Dietmar Schlosser

Untapped potential: exploiting fungi in bioremediation of hazardous chemicals

Fungi possess the biochemical and ecological capacity to degrade environmental organic chemicals and to decrease the risk associated with metals, metalloids and radionuclides, either by chemical modification or by influencing chemical bioavailability. Furthermore, the ability of these fungi to form extended mycelial networks, the low specificity of their catabolic enzymes and their independence from using pollutants as a growth substrate make these fungi well suited for bioremediation processes. However, despite dominating the living biomass in soil and being abundant in aqueous systems, fungi have not been exploited for the bioremediation of such environments. In this Review, we describe the metabolic and ecological features that make fungi suited for use in bioremediation and waste treatment processes, and discuss their potential for applications on the basis of these strengths.

Laccase-Catalyzed Oxidation of Mn2+ in the Presence of Natural Mn3+ Chelators as a Novel Source of Extracellular H2O2 Production and Its Impact on Manganese Peroxidase

ABSTRACT A purified and electrophoretically homogeneous blue laccase from the litter-decaying basidiomycete Stropharia rugosoannulata with a molecular mass of approximately 66 kDa oxidized Mn2+ to Mn3+, as assessed in the presence of the Mn chelators oxalate, malonate, and pyrophosphate. At rate-saturating concentrations (100 mM) of these chelators and at pH 5.0, Mn3+ complexes were produced at 0.15, 0.05, and 0.10 μmol/min/mg of protein, respectively. Concomitantly, application of oxalate and malonate, but not pyrophosphate, led to H2O2 formation and tetranitromethane (TNM) reduction indicative for the presence of superoxide anion radical. Employing oxalate, H2O2 production, and TNM reduction significantly exceeded those found for malonate. Evidence is provided that, in the presence of oxalate or malonate, laccase reactions involve enzyme-catalyzed Mn2+ oxidation and abiotic decomposition of these organic chelators by the resulting Mn3+, which leads to formation of superoxide and its subsequent reduction to H2O2. A partially purified manganese peroxidase (MnP) from the same organism did not produce Mn3+ complexes in assays containing 1 mM Mn2+ and 100 mM oxalate or malonate, but omitting an additional H2O2 source. However, addition of laccase initiated MnP reactions. The results are in support of a physiological role of laccase-catalyzed Mn2+ oxidation in providing H2O2 for extracellular oxidation reactions and demonstrate a novel type of laccase-MnP cooperation relevant to biodegradation of lignin and xenobiotics.

Fungi in freshwaters: ecology, physiology and biochemical potential

Research on freshwater fungi has concentrated on their role in plant litter decomposition in streams. Higher fungi dominate over bacteria in terms of biomass, production and enzymatic substrate degradation. Microscopy-based studies suggest the prevalence of aquatic hyphomycetes, characterized by tetraradiate or sigmoid spores. Molecular studies have consistently demonstrated the presence of other fungal groups, whose contributions to decomposition are largely unknown. Molecular methods will allow quantification of these and other microorganisms. The ability of aquatic hyphomycetes to withstand or mitigate anthropogenic stresses is becoming increasingly important. Metal avoidance and tolerance in freshwater fungi implicate a sophisticated network of mechanisms involving external and intracellular detoxification. Examining adaptive responses under metal stress will unravel the dynamics of biochemical processes and their ecological consequences. Freshwater fungi can metabolize organic xenobiotics. For many such compounds, terrestrial fungal activity is characterized by cometabolic biotransformations involving initial attack by intracellular and extracellular oxidative enzymes, further metabolization of the primary oxidation products via conjugate formation and a considerable versatility as to the range of metabolized pollutants. The same capabilities occur in freshwater fungi. This suggests a largely ignored role of these organisms in attenuating pollutant loads in freshwaters and their potential use in environmental biotechnology.

