Jerzy Długoński

Emulsifier production by steroid transforming filamentous fungus Curvularia lunata. Growth and product characterization

Curvularia lunata IM 2901, the filamentous fungus used for hydrocortisone manufacture, was found to be able to produce an extracellular emulsifying agent on water-soluble compounds with a yield of 2.6 g l(-1). Cell-free culture broth containing the examined agent caused the formation of stable emulsions with hydrophobic compounds of natural and xenobiotic origin. Vegetable and mineral oils were the best substrates for emulsification. Kinetics of surfactant biosynthesis was classified as a mixed growth-associated and non-growth-associated type. The crude emulsifier was isolated from culture fluid by acetone precipitation. Preliminary chemical characterization showed that the studied bioemulsifier contained 34.0% C, 5.7% H, 1.8% N, 0.15% S and was a complex of protein (25%) and polysaccharide (48%). Sugar component was identified as a polymer of D-glucose.

Degradation of tributyltin by the filamentous fungus Cunninghamella elegans, with involvement of cytochrome P-450

Cunninghamella elegans degraded tributyltin (TBT) at 20 mg l−1 when grown in Sabouraud medium. Above this concentration, growth was inhibited. After 7 d 70% TBT (added at 10 mg l−1) was converted to less toxic derivatives: dibutyltin and monobutyltin. TBT metabolism was totally blocked by cytochrome P-450 inhibitors, metyrapone and proadifen. Only in medium with 1-aminobenzotriazole, was dibutyltin (0.42 mg l−1) found after 7 d of culturing. It is postulated that the significant resistance of C. elegans to TBT is associated with the capacity of the fungus to metabolise TBT.

Degradation and toxicity reduction of the endocrine disruptors nonylphenol, 4-tert-octylphenol and 4-cumylphenol by the non-ligninolytic fungus Umbelopsis isabellina.

Nonylphenol (NP), 4-tert-octylphenol (4-t-OP) and 4-cumylphenol (4-CP) are pollutants that are known as endocrine disruptors mainly due to their estrogen-mimicking activity. These phenolic substances are used in a wide range of industrial and commercial applications. In the present study, biodegradation of tNP, 4-t-OP and 4-CP using the non-ligninolytic fungus Umbelopsis isabellina was investigated. After 12h of incubation, more than 90% of initially applied tNP, 4-t-OP and 4-CP (25mgL(-1)) were eliminated. GC-MS analysis revealed several derivatives mainly (hydroxyalkyl)phenols. Moreover, xenobiotic biotransformation led to the formation of intermediates with less harmful effects than the parent compounds. For all xenobiotics, a decrease in growth medium toxicity was observed, using Artemia franciscana and Daphnia magna as bioindicators. The results indicate that U. isabellina has potential in the degradation and detoxification of contaminants with endocrine activity. Moreover, this is the first report demonstrating that a microorganism is capable of effective 4-CP elimination.

Butyltins degradation by Cunninghamella elegans and Cochliobolus lunatus co-culture.

Organotin compounds are ubiquitous in environment. However, biodegradation of tributyltin (TBT) and dibutyltin (DBT) to non toxic metabolites by fungi has been seldom observed. In this study we constructed a fungal co-culture with an efficient ability of TBT and its metabolites removal. The microscopic fungus strain Cunninghamella elegans degraded TBT via hydroxybutyldibutyltin (OHBuDBT) to its metabolites: DBT and monobutyltin (MBT), which were then transformed by Cochliobolus lunatus. The sequential biodegradation resulted in a 10-fold decrease in samples toxicity to Artemia franciscana larvae. With an initial TBT concentration of 5 mg l(-1), the co-culture of both fungi almost completely eliminated butyltins during 12 days of incubation in synthetic medium. To our knowledge, this is the first report that the mixed fungal co-culture could efficiently degrade TBT. This process was associated with glucose utilization, and a cometabolic nature of butyltins removal by selected strains has been suggested.

Cortexolone 11β-hydroxylation in protoplasts of Curvularia lunata

Abstract Transformation of cortexolone to hydrocortisone by the filamentous fungus Curvularia lunata using protoplasts as a research tool was studied. A stable mutant of C. lunata (IM 2901/366) hydroxylated cortexolone at 11 β -position with significantly higher activity than the wild strain (IM 2901) at the similar rate of growth. Protoplasts released from the mycelium of the wild strain and the mutant transformed the steroid substrate with about four and over five times higher yield than the parental mycelium (respectively). Due to the higher hydroxylation ability of the mutant, the activity of mutant protoplasts was 24 times higher than the activity of the mycelium of the wild strain. Hydroxylation of cortexolone was blocked in both strains by the inhibitor of cytochrome P-450 (cyt P-450) activity, ketoconazole. Cyt P-450, as hemoprotein involved in steroid-hydroxylating system, was localized in microsomal fractions of both strains. Estimation of cyt P-450 content in the intact protoplasts revealed that the level of cyt P-450 in the mutant cells was higher and it increased more rapidly in steroid presence than in the wild strain cells. It is suggested that the higher amount of cyt P-450 and higher steroid transformation activity of the mutant result from mutation(s) of genes involved in steroid 11 β -hydroxylase synthesis regulation.

