Consumption of the triazine herbicide atrazine by laccase and laccase-free variants of the soil fungus Mycelia sterilia INBI-2-26

Asporogenic fungus Mycelia sterilia INBI 2-26 isolated from tropical soils with high residual dioxin content (as a result of Agent Orange defoliant treatment during the Vietnamese-American war) and capable of atrazine decomposition was treated to obtain protoplasts. This technique resulted in isolation of laccase-positive and laccase-negative clones. Atrazine consumption by liquid surface cultures of Mycelia sterilia INBI 2-26 was monitored by using enzyme immune assay and reversed phase HPLC. Atrazine (20 micrograms/l) stimulated fungal growth. Laccase-positive clone consumed up to 80% of atrazine within four weeks. However, no correlation of atrazine consumption and laccase activity in the culture medium was observed. Moreover, the laccase-negative clone was also capable of consuming at least 60-70% of atrazine within three weeks. Surprisingly, in the corresponding control set (cultivation of laccase-negative clone without atrazine) an unidentified metabolite having a retention time and UV-spectrum similar to those of atrazine was also found. It was concluded that the presence of laccase was not a crucial factor in atrazine consumption by this fungus.     

Enzyme Immunoassay of Herbicide Decomposition by Soil and Wood Decay Fungi

The effect of herbicide atrazine was studied on the growth and development of a number of soil and wood decay fungi: white-rot basidiomycetes (Cerrena maxima, Coriolopsis fulvocenerea, and Coriolus hirsutus), thermophilic micromycetes from self-heating grass composts (cellulolytic fungus Penicilliumsp. 13 and noncellulolytic ones Humicola lanuginosaspp. 5 and 12), and mesophilic phenol oxidase-producing micromycete Mycelia sterilia INBI 2-26. Detection of atrazine in liquid fungal cultures was performed by using the enzyme immune assay technique. Both stimulation (Humicola lanuginosa 5) and suppression (Humicola lanuginosa 12 and Penicillium sp. 13) of fungal growth with atrazine were observed on solid agar media. HyphomyceteMycelia sterilia INBI 2-26 was almost insensitive to the presence of atrazine. Neither of the thermophilic strains was capable of atrazine consumption in three-week cultivation. In contrast with that, active laccase producers Cerrena maxima, Coriolopsis fulvocenerea, and Coriolus hirsutus consumed up to 50% atrazine in 5-day cultivation in the presence of the xenobiotic and at least 80–92% in 40 days. Mycelia steriliaINBI 2-26, which also forms extracellular laccase, also consumed up to 70% atrazine in 17 days. The degree of atrazine consumption depended on the term of its addition to the fungal culture medium.     

Immunoenzyme analysis of decomposition of herbicides by soil and wood-rot fungi

The effect of herbicide atrazine was studied on the growth and development of a number of soil and wood decay fungi: white-rot basidiomycetes (Cerrena maxima, Coriolopsis fulvocenerea, and Coriolus hirsutus), thermophilic micromycetes from self-heating grass composts (cellulolytic fungus Penicillium sp. 13 and noncellulolytic ones Humicola lanuginosa spp. 5 and 12), and mesophilic phenol oxidase-producing micromycete Mycelia sterilia INBI 2-26. Detection of atrazine in liquid fungal cultures was performed by using enzyme immunoassay technique. Both stimulation (Humicola lanuginosa 5) and suppression (Humicola lanuginosa 12 and Penicillium sp. 13) of fungal growth with atrazine were observed on solid agar media. Hyphomycete Mycelia sterilia INBI 2-26 was almost insensitive to the presence of atrazine. Neither of thermophilic strains was capable of atrazine consumption in three-week cultivation. In contrast with that, active laccase producers Cerrena maxima, Coriolopsis fulvocenerea, and Coriolus hirsutus consumed up to 50% atrazine in 5-day cultivation in the presence of the xenobiotic and at least 80-90% in 40 days. Mycelia sterilia INBI 2-26, which also forms extracellular laccase, also consumed up to 70% atrazine in 17 days. The degree of atrazine consumption depended on the term of its addition to the fungal culture medium.     