Hydroxylated Metabolites of 2,4-Dichlorophenol Imply a Fenton-Type Reaction in Gloeophyllum striatum

ABSTRACT While degrading 2,4-dichlorophenol, two strains ofGloeophyllum striatum, a basidiomycetous fungus causing brown rot decay of wood, simultaneously produced 4-chlorocatechol and 3,5-dichlorocatechol. These metabolites were identified by comparing high-performance liquid chromatography retention times and mass spectral data with those of chemically synthesized standards. Under similar conditions, 3-hydroxyphthalic hydrazide was generated from phthalic hydrazide, a reaction assumed to indicate hydroxyl radical formation. Accordingly, during chemical degradation of 2,4-dichlorophenol by Fentons reagent, identical metabolites were formed. Both activities, the conversion of 2,4-[U-14C]dichlorophenol into14CO2 and the generation of 3-hydroxyphthalic hydrazide, were strongly inhibited by the hydroxyl radical scavenger mannitol and in the absence of iron. These results provide new evidence in favor of a Fenton-type degradation mechanism operative inGloeophyllum.

Prospects for microbiological solutions to environmental pollution with plastics

Synthetic polymers, commonly named plastics, are among the most widespread anthropogenic pollutants of marine, limnic and terrestrial ecosystems. Disruptive effects of plastics are known to threaten wildlife and exert effects on natural food webs, but signs for and knowledge on plastic biodegradation are limited. Microorganisms are the most promising candidates for an eventual bioremediation of environmental plastics. Laboratory studies have reported various effects of microorganisms on many types of polymers, usually by enzymatic hydrolysis or oxidation. However, most common plastics have proved to be highly recalcitrant even under conditions known to favour microbial degradation. Knowledge on environmental degradation is yet scarcer. With this review, we provide a comprehensive overview of the current knowledge on microbiological degradation of several of the most common plastic types. Furthermore, we illustrate the analytical challenges concerning the evaluation of plastic biodegradation as well as constraints likely standing against the evolution of effective biodegradation pathways.

Chlorobenzene biodegradation under consecutive aerobic–anaerobic conditions

The biodegradation of monochlorobenzene, the main contaminant in a quaternary aquifer at Bitterfeld, Central Germany, was studied in microcosm experiments employing either original groundwater or defined mineral media together with the indigenous microbial community from the polluted site. The impact of consecutive aerobic-anaerobic-aerobic incubations on monochlorobenzene biodegradation, microbial diversity, and pH development was examined. The related changes in microbial community composition were analyzed by 16S rRNA gene-based single-strand conformation polymorphism (SSCP) fingerprints and sequencing of dominant bands and by quantitative analysis of bacterial respiratory chain quinones as biomarkers. Under aerobic conditions, the indigenous microbial community of the groundwater degraded monochlorobenzene mainly via the modified ortho-pathway. Respiratory chain quinones and SSCP analysis suggested dominance of the genera Acidovorax and Pseudomonas. A shift to anoxic conditions resulted in monochlorobenzene biotransformation but no dechlorination. The ability to degrade monochlorobenzene aerobically remained preserved throughout a fortnightly anoxic period at sufficiently high buffer capacity. Acidification, caused by monochlorobenzene biodegradation, was alkalinity-controlled. At low initial alkalinity a substantial decrease in pH, monochlorobenzene degradation, and total counts of live cells, accompanied by a change of the microbial community composition, was observed.

Production of a robust nanobiocatalyst for municipal wastewater treatment

Immobilization is a fundamental method to improve both enzyme activity and stability. In the present work, the process previously described for immobilizing laccase - an enzyme oxidizing phenolic compounds - onto fumed silica was optimized, in order to efficiently produce industrially relevant amounts of a nanobiocatalyst for biological micropollutant elimination, whilst saving 80% of surface modification agent (3-aminopropyl triethoxy silane) and 90% of cross-linker (glutaraldehyde). Minimized losses during preparation and favorable effects of immobilization yielded conjugates with drastically increased enzymatic activity (164% of invested activity). Long-term stability and activity regarding bisphenol A (2,2-bis(4-hydroxyphenyl)propane) removal of the synthesized biocatalyst were assessed under application-relevant conditions. With 81.1±0.4% residual activity after 7 days, stability of conjugates was drastically higher than of free laccase, which showed virtually no activity after 1.5 days. These results illustrate the huge potential of fumed silica nanoparticles/laccase-composites for innovative biological wastewater treatment.

