The original strain f6 and isolates B13 and B26 successfully degraded all seven tested PAH compounds present in experimental soil samples, but the higher or lower ligninolytic enzyme production of isolates tested had no substantial effect on the extent of the degradation. In our screening, six basidiospore-derived isolates growing well in nonsterile soil were found, which could be suitable for the prospective biotechnological exploitation.

Extracellular oxidative enzyme production and PAH removal in soil by exploratory mycelium of white rot fungi.

Selected strains of three species of white rot fungi, Pleurotus ostreatus, Phanerochaete chrysosporium and Trametes versicolor, were grown in sterilized soil from straw inocula. The respective colonization rates and mycelium density values decreased in the above mentioned order. Three- and four-ringed PAHs at 50 ppm inhibited growth of fungi in soil to some extent. The activities of fungal MnP and laccase (units per g dry weight of straw or soil), extracted with 50 mM succinate-lactate buffer (pH 4.5), were 5 to 20-fold higher in straw compared to soil. The enzyme activities per g dry soil in P. ostreatus and T. versicolor were similar, in contrast to P. chrysosporium, where they were extremely low. Compared to the aerated controls, P. ostreatus strains reduced the levels of anthracene, pyrene and phenanthrene by 81-87%, 84-93% and 41-64% within 2 months, respectively. During degradation of anthracene, all P. ostreatus strains accumulated anthraquinone. PAH removal rates in P. chrysosporium and T. versicolor soil cultures were much lower.

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

Soil samples from an agricultural field contaminated with 10 ppm 14C-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 mineralize 14C-benz(a)anthracene to 14CO2 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 genus Pleurotus, colonized the soil. During the experiment the non-colonizing 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 as 14CO2 whereas the samples with the colonizing fungi respired only 15-25% as 14CO2. This was 3-5 times more 14CO2 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 ppm 14C-pyrene. However, in pure culture studies in which 14C-pyrene was added to the straw substrate, Pleurotus sp. (P2), as a representative of the colonizing fungi, mineralized 40.3% of the added radioactivity to 14CO2. The noncolonizing fungi Dichomitus squalens and Flammulina velutipes liberated only 17.2 or 1.7%, respectively, as 14CO2. 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.

Comparison of phenanthrene and pyrene degradation by different wood-decaying fungi.

The degradation of phenanthrene and pyrene was investigated by using five different wood-decaying fungi. After 63 days of incubation in liquid culture, 13.8 and 4.3% of the [ring U-14C]phenantherene and 2.4 and 1.4% of the [4,5,9,10-14C]pyrene were mineralized by Trametes versicolor and Kuehneromyces mutabilis, respectively. No 14CO2 evolution was detected in either [14C]phenanthrene or [14C]pyrene liquid cultures of Flammulina velutipes, Laetiporus sulphureus, and Agrocybe aegerita. Cultivation in straw cultures demonstrated that, in addition to T. versicolor (15.5%) and K. mutabilis (5.0%), L. sulphureus (10.7%) and A. aegerita (3.7%) were also capable of mineralizing phenanthrene in a period of 63 days. Additionally, K. mutabilis (6.7%), L. sulphureus (4.3%), and A. aegerita (3.3%) mineralized [14C]pyrene in straw cultures. The highest mineralization of [14C] pyrene was detected in straw cultures of T. versicolor (34.1%), which suggested that mineralization of both compounds by fungi may be independent of the number of aromatic rings. Phenanthrene and pyrene metabolites were purified by high-performance liquid chromatography and identified by UV absorption, mass, and 1H nuclear magnetic resonance spectrometry. Fungi capable of mineralizing phenanthrene and pyrene in liquid culture produced enriched metabolites substituted in the K region (C-9,10 position of phenanthrene and C-4,5 position of pyrene), whereas all other fungi investigated produced metabolites substituted in the C-1,2, C-3,4, and C-9,10 positions of phenanthrene and the C-1 position of pyrene.

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

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 the fluoroquinolone enrofloxacin by wood-rotting fungi.

The veterinary fluoroquinolone enrofloxacin was degraded in vitro by four species of wood-rotting fungi growing on wetted wheat straw containing carbonyl-14C-labeled drug. A maximum 14CO2 production of 17% per week was observed with the brown rot fungus Gloeophyllum striatum, resulting in up to 53% after 8 weeks. However, rates reached at most 0.2 and 0.9% per week, if enrofloxacin was preadsorbed to native or gamma ray-sterilized soil, respectively.

Effects of different heat pre-treatments of wheat straw on its microbial activity and colonization by different tropical and sub-tropical edible mushrooms.

Quantitative differentiation of microbial activity in wheat straw substrate is described after different heat pre-treatments and addition of water during solid-state fermentation. All the 28 tested strains of tropical and sub-tropical edible mushrooms colonized sterile wheat straw. Substrate pre-treated at 25°C was primarily colonized by Coprinus sp. and other competitive microorganisms, and had the highest pH values. With some exceptions, increasing rates of growth occurred with substrate pre-treatment at 60 and 90°C. Best growth and highest speed of colonization were on sterilized straw. Heat pre-treatment of cereal straw at 60 and 90°C should be sufficient for commercial cultivation processes. Thus, a short fermentative pre-treatment could reduce the risk of infection. The strains tested do not differ significantly from those of temperate climates.

Bamboo as a support material in anaerobic reactors.

The changes in 2.5-cm thick bamboo rings during their use as a support material in an anaerobic reactor show that the wash out and degradation processes are largely completed within 6 months to a year. The dry mass of the bamboo used decreased more quickly in the first half of the year than during the second half and asymptotically approached a loss limit of approximately 15% (w/w). The decrease in substances contained in the bamboo during the 2-year experimental period led to reductions in wall thickness of the rings of 0.8 mm and in the absorbable pressure of 21%. Bamboo thus appears to be suitable for long-term use in an anaerobic reactor.