Fungal Decomposition of Oat Straw during Liquid and Solid-State Fermentation

White rot fungi (Coriolus hirsutus, Coriolus zonatus, and Cerrena maxima from the collection of the Komarov Botanical Institute of the Russian Academy of Sciences) and filamentous fungi (Mycelia sterilia INBI 2-26 and Trichoderma reesei6/16) were grown on oat straw–based liquid and solid media, as well as in a bench-scale reactor, either individually or as cocultures. All fungi grew well on solid agar medium supplemented with powdered oat straw as the sole carbon source. Under these conditions, the mold Trichoderma reesei fully suppressed the growth of all basidiomycetes studied; conversely,Mycelia sterilia neither affected the development of any of the cultures, nor did it show any substantial susceptibility to suppression by their presence. Pure solid cultures of basidiomycetes, as well as the coculture of Coriolus hirsutus andCerrena maxima,caused a notable bleaching of the oat straw during its consumption. When grown on the surface of oat straw–based liquid medium, the basidiomycetes consumed up to 40% of the polysaccharides without measurable lignin degradation (a concomitant process). Under these conditions, Mycelia sterilia decomposed no more than 25% of the lignin in 60 days, but this was observed only after polysaccharide exhaustion and biomass accumulation. In contrast, during solid-state straw fermentation, white rot fungi consumed up to 75% of cellulose and 55% of lignin in 83 days (C. zonarus), whereas the corresponding consumption levels for cocultures ofMycelia sterilia and Trichoderma reesei equaled 70 and 45%, respectively (total loss of dry weight ranged from 55 to 60%). Carbon dioxide–monitored solid-state fermentation of oat straw by the coculture of filamentous fungi was successfully performed in an aerated bench-scale reactor.     

Cellobiose dehydrogenase formation by filamentous fungus Chaetomium sp. INBI 2-26(-)

Laccase-negative filamentous fungus INBI 2-26(-) isolated from non-sporulating laccase-forming fungal association INBI 2-26 by means of protoplast technique was identified as Chaetomium sp. based on partial sequence of its rRNA genes. In the presence of natural cellulose sources, the strain secreted neutral cellobiose dehydrogenase (CDH) activity both in pure culture and in co-culture with laccase-positive filamentous fungus INBI 2-26(+) isolated from the same association. INBI 2-26(-) also secreted CDH during submerged cultivation in minimal medium with glucose as the sole carbon source. Maximal CDH activity of 1IU/ml at pH 6 with 2,6-dichlorophenolindophenol (DCPIP) as an acceptor was obtained on 12th day of submerged cultivation with filter paper as major cellulose source. Cellulase system of Chaetomium sp. INBI 2-26(-) capable of adsorption onto H(3)PO(4)-swollen filter paper consisted of four major proteins (Mr 200, 95, 65 and 55K) based on SDS-polyacrylamide gel electrophoresis and was capable of DCPIP reduction without exogenous cellobiose.     

Degradation of a Lignin–Carbohydrate Substrate by Soil Fungi Producing Laccase and Cellobiose Dehydrogenase

The growth of nonsporulating mycelial fungi INBI 2-26(+), a producer of laccase; INBI 2-26(–), a producer of cellobiose dehydrogenase; and their mixed culture on lignin–carbohydrate substrates under conditions of submerged fermentation was studied. The degrees of degradation of lignin, cellulose, and hemicellulose of cut straw over 23 days amounted to 29.8, 51.4, and 72% for the laccase producer; 15.8, 33.9, and 59.1% for the cellobiose dehydrogenase producer; and 15.8, 39.4, and 64.5% for the mixed culture, respectively. The laccase activity in the medium when strain 2-26(+) was cultivated individually reached its maximum on day 28; the activity of cellobiose dehydrogenase of strain 2-26(–), on days 14–28. A method for determining cellobiose dehydrogenase activity in the presence of laccase was developed. In the mixed culture, both enzymes were formed; however, the level of laccase synthesis was 1.5-fold lower compared to that of strain 2-26(+), while synthesis of cellobiose dehydrogenase was similar to that of the corresponding producer. Cellobiose dehydrogenase failed to boost the action of laccase while degrading the lignin of straw.     

