Evaluación del consorcio entre Pleurotus ostreatus, Trametes versicolor y bacterias aeróbicas para remoción de colorantes sintéticos / Evaluation of the consortium between Pleurotus ostreatus, Trametes versicolor and aerobic bacteria for removal of synthetic dye

RESUMEN Los residuos líquidos producidos al elaborar tinciones biológicas contienen mezclas de compuestos químicos y microorganismos, que generan un elevado impacto ambiental si no son tratados adecuadamente. Por esta razón, en el presente trabajo se evaluaron a Pleurotus ostreatus, Trametes versicolor, Enterobacter xianfangensis, Pseudomonas azotoformans, Pseudomonas sp., Bacillus subtilis y Pseudomonas fluorescens, para el tratamiento de un residuo líquido que contenía colorantes trifenilmetánicos y azóicos, a escala de laboratorio. Inicialmente, se seleccionaron las cepas con menor efecto antagónico y se determinó su potencial para producir las enzimas Lacasa, Manganeso Peroxidasa y Lignino Peroxidasa, al emplear sustratos inductores y mezclas de colorantes. Para el consorcio fúngico/bacteriano la disminución de las unidades de color y demanda química de oxígeno fueron del 99 % y 70 % a las 96 h. La remoción de estos parámetros se relacionó con la interacción positiva e incremento de las poblaciones de hongos, bacterias y la producción de enzimas ligninolíticas, obteniendo valores a las 96 h de 7.0 y 14.0 unidades logarítmicas para hongos y bacterias, con unas actividades enzimáticas de 75 U/L, 205 U/L y 0.63 U/L para Lacasa, MnP y LiP, respectivamente. Con el presente trabajo se demostró que con el uso consorcios fúngicos/bacterianos se incrementa la remoción de colorantes y se disminuye el tiempo de proceso. Sugiriendo que estos microorganismos podrían ser evaluados en plantas de tratamiento que integren diferentes unidades de tratamiento para optimizar la remoción de contaminantes con baja biodegradabilidad.

ABSTRACT The liquid wastes generated when biological stains are prepared, contain a mixture of chemical compounds and microorganisms, with high environmental impact. For this reason, Pleurotus ostreatus, Trametes versicolor, Enterobacter xianfangensis, Pseudomonas azotoformans, Pseudomonas sp., Bacillus subtilis and Pseudomonas fluorescens, were used to evaluate the treatment of a liquid waste containing triphenylmethane and azo dyes, on a laboratory scale. Initially, the strains with less antagonistic effect among them were selected for their potential to produce enzymes as Laccase, Manganese Peroxidase and Lignin Peroxidase. The enzymatic activity was determined by using inducing substrates and dye mixtures. For fungal/bacterial consortium, the decrease in color, Chemical Oxygen Demand and in Biochemical Oxygen demand was of 99 %, 70 % and 65 % at 96 h, respectively. The removal of these parameters was related to the positive interaction between the populations of fungi, bacteria and the production of ligninolytic enzymes, obtaining values of 7.0 and 14.0 logarithmic units for fungi and total bacteria at 96 h with enzymatic activities of 75 U/ L, 205 U/L and 0.63 U/L for Laccase, MnP and LiP. The present work demonstrates that using of fungal/bacterial consortia, the removal of dyes is increased, and the process time is decreased. Suggesting that these microorganisms could be evaluated in treatment plants that integrate different treatment units to optimize the removal of contaminants with low biodegradability.

Morphological Characteristic Regulation of Ligninolytic Enzyme Produced by Trametes polyzona

A newly isolated white rot fungal strain KU-RNW027 was identified as Trametes polyzona, based on an analysis of its morphological characteristics and phylogenetic data. Aeration and fungal morphology were important factors which drove strain KU-RNW027 to secrete two different ligninolytic enzymes as manganese peroxidase (MnP) and laccase. Highest activities of MnP and laccase were obtained in a continuous shaking culture at 8 and 47 times higher, respectively, than under static conditions. Strain KU-RNW027 existed as pellets and free form mycelial clumps in submerged cultivation with the pellet form producing more enzymes. Fungal biomass increased with increasing amounts of pellet inoculum while pellet diameter decreased. Strain KU-RNW027 formed terminal chlamydospore-like structures in cultures inoculated with 0.05 g/L as optimal pellet inoculum which resulted in highest enzyme production. Enzyme production efficiency of T. polyzona KU-RNW027 depended on fungal pellet morphology as size, porosity, and formation of chlamydospore-like structures.