Biotransformation of 1-Naphthol by a Strictly Aquatic Fungus

The aquatic hyphomycete Heliscus lugdunensis belongs to a group of exclusively aquatic mitosporic fungi with an only scarcely explored potential to oxidatively attack xenobiotic compounds, and was used to study the biotransformation of the environmental pollutant metabolite 1-naphthol. H. lugdunensis metabolized approximately 74% of 1-naphthol within 5 days. The identification and quantification of degradation products using gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, and high performance liquid chromatography revealed that approximately 12% of the parent compound was converted into 1-naphthylsulfate, 3% was transformed into 1-methoxy-naphthalene, and less than 1% was converted into 1,4-naphthoquinone. A further metabolite, most likely 4-hydroxy-1-naphthylsulfate, was also detected. In contrast to sulfate conjugate metabolites, no glucuronide and glucoside conjugates of 1-naphthol were found, and neither UDP-glucuronyltransferase nor UDP-glucosyltransferase present in H. lugdunensis showed activity towards 1-naphthol. These results support a role of fungi adapted to aquatic environments in affecting the environmental fate of pollutants in aquatic ecosystems.

Quantification of the Influence of Extracellular Laccase and Intracellular Reactions on the Isomer-Specific Biotransformation of the Xenoestrogen Technical Nonylphenol by the Aquatic Hyphomycete Clavariopsis aquatica

ABSTRACT The aquatic hyphomycete Clavariopsis aquatica was used to quantify the effects of extracellular laccase and intracellular reactions on the isomer-specific biotransformation of technical nonylphenol (t-NP). In laccase-producing cultures, maximal removal rates of t-NP and the isomer 4-(1-ethyl-1,4-dimethylpentyl)phenol (NP112) were about 1.6- and 2.4-fold higher, respectively, than in laccase-lacking cultures. The selective suppression of either laccase or intracellular reactions resulted in essentially comparable maximal removal rates for both compounds. Evidence for an unspecific oxidation of t-NP isomers was consistently obtained from laccase-expressing fungal cultures when intracellular biotransformation was suppressed and from reaction mixtures containing isolated laccase. This observation contrasts with the selective degradation of t-NP isomers by bacteria and should prevent the enrichment of highly estrogenic isomers in remaining t-NP. In contrast with laccase reactions, intracellular fungal biotransformation caused a significant shift in the isomeric composition of remaining t-NP. As a result, certain t-NP constituents related to more estrogenic isomers were less efficiently degraded than others. In contrast to bacterial degradation via ipso-hydroxylation, the substitution pattern of the quaternary α-carbon of t-NP isomers does not seem to be very important for intracellular transformation in C. aquatica. As-yet-unknown intracellular enzymes are obviously induced by nonylphenols. Mass spectral data of the metabolites resulting from the intracellular oxidation of t-NP, NP112, and 4-(1-ethyl-1,3-dimethylpentyl)phenol indicate nonyl chain hydroxylation, further oxidation into keto or aldehyde compounds, and the subsequent formation of carboxylic acid derivatives. Further metabolites suggest nonyl chain desaturation and methylation of carboxylic acids. The phenolic moieties of the nonylphenols remained unchanged.

Extracellular laccase activity and transcript levels of putative laccase genes during removal of the xenoestrogen technical nonylphenol by the aquatic hyphomycete Clavariopsis aquatica.

We investigated the influence of potential laccase inducers with environmental relevance on extracellular laccase activity and removal of the xenoestrogen technical nonylphenol (tNP) by the aquatic hyphomycete Clavariopsis aquatica. Concomitantly, we identified two putative laccase gene fragments (Icc1 and Icc2) and have followed their expression during removal of tNP under different conditions. Our results indicate a significant effect of copper on extracellular laccase activity in supernatants of fungal cultures. Laccase activity was highest in the presence of copper when added together with vanillic acid, followed by copper when used alone. Only slight laccase activities were recorded in the presence of only vanillic acid, whereas in the absence of either compound laccase activities were negligible. Laccase activity was well correlated with the removal efficiency of tNP, indicating the involvement of laccase in tNP bioconversion. Overall, Icc2 was less expressed than Icc1. The expression of Icc1 and Icc2 correlated only partially with the measured laccase activity, suggesting the existence of cell-associated laccase fractions not detectable in fungal culture supernatants and/or the existence of additional laccase genes.