Isolation and characterization of a micromycete, a producer of neutral catechol oxidases, from tropical soils with elevated dioxine content

—Samples of South Vietnamese soils intensely treated with Agent Orange defoliant were tested for the presence of fungi and actinomycetes with an elevated phenol oxidase activity. As a result, a fast-growing nonsporulating strain producing neutral phenol oxidases was isolated and identified asMycelia sterilia INBI2-26. The strain formed extracellular phenol oxidases during surface growth on a liquid medium in the presence of guayacol and copper sulfate, as well as during submerged cultivation in liquid medium containing wheat bran and sugar beet pulp. Isoelectric focusing of the culture liquid revealed two major catechol oxidases (PO1 and PO2) with pI 3.5 and 8, respectively. The enzymes were purified by Ultrafiltration, ion exchange chromatography, and exclusion HPLC. Both were stable between pH 3 and 8. At pH 8 and 40°C., they retained at least 50% of activity after incubation for 50 h. At 50°C., PO2 was more stable and retained 40% of activity after 50 h, whereas PO1 was inactivated in 3–6 h. The pH-optimutns for PO1 and PO2 toward catechol were 6 and 6.5; and theK _m values were 1.5±0.35 and 1.25±0.2 mM, respectively. PO1 and PO2 most optimally oxidized 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) at pH 3 withK _m values 1.6±0.18 and 0.045±0.01 mM, respectively, but displayed no activity toward tyrosine. The PO2 absorbance spectrum had a peak at 600 nm, thus indicating the enzyme to be a member of the laccase family.     

Overproduction of recombinant laccase using a homologous expression system in <Emphasis Type="Italic">Coriolus versicolor</Emphasis>

One of the major extracellular enzymes of the white-rot fungus Coriolus versicolor is laccase, which is involved in the degradation of lignin. We constructed a homologous system for the expression of a gene for laccase III (cvl3) in C. versicolor, using a chimeric laccase gene driven by the promoter of a gene for glyceraldehyde-3-phosphate dehydrogenase (gpd) from this fungus. We transformed C. versicolor successfully by introducing both a gene for hygromycin B phosphotransferase (hph) and the chimeric laccase gene. In three independent experiments, we recovered 47 hygromycin-resistant transformants at a transformation frequency of 13 transformants g^–1 of plasmid DNA. We confirmed the introduction of the chimeric laccase gene into the mycelia of transformants by a polymerase chain reaction in nine randomly selected transformants. Overproduction of extracellular laccase by the transformants was revealed by a colorimetric assay for laccase activity. We examined the transformant (T2) that had the highest laccase activity and found that its activity was significantly higher than that of the wild type, particularly in the presence of copper (II). Our transformation system should contribute to the efficient production of the extracellular proteins of C. versicolor for the accelerated degradation of lignin and aromatic pollutants.     

Direct somatic embryogenesis fromBegonia gracilis explants

Direct somatic embryogenesis ofBegonia gracilis was achieved from microcultured laminar segments and petioles on Murashige and Skoog medium with 0.5 mg 1^–1 kinetin and 2% coconut water. Somatic embryos were obtained with greater frequency from petiole explants than from leaf blade sections. Under red light (45 mol m^–2 s^–1), approximately 80% of the petiole explants successfully produced somatic embryos but only 30% of the leaf blade sections responded. However, somatic embryos were significantly more abundant on responding lamina explants (60–70 embryos/leaf section) than on petioles (40–50 embryos/petiole). These trends were similar for explants kept in the dark, but overall production was lower. Somatic embryos were produced more quickly (5 weeks) from petioles than from lamina explants (8 weeks). The somatic embryos germinated to produce plantlets and subsequently shoot cultures with the same appearance as the parental clone.Abbreviations BA benzyladenine - MS Murashige and Skoog (1962) - NAA naphthalene acetic acid - SE somatic embryo     