Ligninolytic fungus Polyporus sp. S133 mediated metabolic degradation of fluorene

ABSTRACT This study aimed to investigate the impact of nonionic surfactants on the efficacy of fluorine degradation by Polyporus sp. S133 in a liquid culture. Fluorene was observed to be degraded in its entirety by Polyporus sp. S133 subsequent to a 23-day incubation period. The fastest cell growth rate was observed in the initial 7 days in the culture that was supplemented with Tween 80. The degradation process was primarily modulated by the activity of two ligninolytic enzymes, laccase and MnP. The highest laccase activity was stimulated by the addition of Tween 80 (2443 U/L) followed by mixed surfactant (1766 U/L) and Brij 35 (1655 U/L). UV-vis spectroscopy, TLC analysis and mass spectrum analysis of samples subsequent to the degradation process in the culture medium confirmed the biotransformation of fluorene. Two metabolites, 9-fluorenol (λmax 270, tR 8.0 min and m/z 254) and protocatechuic acid (λmax 260, tR 11.3 min and m/z 370), were identified in the treated medium.

Screening of ligninase-producing bacteria from south east Pahang peat swamp forest soil

Aims: Research on lignin degradation capability is previously restricted exclusively to fungal enzymes. However, recent studies had successfully revealed several soil bacterial strains that were able to produce ligninolytic enzymes. These bacterial ligninolytic enzymes were claimed to be more specific in catalysing cleavage of certain linkages between phenolic units of lignin polymers as compared to fungal enzymes. The present study focuses on screening for ligninaseproducing bacteria isolated from South East Pahang Peat Swamp Forest (SEPPSF) soil using agar-based assay. Methodology and results: Thirteen isolates used in this study, which were selected based on distinctive colony morphology from our previous isolation work, showed decolourisation zone on Azure B plates screening. The ratio of decolourisation zones were measured to the ratio of the colony size and the biggest ratio was 2.22 by isolate AR1. Only 4 out of the 13 isolates were able to grow on lignin plates. Subsequently, the 4 isolates, AR3, AR8, AR10 and AR13 were tested on M1 agar supplemented with 3 ligninolytic enzyme indicator compounds which were tannic acid (TA), guaiacol and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) respectively. All four isolates showed growth on TA plates while only AR10 showed a clear brown coloration. An Intense reddish-brown colour formation was observed around the colony of isolates AR3 and AR10 on guaiacol plates while none exhibited green coloration around the colonies when tested on ABTS plates. Conclusions, significance and impacts of study: Isolate AR10 that was identified as Serratia sp. was perceived to be a potential ligninase-producer, though in-depth analysis has to be conducted in the future to determine the specific ligninolytic enzyme activities and characteristics. The application of different substrates is essential to investigate the ligninolytic potential and reaction of those bacterial enzymes towards different indicator compounds. This study is a preliminary endeavour concerning potential ligninolytic enzymes from bacteria as biocatalysts in various industrial processes. This is the first report on preliminary study for ligninolytic activities of soil bacteria from SEPPSF soil.

Screening and identification of Trametes lactinea and Pycnoporouscoccineus isolated from Kampar for laccase production