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.

Malachite green decolorization by the filamentous fungus Myrothecium roridum--Mechanistic study and process optimization.

The filamentous fungus Myrothecium roridum isolated from a dye-contaminated area was investigated in terms of its use for the treatment of Malachite green (MG). The mechanisms involved in this process were established. Peroxidases and cytochrome P-450 do not mediate MG elimination. The laccase of M. roridum IM 6482 was found to be responsible for the decolorization of 8-11% of MG. Thermostable low-molecular-weight factors (LMWF) resistant to sodium azide were found to be largely involved in dye decomposition. In addition, MG decolorization by M. roridum IM 6482 occurred in a non-toxic manner. Data from antimicrobial tests showed that MG toxicity decreased after decolorization. To optimize the MG decolorization process, the effects of operational parameters (such as the medium pH and composition, process temperature and culture agitation) were examined. The results demonstrate that M. roridum IM 6482 may be used effectively as an alternative to traditional decolorization agents.

Isolation of Streptomyces sp. strain capable of butyltin compounds degradation with high efficiency.

Dibutyltin (DBT), a widely used plastic stabilizer, has been detected in the environment as well as in human tissues. DBT is considered to be highly neurotoxic and immunotoxic. Hence, DBT needs to be considered as a potential toxic chemical. Degradation of butyltin compounds by Streptomyces sp. isolated from plant waste composting heaps was studied. Glucose grown cells degraded organotin from 10 to 40 mg l(-1). After 1 day of incubation 90% of DBT (added at 20 mg l(-1)) was converted to less toxic derivative--monobutyltin (MBT). DBT metabolism was inhibited by metyrapone addition, a known cytochrome P-450 inhibitor. It could provide evidence that cytochrome P-450 system is involved in DBT metabolism in Streptomyces sp. IM P102. Moreover, according to our knowledge, the degradation of DBT by actinobacterium has not been previously described.

Biodegradation and utilization of 4-n-nonylphenol by Aspergillus versicolor as a sole carbon and energy source

4-n-Nonylphenol (4-n-NP) is an environmental pollutant with endocrine-disrupting activities that is formed during the degradation of nonylphenol polyethoxylates, which are widely used as surfactants. Utilization of 4-n-NP by the filamentous fungus Aspergillus versicolor as the sole carbon and energy source was investigated. By means of gas chromatography-mass spectrometry, we showed that in the absence of any carbon source other than 4-n-NP in the medium, A. versicolor completely removed the xenobiotic (100 mg L(-1)) after 3 d of cultivation. Moreover, mass spectrometric analysis of intracellular extracts led to the identification of eight intermediates. The mineralization of the xenobiotic in cultures supplemented with 4-n-NP [ring-(14)C(U)] as a growth substrate was also assessed. After 3 d of incubation, approximately 50% of the initially applied radioactivity was recovered in the form of (14)CO2, proving that this xenobiotic was completely metabolized and utilized by A. versicolor as a carbon source. Based on microscopic analysis, A. versicolor is capable of germinating spores under such conditions. To confirm these observations, a microcalorimetric method was used. The results show that even the highest amount of 4-n-NP initiates heat production in the fungal samples, proving that metabolic processes were affected by the use of 4-n-NP as an energetic substrate.

Action of tributyltin (TBT) on the lipid content and potassium retention in the organotins degradating fungus Cunninghamella elegans.

The purpose of the presented paper was to study the effect of high concentrations of tributyltin (TBT) on the potassium retention and fatty acid (FA) composition of the fungus Cunninghamella elegans recognized as a very efficient TBT degrader. An increase in TBT had a strong influence on the potassium concentration in the fungus. In growth medium without TBT, the potassium content of the fungal cells was 5.8xa0mgxa0K+xa0gxa0dry weight−1. The maximum concentration of K+ was 15.06xa0mgxa0g−1 dry weight at 30xa0mgxa0l−1 of TBT. The major FAs that characterized the tested strain were C16:0, C18:1, C18:2, C18:3 and C18:0. TBT in the concentration range 5–30xa0mgxa0l−1 strongly influenced the FA composition. In the presence of the organotin, the degree of saturation increased. It suggests that the observed changes promote an increase in the lipid ordering of the membrane by reducing its permeability and inhibiting potassium ion efflux.