Angélica M. Arambarri

Induction, Isolation, and Characterization of Two Laccases from the White Rot Basidiomycete Coriolopsis rigida

ABSTRACT Previous work has shown that the white rot fungus Coriolopsis rigida degraded wheat straw lignin and both the aliphatic and aromatic fractions of crude oil from contaminated soils. To better understand these processes, we studied the enzymatic composition of the ligninolytic system of this fungus. Since laccase was the sole ligninolytic enzyme found, we paid attention to the oxidative capabilities of this enzyme that would allow its participation in the mentioned degradative processes. We purified two laccase isoenzymes to electrophoretic homogeneity from copper-induced cultures. Both enzymes are monomeric proteins, with the same molecular mass (66 kDa), isoelectric point (3.9), N-linked carbohydrate content (9%), pH optima of 3.0 on 2,6-dimethoxyphenol (DMP) and 2.5 on 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), absorption spectrum, and N-terminal amino acid sequence. They oxidized 4-anisidine and numerous phenolic compounds, including methoxyphenols, hydroquinones, and lignin-derived aldehydes and acids. Phenol red, an unusual substrate of laccase due to its high redox potential, was also oxidized. The highest enzyme affinity and efficiency were obtained with ABTS and, among phenolic compounds, with 2,6-dimethoxyhydroquinone (DBQH2). The presence of ABTS in the laccase reaction expanded the substrate range of C. rigida laccases to nonphenolic compounds and that of MBQH2 extended the reactions catalyzed by these enzymes to the production of H2O2, the oxidation of Mn2+, the reduction of Fe3+, and the generation of hydroxyl radicals. These results confirm the participation of laccase in the production of oxygen free radicals, suggesting novel uses of this enzyme in degradative processes.

Biodegradation of aliphatic and aromatic hydrocarbons by natural soil microflora and pure cultures of imperfect and lignolitic fungi.

The biodegradation of aliphatic and aromatic hydrocarbons by natural soil microflora and seven fungi species, including imperfect strains and higher level lignolitic species, is compared in a 90-day laboratory experiment using a natural, not-fertilized soil contaminated with 10% crude oil. The natural microbial soil assemblage isolated from an urban forest area was unable to significantly degrade crude oil, whereas pure fungi cultures effectively reduced the residues by 26-35% in 90 days. Normal alkanes were almost completely degraded in the first 15 days, whereas aromatic compounds (phenanthrene and methylphenanthrenes) exhibited slower kinetics. Aspergillus terreus and Fusarium solani, isolated from oil-polluted areas, produced the more efficient attack of aliphatic and aromatic hydrocarbons, respectively. Overall, imperfect fungi isolated from polluted soils showed a somewhat higher efficiency, but the performance of unadapted, indigenous, lignolitic fungi was comparable, and all three species, Pleurotus ostreatus, Trametes villosus and Coriolopsis rigida, effectively degraded aliphatic and aromatic components. The simultaneous, multivariate analysis of 22 parameters allowed the elucidation of a clear reactivity trend of the oil components during biodegradation: lower molecular weight n-alkanes > phenanthrene > 3-2-methylphenanthrenes > intermediate chain length n-alkanes > longer chain length n-alkanes > isoprenoids approximately 9-1-methylphenanthrenes. Irrespective of the individual degrading capacities, all fungi species tested seem to follow this decomposition sequence.

Phenanthrene degradation by microorganisms isolated from a contaminated stream

Autochthonous yeast and bacteria strains were isolated from a contaminated stream and studied for their potential to degrade phenanthrene, a three-ring polycyclic aromatic hydrocarbon (PAH), as their only source of carbon and energy. Rhodotorula glutinis and Pseudomonas aeruginosa were the prevailing microorganisms utilizing phenanthrene. Cells of these microorganisms were inoculated in liquid mineral basal medium at 25, 50, 100 and 200 mg phenanthrene liter−1 in ethanolic solution. To volatilize the ethanol the medium was dried for 40 min at 35°C with UV exposure. Almost complete phenanthrene degradation was observed during the 1-month incubation period. R. glutinis grew exponentially to a maximum of 9.5×106 colony-forming units (CFUs) ml−1, with a short lag time only at 200 mg liter−1. The highest density (1.4×107 CFU ml−1) without a lag time was obtained at 50 mg liter−1; lower cell numbers were observed at 25 and 100 mg liter−1. During the logarithmic phase the specific growth rate (μ) was 0.022 h−1 and the corresponding doubling time (td) was 31.5 h (50 mg liter−1). A reduction to one-tenth of cell yeast numbers was observed at the lowest PAH levels on the 28th incubation day and a decrease of 100-fold was observed at 200 mg liter−1. Higher μ-values (μ=d [ln x/x0]/dt), shorter td (td=[ln 2]/μ), and a maximum of 7.7–7.4×108 CFU ml−1 were observed in P. aeruginosa cultures at 50–100 mg liter−1, respectively, on the 22nd day. At 25 and 200 mg liter−1 lower bacteria numbers were obtained (2.1–3.0×108 CFU ml−1) on the 28th day. R. glutinis was as active as P. aeruginosa at growing on phenanthrene; the aromatic hydrocarbon degradation correlated directly to microbial density and biomass increase, the highest biomass reaching 238.0 and 50.0 mg liter−1 for the yeast and bacteria species, respectively.