Aims: Laccase is a blue copper oxidase that catalyses four electron reduction of molecular oxygen to water. It is able to oxidise aromatic compounds with molecular oxygen as the terminal electron acceptor. The aim of this study was to screen for laccase producing basidiomycetes isolated from decaying woods and tree trunks around Kampar, Perak. Methodology and results: The isolated basidiomycetes were screened for their laccase activity on different agar plates supplemented with 2, 2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), guaiacol and Remazol Brillant Blue R (RBBR), respectively. In the presence of laccase, the colourless ABTS and guaiacol were oxidised to form blue-green and reddish-brown coloured zone around the fungal colony, respectively; whereas the blue RBBR was decolourised by the enzyme. Colour or colourless halo zones that are formed on the agar plates indicate the presence of ligninolytic enzyme activities. Isolates KA1 and TR9 indicated the highest enzymatic hydrolysis on ABTS plates with the halo zone ratio of 1.43  0.04 and 0.98  0.01, respectively. Based on the BLAST results from the amplicon of ITS1 and ITS4 primers, Isolates KA1 and TR9 were identified as Trametes lactinea and Pycnoporous coccineus, respectively. Under submerged fermentation, P. coccineus has higher laccase production (0.72 U/mL) compared with T. lactinea (0.16 U/mL). Conclusion, significance and impact of study: Both T. lactinea and P. coccineus are potential strains for laccase production which can be used for dye decolourisation and degradation. Future studies will focus on the application of the laccase in textile dye degradation.

Selection of white-rot fungi to formulate complex and coated pellets for Reactive Orange 165 decolourization

Six strains of white-rot fungi isolated from southern Chile were evaluated for their ergosterol/biomass correlation and ligninolytic potential in solid medium to formulate pellets for Reactive Orange 165 (RO165) decolourization. The fungus Anthracophyllum discolor was selected to formulate complex pellets (fungal mycelium, sawdust, and activated carbon), coated pellets (complex pellet + alginate) and simple pellets (fungal mycelium). The activity of ligninolytic enzymes (laccase, manganese peroxidase, manganese-independent peroxidase, and lignin peroxidase) was evaluated in both the complex and coated pellets in modified Kirk medium, and the morphology of the pellets was studied using scanning electron microscopy (SEM). Complex pellets of A. discolor showed a higher enzymatic production mainly MnP (38 U L-1 at day 15) compared to coated and simple pellets. Examinations using SEM showed that both pellets produced a black core that was entrapped by a layer of fungal mycelium. Decolourization of RO165 was demonstrated with all the pellets formulated. However, the highest and fastest decolourization was obtained with complex pellets (100 percent at day 8). Therefore, complex pellets of A. discolor can be used for the biological treatment of wastewater contaminated with RO165.

A practical culture technique for enhanced production of manganese peroxidase by Anthracophyllum discolor Sp4

In this study, different growth conditions of Anthracophyllum discolor Sp4 including the effect of agitation, additions of lignocellulosic support, inducer and surfactant were evaluated on the MnP production in Kirk medium using a culture system made up of the tubes containing the glass bead . The highest MnP production (1,354 U/L on day 13) was obtained when the medium was supplemented with wheat grain and 0.25 mM MnSO4 as inducer, under static conditions at 30°C. Two isoenzymes were purified (35 and 38 kDa respectively). MnP presented a maximal activity in the pH range between 4.5 and 5.5, a relatively high temperature tolerance (50ºC) and a high catalytic activity for 2,6-dimethoxyphenol and hydrogen peroxide.

Size exclusion chromatography for the removal of pigments from extracellular ligninolytic enzyme extracts from decayed wheat straw.

Solid-state fermentation of wheat straw was carried out by a native white rot basidiomycete Daedaleopsis flavida strain 5A. Extract prepared from the 12-day decayed wheat straw contained extracellular ligninolytic enzymes like manganese peroxidase (MnP), manganese-independent peroxidase (MIP), lignin peroxidase (LiP) and laccase along with straw-degraded products and pigments. Sephacryl S-200 size exclusion chromatography in 16/100 column was used for the separation of these ligninolytic enzymes and strawdegraded products and pigments. Recovery of pigment-free ligninolytic enzyme activities as protein was 40% of the total proteins loaded and specific LiP activity increased 34 fold after size exclusion chromatography. Thus accurate estimation of LiP by veratryl alcohol oxidation assay was possible only after the removal of interfering pigments. The reproducibility of size exclusion chromatography is adjudged satisfactory from the consistent results obtained after seven repetitive uses of matrices.