Degradation of the Herbicide Atrazine by the Soil Mycelial Fungus INBI 2-26 (–), a Producer of Cellobiose Dehydrogenase

Nonsporulating mycelial fungi producing cellobiose dehydrogenase (CDH) and isolated from soils of South Vietnam with a high residual content of dioxins are capable of growing on a solid medium in the presence of high atrazine concentrations (to 500 mg/l). At 20 and 50 mg/l atrazine, the area of fungal colonies was 1.5–1.2-fold larger, respectively, than the control colonies of the same age, whereas development of the colonies at 500 mg/l atrazine was delayed by 5 days, compared with controls grown in the absence of atrazine. Surface cultivation of the fungus on a minimal medium with glucose as a sole source of carbon and energy decreased the initial concentration of atrazine (20 mg/l) 50 times in 40 days; in addition, no pronounced sorption of atrazine by mycelium was detected. This was paralleled by an accumulation in the culture medium of extracellular CDH; atrazine increased the synthesis of this enzyme two- to threefold. Accumulation of -glucosidase (a mycelium-associated enzyme) and cellulases preceded the formation of CDH.     

Loss of Cytochrome c Oxidase Activity and Acquisition of Resistance to Quinone Analogs in a Laccase-Positive Variant of Azospirillum lipoferum

Laccase, a p-diphenol oxidase typical of plants and fungi, has been found recently in a proteobacterium, Azospirillum lipoferum. Laccase activity was detected in both a natural isolate and an in vitro-obtained phase variant that originated from the laccase-negative wild type. In this study, the electron transport systems of the laccase-positive variant and its parental laccase-negative forms were compared. During exponential (but not stationary) growth under fully aerobic (but not under microaerobic) conditions, the laccase-positive variant lost a respiratory branch that is terminated in a cytochrome c oxidase of the aa(3) type; this was most likely due to a defect in the biosynthesis of a heme component essential for the oxidase. The laccase-positive variant was significantly less sensitive to the inhibitory action of quinone analogs and fully resistant to inhibitors of the bc(1) complex, apparently due to the rearrangements of its respiratory system. We propose that the loss of the cytochrome c oxidase-containing branch in the variant is an adaptive strategy to the presence of intracellular oxidized quinones, the products of laccase activity.     

Keratinophilic fungi isolated from antarctic soil

In the present study, 10 soil samples were collected aseptically from an equal number of areas of the Antarctic in the zone occupied by the 1986–1987 Italian expedition for research on keratinophilic fungi.Of particular interest was the isolation of a pathogenic fungus, Microsporum gypseum, from two sites in the base camp occupied by men and by skuas. Trichophyton terrestre was isolated from a site in which people worked and through which penguins and skuas passed.The most widespread fungal species were members of the genus Chrysosporium. Some of these species were isolated but not identified and this part of the study was still be completed.Another significant finding was the absence of fungi in one sample, while in another the widespread and abundant growth in all the seeded dishes of a single species of Chrysosporium.Other species in genera of general diffusion in many environments were also isolated : Aspergillus spp., Malbranchea sp., Mycelia sterilia spp., Paecilomyces sp., Penicillium spp. and Scopulariopsis spp.     

Fungal decomposition of oat straw during liquid and solid state fermentation

White rot fungi (Coriolus hirsutus, Coriolus zonatus, and Cerrena maxima from the collection of the Komarov Botanical Institute of the Russian Academy of Sciences) and filamentous fungi (Mycelia sterilia INBI 2-26 and Trichoderma reesei 6/16) were grown on oat straw-based liquid and solid media, as well as in a bench-scale reactor, either individually or as co-cultures. All fungi grew well on solid agar medium supplemented with powdered oat straw as the sole carbon source. Under these conditions, the mould Trichoderma reesei fully suppressed the growth of all basidiomycetes studied; conversely, Mycelia sterilia neither affected the development of any of the cultures, nor did it show any substantial susceptibility to suppression by their presence. Pure solid cultures of basidiomycetes, as well as the co-culture of Coriolus hirsutus and Cerrena maxima caused a notable bleaching of the oat straw during its consumption. When grown on the surface of oat straw-based liquid medium, the basidiomycetes consumed up to 40% polysaccharides without measurable lignin degradation (a concomitant process). Under these conditions, Mycelia sterilia decomposed no more than 25% lignin in 60 days, but this was observed only after polysaccharide exhaustion and biomass accumulation. In contrast, during solid state straw fermentation, white rot fungi consumed up to 75% cellulose and 55% lignin in 83 days (C. zonarus), whereas the corresponding consumption levels for co-cultures of Mycelia sterilia and Trichoderma reesei equaled 70 and 45%, respectively (total loss of dry weight ranged from 55 to 60%). Carbon dioxide-monitored solid-state fermentation of oat straw by the co-culture of filamentous fungi was successfully performed in an aerated bench-scale reactor.     