Pyrene degradation by yeasts and filamentous fungi

The saprotrophic soil fungi Fusarium solani (Mart.) Sacc., Cylindrocarpon didymum (Hartig) Wollenw, Penicillium variabile Sopp. and the yeasts Rhodotorula glutinis (Fresenius) Harrison and Rhodotorula minuta (Saito) Harrison were cultured in mineral medium with pyrene. The remaining pyrene concentrations were periodically determined during 20 incubation days, using HPLC. To assess the metabolism of pyrene degradation we added 0.1 microCi of [4,5,9,10] 14C-pyrene to each fungi culture and measured the radioactivity in the volatile organic substances, extractable, aqueous phase, biomass and 14CO2 fractions. The assays demonstrated that F. solani and R. glutinis metabolized pyrene as a sole source of carbon. Differences in their activities at the beginning of the cultures disappeared by the end of the experiment, when 32 and 37% of the original pyrene concentration was detected, for the soil fungi and yeasts, respectively. Among the filamentous fungi, F. solani was highly active and oxidized pyrene; moreover, small but significant degradation rates were observed in C. didymum and P. variahile cultures. An increase in the 14CO2 evolution was observed at the 17th day with cosubstrate. R. glutinis and R. minuta cultures showed similar ability to biotransform pyrene, and that 35% of the initial concentration was consumed at the end of the assay. The same results were obtained in the experiments with or without glucose as cosubstrate.

Diversity in soil fungi from undisturbed and disturbed Celtis tala and Scutia buxifolia forests in the eastern Buenos Aires province (Argentina).

The rhizospheric soil microfungi from a native forest (undisturbed and disturbed) were studied using soil dilution plate and soil washing methods. Fungi were isolated using slightly acid and alkaline culture media. 54 taxa were isolated: 49 from undisturbed forest soil and 37 from disturbed forest soil. Acremonium sp., Aspergillus ustus, Coemansia pectinata, Doratomyces stemonitis, Fusarium solani, F. oxysporum, Gliocladium roseum, Humicola fusco-atra, Mortierella sp., Penicillium lilacinum, Trichoderma harzianum, and T koningii, showed the highest frequency, in both, undisturbed and disturbed forests. In undisturbed soil forest the biodiversity index was 3.97 whereas in disturbed ones was 3.89.

Production of ligninolytic enzymes by Fusarium solani strains isolated from different substrata

A comparative study on the extracellular ligninolytic enzymatic activity of five strains of Fusarium solani in a carbon-limited medium under shaking, revealed a differential production of these enzymes. Aryl alcohol oxidase (AAO) activity was observed only in the supernatant of strain CLPS no. 568 with levels higher than 57 mU ml−1. Free extracellular laccase activity was detected in strains CLPS nos. 493, 568 and 570, strain no. 568 being the one which showed the highest activity (over 8.6 mU ml−1). Free extracellular lignin peroxidase (LiP) activity was not detected in any isolate tested, whereas low levels of manganese-dependent peroxidase (MnP) and manganese-independent peroxidase (MIP) activities were detected in certain isolates used. The AAO activity of F. solani on primary α-alcohols such as veratryl alcohol, is reported for the first time; this enzyme activity is hydrogen-peroxide independent. This is also the first report for extracellular MnP and MIP activities of F. solani.