Degradation of lignosulfonic and tannic acids by ligninolytic soil fungi cultivated under icroaerobic conditions

Soil fungi were evaluated regarding their ability to degrade lignin-related compounds by producing the ligninolytic enzymes. Lignosulfonic and tannic acids were used as sole carbon sources during 30 days under microaerobic and very-low-oxygen conditions. The fungi produced lignin-peroxidase, manganese-peroxidase and laccase . Expressive degradations was observed by C18 reversed-phase HPLC, indicating the biodegradation potential of these fungi, showing more advantages than obligate anaerobes to decontaminate the environment when present naturally.

Fungos isolados de solo foram avaliados quanto à habilidade em degradarem compostos derivados de lignina pela produção de enzimas ligninolíticas. Os ácidos lignosulfônicos e tânico foram usados separadamente como única fonte de carobono para cultivo dos fungos em 30 dias sob condições microaeróbias. Os fungos foram capazes de crescer e usar tais compostos como fonte de carbono e mostraram produção de lignina-peroxidase, manganês-peroxidase e lacase. Degradações expressivas dos ácidos lignosulfônico e tânico foram verificadas por Cromatografia Liquida de Alta Eficiência (CLAE), indicando grande potencial de uso em processos de biorremediação de macromoléculas aromáticas similares à lignina em ambientes naturais sob condições baixas de oxigenação.

Screening for ligninolytic enzymes from autochthonous fungi and applications for decolorization of Remazole Marine Blue / Triagem de enzimas ligninolíticas de fungos autóctones e aplicações para descoloramento de Remazole Marine Blue

This study presents new and alternative fungal strains for the production of ligninolytic enzymes which have great potential to use in industrial and biotechnological processes. Thirty autochthonous fungal strains were harvested from Bornova-Izmir in Turkiye. In the fresh fruitbody extracts laccase, manganese peroxidase and lignin peroxidase activities, which are the principal enzymes responsible for ligninocellulose degradation by Basidiomycetes, were screened. Spores of some of the basidiomycetes species such as Cortinarius sp., Trametes versicolor, Pleurotus ostreatus, Abortiporus biennis, Lyophyllum subglobisporium, Ramaria stricta, Ganoderma carnosum, Lactarius delicious ve Lepista nuda were isolated and investigated optimum cultivation conditions in submerged fermentation for high yields of ligninolytic enzyme production. In addition, isolated fungal strains were monitored on agar plates whether having the capability of decolorization of a textile dye Remazol Marine Blue.

Este estudo apresenta novas cepas de fungos produtores de enzimas ligninolíticas com potencial de aplicação em processos industriais e biotecnológicos. Trinta cepas de fungos autóctones foram obtidos em Bornova-Izmir, Turquia. Os extratos frescos dos corpos de frutificação foram submetidos à triagem de atividade de lacase, manganês peroxidase e lignina peroxidase, que são as principais enzimas de degradação de ligninocelulose pelos Basidiomycetes. Foram isolados esporos de Cortinarius sp, Tramnetes versicolor, Pleorotus ostreatus, Abortiporus biennis, Lyophyllum subglobisporium, Ramaria stricta, Ganoderma carnosum, Lactarius delicius ve Lepista desnuda, investigando-se as condições ótimas de cultivo em fermentação submersa para produção de enzimas ligninolíticas com elevado rendimento. Além disso, as cepas fúngicas isoladas foram monitoradas em placas de ágar quanto a capacidade de descoloramento do corante têxtil Remazole Marine Blue.

Degradation of Three Aromatic Dyes by White Rot Fungi and the Production of Ligninolytic Enzymes