Atrazine,desethylatrazine (DEA) and desisopropylatrazine (DIA) degradation by Pleurotus ostreatus INCQS 40310

Abstract

Atrazine is the most common herbicide applied in crops of economic relevance, such as sugar cane, soybean, and corn. Atrazine and its derivatives desethylatrazine (DEA) and desisopropylatrazine (DIA) are toxic to the environment, affecting animal and human health. Thus, this study aimed to evaluate the degradation of atrazine and its derivatives by the fungus Pleurotus ostreatus INCQS 40310, as well as the potential of the enzymes involved in this process. P. ostreatus INCQS 40310 was able to degrade atrazine (82%), DEA (71%), and DIA (56%) over 22?days of fungal cultivation. Proteomic analysis indicated the participation of hydrolases and peroxidases during the degradation process. Additionally, resting cells of the fungus were tested to verify the action of intracellular enzymes in the degradation process, suggesting the participation of cytochrome P450 enzymatic complex. Resting cells experiments promoted the degradation of 50% of atrazine, 36% of DIA, 30% of DEA. So far, this is the first work evaluating the biodegradation of DEA and DIA by fungus.     

Degradation of lignin-carbohydrate substrate by soil fungi--producers of laccase and cellobiose dehydrogenase

The growth of nonsporulating mycelial fungi INBI 2-26(+), producer of laccase; INBI 2-26(-), producer of cellobiose dehydrogenase; and their mixed culture on lignin-carbohydrate substrates under conditions of submerged fermentation were studied. The degrees of degradation of lignin, cellulose, and hemicellulose of cut straw over 23 days amounted to 29.8, 51.4, and 72% for the laccase producer; 15.8, 33.9, and 59.1% for the cellobiose dehydrogenase producer; and 15.8, 39.4, and 64.5% for the mixed culture, respectively. The laccase activity in the medium when strain 2-26(+) was cultivated individually reached its maximum on day 28; the activity of cellobiose dehydrogenase of strain 2-26(-), on days 14 to 28. A method for determining cellobiose dehydrogenase activity in the presence of laccase was developed. In the mixed culture, both enzymes were formed; however, the level of laccase synthesis was 1.5-fold lower compared to that of strain 2-26(+), while synthesis of cellobiose dehydrogenase was similar to that of the corresponding producer. Cellobiose dehydrogenase failed to boost the action of laccase while degrading the lignin of straw.     

Solid state fermentation of a Mycelia Sterilia laccase using steam-exploded wheat straw

Mycelia Sterilia YY-5, an endophytic fungus isolated from Rhus Chinensis Mill, was used in SSF for laccase production using steam-exploded wheat straw (SEWS). The fermentation period of YY-5 in solid state fermentation (SSF) shortened to 4 days compared with 5 days of submerged liquid fermentation (SmF) and the maximum laccase activity was 678.1 IU g⁻¹ substrate. The steam-explosion intensity (Log₁₀ R ₀) of SEWS had a significant effect on the growth of YY-5 and laccase activity, since SEWS could provide enough carbon source for YY-5 and inducers for laccase. The optimum SSF conditions using SEWS with Log₁₀ R ₀ = 3.597 as substrate were: inoculating with liquid inocula, keeping the solid-to-liquid ratio (S/L) for 1:4 and cultivating at 26°C. Under the optimum fermentation condition the laccase activity of YY-5 reached 849.5 ± 42.5 IU g⁻¹ substrate. The enzyme composition analysis indicated that laccase was the dominant enzyme of YY-5. Assayed with SDS-PAGE and active PAGE electrophoresis, the molecular weight of YY-5 laccase was approximately 45 kDa.     