Occurrence of Different Species of Fusarium from Wheat in Relation to Disease Levels Predicted by a Weather-Based Model in Argentina Pampas Region

Fusarium head blight (FHB) is an important disease throughout many of the world wheat-growing areas that have humid to semi-humid climate. The infection happens mainly during the anthesis of the wheat, when there have been favorable conditions of moisture and temperature. The direct relation of the infection to environmental factors makes possible the formulation of mathematical models that predict the disease. The causal agent of the FHB of the spike of wheat is attributed principally to Fusarium graminearum. High economic losses due yield decrease have been recorded in Argentina. In the present work, 67 isolates of Fusarium spp. were obtained from samples of wheat grains from Pampas region from 15 locations distributed in Buenos Aires, Entre Ríos, Santa Fe and Córboba provinces during 2006 and 2007 wheat-growing seasons. The identification of species from monosporic isolates was carried out by morphological characterization and use of species-specific PCR-based assays. Both identification criteria were necessary and complementary for the species determination, since in some cases the molecular identification was not specific. Scanty presence of F. graminearum was observed in 2006 wheat-growing season coinciding with the lack of favorable meteorological conditions for producing FHB infection events. High presence of F. graminearum isolates was observed in 2007 wheat-growing season, in accordance with moderate incidence of the disease according to spatial distribution of FHB incidence values. The aim of this report was to identify the causal agent of the FHB disease by different taxonomic criteria and to relate its occurrence with disease incidence values predicted by a weather-based model in Argentina.

Selection of autochthonous yeast strains able to degrade biphenyl

In order to assess the role of yeasts in the natural detoxification process of sediments polluted with biaryl compounds, indigenous yeast species able to degrade biphenyl (BP) were isolated and identified. The degradation ability of 24 strains of the genera Candida spp., Cryptococcus spp., Pichia spp., Rhodotorula spp., Trichosporon spp. and Yarrowia spp. was evaluated by the identification of the BP-metabolites, by HPLC analysis. 4-Hydroxybiphenyl was the main derivative in the Candida krusei, C. tenuis, C. tropicalis, Pichia haplophila, Rhodotorula glutinis, Trichosporon pullulans and Yarrowia lipolytica cultures. 3-Hydroxybiphenyl was detected in minor amounts in the culture supernatant of C. tropicalis, C. krusei strains and R. glutinis. Further hydroxylation led to 3,4-dihydroxy and 2,3-dihydroxybiphenyl; the former in C. tropicalis, C. krusei and R. glutinis cultures, and the latter only in the R. glutinis assays. The cleavage product 4-phenyl-2-pyrone-6-carboxylic acid, was observed in R. glutinis and Y. lipolytica cultures. The degradation ability of the R. glutinis isolates was noteworthy; as four hydrolxylated intermediates and a ring-cleavage product were obtained in both strain cultures. The species studied in this report were dominant in polluted sediments; furthermore, R. glutinis had been mentioned as able to degrade other aromatic hydrocarbons and had high relevance in bioremediation experiments.

Dematiocladium celtidis gen. sp. nov. (Nectriaceae, Hypocreales), a new genus from Celtis leaf litter in Argentina

A Cylindrocladium-like hyphomycete collected on leaf litter of Celtis tala in Argentina had rDNA sequence data (ITS and LSU) that showed it resides in the Hypocreales, and is a member of the Nectriaceae, closely related to, but distinct from Cylindrocladium. A new genus, Dematiocladium and species, D. celtidis gen. sp. nov. is, therefore, introduced to accommodate this fungus. Based on morphology, it can be distinguished from other conidial hypocrealean genera with hyaline, penicillate conidiophores and cylindrical conidia by lacking stipe extensions and vesicles, and by the presence of brown to dark brown, thick-walled setae.

Coriolopsis rigida, a potential model of white-rot fungi that produce extracellular laccases.

In the last two decades, a significant amount of work aimed at studying the ability of the white-rot fungus Coriolopsis rigida strain LPSC no. 232 to degrade lignin, sterols, as well as several hazardous pollutants like dyes and aliphatic and aromatic fractions of crude oil, including polycyclic aromatic hydrocarbons, has been performed. Additionally, C. rigida in association with arbuscular mycorrhizal fungi appears to enhance plant growth, albeit the physiological and molecular bases of this effect remain to be elucidated. C. rigidas ability to degrade lignin and lignin-related compounds and the capacity to transform the aromatic fraction of crude oil in the soil might be partially ascribed to its ligninolytic enzyme system. Two extracellular laccases are the only enzymatic components of its lignin-degrading system. We reviewed the most relevant findings regarding the activity and role of C. rigida LPSC no. 232 and its laccases and discussed the work that remains to be done in order to assess, more precisely, the potential use of this fungus and its extracellular enzymes as a model in several applied processes.