This study was conducted to evaluate the degradation of aromatic dyes and the production of ligninolytic enzymes by 10 white rot fungi. The results of this study revealed that Pycnoporus cinnabarinus, Pleurotus pulmonarius, Ganoderma lucidum, Trametes suaveolens, Stereum ostrea and Fomes fomentarius have the ability to efficiently degrade congo red on solid media. However, malachite green inhibited the mycelial growth of these organisms. Therefore, they did not effectively decolorize malachite green on solid media. However, P. cinnabarinus and P. pulmonarius were able to effectively decolorize malachite green on solid media. T. suaveolens and F. rosea decolorized methylene blue more effectively than any of the other fungi evaluated in this study. In liquid culture, G. lucidum, P. cinnabarinus, Naematoloma fasciculare and Pycnoporus coccineus were found to have a greater ability to decolorize congo red. In addition, P. cinnabarinus, G. lucidum and T. suaveolens decolorized methylene blue in liquid media more effectively than any of the other organisms evaluated in this study. Only F. fomentarius was able to decolorize malachite green in liquid media, and its ability to do so was limited. To investigate the production of ligninolytic enzymes in media containing aromatic compounds, fungi were cultured in naphthalene supplemented liquid media. P. coccineus, Coriolus versicolor and P. cinnabarinus were found to produce a large amount of laccase when grown in medium that contained napthalene.

Production of phenol-oxidases and peroxidases by fungi isolated from irrigated rice

The aim of this work was to study the potential of fungus strains considered as prospective degraders for the herbicides quinclorac and propanil, for ligninolytic enzyme production. Eight fungal strains were grown in King's B liquid culture medium supplemented with 0.05% Remazol Brilliant Blue R (RBBR) and in liquid culture medium containing wheat bran as substrate. The enzymatic system assessment were: lignin peroxidase, manganese peroxidase and laccases. Results indicated differential patterns of ligninolytic enzyme production; the highest enzymatic activities were related to the production of lignin peroxidase. Among the strains, two (P3SA1F and P11SA2F) showed RBBR discoloration, suggesting the possibility of their application in bioremediation studies.

A proposta deste trabalho foi estudar o potencial das linhagens fúngicas, consideradas potenciais degradadoras dos herbicidas quinclorac e propanil, para produção de enzimas ligninolíticas. Oito linhagens fúngicas foram cultivadas em meio de cultura líquido King's B suplementado com 0,05% de Remazol Brilliant Blue R (RBBR) e em meio de cultura líquido contendo farelo de trigo como substrato. Os sistemas enzimáticos avaliados foram: lignina peroxidase, manganês peroxidase e lacases. Os resultados demonstraram padrões diferenciados quanto à produção de enzimas ligninolíticas entre as linhagens, sendo que as maiores atividades enzimáticas estiveram relacionadas à produção de lignina peroxidase. Das oito linhagens, duas (P3SA1F e P11SA2F) apresentaram descoloração do RBBR, sugerindo a possibilidade de sua aplicabilicação em estudos de biorremediação

Production of phenol-oxidases and peroxidases by fungi isolated from irrigated rice

The aim of this work was to study the potential of fungus strains considered as prospective degraders for the herbicides quinclorac and propanil, for ligninolytic enzyme production. Eight fungal strains were grown in King's B liquid culture medium supplemented with 0.05% Remazol Brilliant Blue R (RBBR) and in liquid culture medium containing wheat bran as substrate. The enzymatic system assessment were: lignin peroxidase, manganese peroxidase and laccases. Results indicated differential patterns of ligninolytic enzyme production; the highest enzymatic activities were related to the production of lignin peroxidase. Among the strains, two (P3SA1F and P11SA2F) showed RBBR discoloration, suggesting the possibility of their application in bioremediation studies.

A proposta deste trabalho foi estudar o potencial das linhagens fúngicas, consideradas potenciais degradadoras dos herbicidas quinclorac e propanil, para produção de enzimas ligninolíticas. Oito linhagens fúngicas foram cultivadas em meio de cultura líquido King's B suplementado com 0,05% de Remazol Brilliant Blue R (RBBR) e em meio de cultura líquido contendo farelo de trigo como substrato. Os sistemas enzimáticos avaliados foram: lignina peroxidase, manganês peroxidase e lacases. Os resultados demonstraram padrões diferenciados quanto à produção de enzimas ligninolíticas entre as linhagens, sendo que as maiores atividades enzimáticas estiveram relacionadas à produção de lignina peroxidase. Das oito linhagens, duas (P3SA1F e P11SA2F) apresentaram descoloração do RBBR, sugerindo a possibilidade de sua aplicabilicação em estudos de biorremediação