Micropropagation and ecorestoration of Vanda spathulata,an exquisite orchid

Node explants collected from flowering plants of Vanda spathulata, an endemic and exquisite orchid of Peninsular India and Sri Lanka, were cultured in Mitra medium with combinations of 4.4–88.8 m 6-benzyl adenine (BA) and 0.0–114.2 m indole-3-acetic acid (IAA). Combinations of 44.4 m BA with 17.1 or 28.5 m IAA and 66.6 mM BA with 28.5 or 40.0 m IAA induced maximum formation of 12.6 and 12.1 shoots / node, respectively, in a 6-month period. Subcultured nodal explants produced maximum of 6.1 shoots at combinations of 22.2–44.4 m 21 BA and 5.7–28.5 m IAA. Rooting of shoots occurred in medium containing 75 g l^–1 banana pulp and 5.7 m IAA within 3–9 weeks. Plantlets of 2–5 cm length possessing two to five roots established easily in community pots at 80–90% rates without hardening. Community potted plants introduced into forest segments at Ponmudi and Palode in Southern Western Ghats of India established at a rate of 50–70%.     

Cumulative effect of low and high atrazine concentrations on <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants

Atrazine belongs to the widely used herbicides blocking the electron transport chain in chloroplasts, thus resulting in the generation of active oxygen species. In the present work, we demonstrated that, at low concentrations mimicking residual amounts, atrazine enhanced the susceptibility of Arabidopsis plants to further treatments with the same herbicide applied at the recommended field rate. Arabidopsis thaliana plants were treated three times (at five-day intervals) with 1 µM atrazine. Five days after the last treatment, the plants were sprayed with 5 mM atrazine. Atrazine increased the levels of lipid peroxidation products, hydrogen peroxide, and ion leakage, and caused changes in the activities of antioxidant enzymes, such as superoxide dismutase, guaiacol peroxidase, and catalase.From Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 243–249.Original English Text Copyright © 2005 by Ivanov, Alexieva, Karanov.This article was submitted by the authors in English.This revised version was published online in April 2005 with a corrected cover date.     

Mechanism of Overproduction of Secreted Enzymes in the Mycelial Fungus Penicillium canescens

The fungus Penicillium canescens strain F178 (VKPM) and its niaD ^– mutant exhibited an increased capability of synthesizing extracellular enzymes -galactosidase (50–60 U/ml) and xylanase (100 U/ml). The synthesis was induced by arabinose and its catabolite, arabitol. A deficiency in arabitol dehydrogenase, leading to arabitol accumulation in the cell, was detected in the chain of reactions of arabinose catabolism. The increased synthesis of -galactosidase and xylanase in P. canescens is accounted for by (1) cellular accumulation of the inducer (arabitol) at low concentrations of arabinose in the medium and (2) prevalence of induction over repression.     

Demethylation and delignification of kraft pulp by Trametes versicolor laccase in the presence of 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate)

Summary Bleaching of hardwood kraft pulp by Trametes versicolor was accompanied by release and accumulation of methanol, which was produced by demethylation of the pulp. A partial demethylation of the pulp was observed with isolated laccase I from T. versicolor. The extent of demethylation by laccase was increased to the level released by the fungus by addition of 2,2-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS). Methanol release by the laccase/ABTS combination was followed by slower kappa reduction. Both methanol release and kappa reduction were dependent on laccase and ABTS concentrations. The fungus did not produce a stable equivalent of ABTS during bleaching, because extracellular culture fluid from bleaching cultures gave only the same methanol release from pulp as laccase I. Pulp viscosity, an indicator of cellulose chain length, was decreased only slightly by laccase. Thus the enzyme in the presence of ABTS, unlike the fungus, specifically attacks lignin.Offprint requests to: R. Bourbonnais