Antarctic marine sediment as a source of filamentous fungi-derived antimicrobial and antitumor compounds of pharmaceutical interest.

Antarctica harbors a microbial diversity still poorly explored and of inestimable biotechnological value. Cold-adapted microorganisms can produce a diverse range of metabolites stable at low temperatures, making these compounds industrially interesting for biotechnological use. The present work investigated the biotechnological potential for antimicrobial and antitumor activity of filamentous fungi and bacteria isolated from marine sediment samples collected at Deception Island, Antarctica. A total of 89 microbial isolates were recovered from marine sediments and submitted to an initial screening for L-glutaminase with antitumoral activity and for antimicrobial metabolites. The isolates Pseudogymnoascus sp. FDG01, Pseudogymnoascus sp. FDG02, and Penicillium sp. FAD33 showed potential antiproliferative action against human pancreatic carcinoma cells while showing no toxic effect on non-tumor cells. The microbial extracts from unidentified three bacteria and four filamentous fungi showed antibacterial activity against at least one tested pathogenic bacterial strain. The isolate FDG01 inhibited four bacterial species, while the isolate FDG01 was active against Micrococcus luteus in the minimal inhibitory concentration of 0.015625 µg mL -1. The results pave the way for further optimization of enzyme production and characterization of enzymes and metabolites found and reaffirm Antarctic marine environments as a wealthy source of compounds potentially applicable in the healthcare and pharmaceutical industry.

Pharmaceutical biotechnological potential of filamentous fungi isolated from textile industry.

The need for more effective drugs for the treatment of infectious diseases as well as for general applications including wound healing and burn surgery, has guided efforts for the discovery of new compounds of medical interest. Microorganisms found in textile industrial waste have the ability to produce a variety of enzymes and/or secondary metabolites including molecules of pharmaceutical interest. The present work investigated the biotechnological potential of filamentous fungi isolated from textile industry wastewater for the production of collagenase and antimicrobial metabolites. From 28 isolates assayed, Sarocladium sp. ITF33 showed specific collagenolytic activity with values of 7.62 and 9.04 U mg-1 for the intracellular and extracellular fractions, respectively. The isolate Penicillium sp. ITF28 showed the best antimicrobial activity, reaching MIC ranging from 1.0 to 0.0625 mg mL-1 against five pathogenic bacteria. Molecular analyzes suggest that the isolate Sarocladium sp. ITF 33 can be considered a species not yet described. The results of the present work encourage studies of characterization and purification of the enzymes and secondary metabolites produced by the isolates found aiming future applications in the medical and pharmaceutical fields.

Tolerância de microrganismos eucariotos ao herbicida glifosato / Tolerance of eukaryotic microorganisms to glyphosate herbicide

O glifosato é um herbicida amplamente utilizado. A Organização Mundial da Saúde (OMS) reclassificou o glifosato como "provavelmente cancerígeno a humanos". A remoção do glifosato do ambiente pode ser realizada por ação enzimática microbiana. O presente trabalho enfocou o isolamento de microrganismos do solo capazes de tolerar glifosato como única fonte de carbono. As células foram isoladas em meio de cultivo mínimo suplementado com glifosato. Foram isoladas 17 bactérias, 14 fungos e 1 levedura. Foi verificada a produção da biomassa microbiana na presença e na ausência do glifosato. Um fungo (F3) e uma levedura (L1) foram selecionados após teste de tolerância ao glifosato em meio líquido. Os microrganismos toleraram o glifosato, entretanto, o metabolismo foi afetado pelo herbicida, comparado ao controle sem glifosato. Estatisticamente, o tempo de crescimento apresentou diferenças significativas. Microrganismos eucarióticos isolados de solo com glifosato são tolerantes ao composto e podem ser úteis como biorremediadores de ambientes afetados por este herbicida.(AU)

Glyphosate is one of the most widely used herbicides. The World Health Organization (WHO) has reclassified glyphosate as "probably carcinogenic to humans". Glyphosate removal from the environment can be performed by microbial enzymatic action. The present work focused on the isolation of soil microorganisms that can tolerate glyphosate as the sole carbon source. Cells were isolated in minimal culture medium supplemented with glyphosate. Microbial biomass production was verified in the presence and absence of glyphosate. Seventeen, fourteen and one bacteria, fungi and yeast were isolated, respectively. One fungus (F3) and one yeast (L1), were selected after glyphosate tolerance test in liquid medium. Eukaryotic microorganisms tolerate glyphosate, however metabolism was affected by herbicide compared to control without glyphosate. Statistically growth time showed significant differences. Eukaryotic microorganisms isolated from soil with glyphosate are tolerant to the compound and may be useful as bioremediators of environments affected by this herbicide.(AU)

Antifungal activity of homeopathic medicines against the white mold causing agent Sclerotinia sclerotiorum

White mold disease, caused by fungus Sclerotinia sclerotiorum (Lib.) de Bary., is a disease hard to control due to the high amount of sclerotia produced, which guarantees its survival in the soil for years leading to significant yield losses. Alternative techniques to control the pathogen have been researched, including homeopathy. The present work aimed to evaluate the in vitro antifungal effect of homeopathic medicines on S. sclerotiorum mycelial growth. Homeopathic medicines Sulphur, fungal sclerotium Nosode and Calcarea carbonica, in 30CH, 200CH and 1000CH dynamizations were tested. Assays were carried out in a completely randomized design, with four repetitions. Experiments were performed through the addition of homeopathic medicines on the surface of plates containing culture medium, followed by insertion of a disc containing fungus mycelia and incubation. Control treatment received no homeopathic medicine. The mycelial progression was monitored by seven halo diameter measurements during experiment period. All homeopathic medicines tested and their dynamizations were able to inhibit partially the development of the fungus. Calcarea carbonica at the dynamization of 1000 CH showed the best inhibitory effect on S. sclerotiorum, which under its effect produced a mycelial halo 40% smaller than the control treatment.

Uso de microrganismos de efluente industrial no controle biológico de vetores / Use of industrial effluent microorganisms in biological vector control / Uso de microorganismos de efluentes industriales en el control biológico de vectores

Justificativa e Objetivos: a utilização de microrganismos como controle biológico de vetores sanitários pode ser considerada uma prática menos agressiva ao ambiente, em comparação com os produtos químicos utilizados. O presente estudo avaliou a eficiência de suspensões celulares de fungos e bactérias isolados de efluentes industriais têxteis no controle sanitário dos vetores naturais Aedes aegypti e Dermacentor nitens como alternativa sustentável de controle biológico. Métodos: foram avaliadas sete linhagens de fungos e seis de bactérias. Os isolados foram cultivados em caldo nutriente e caldo de batata, para bactérias e fungos, respectivamente. Alíquotas de 2 mL de cada suspensão microbiana foram adicionadas diretamente nas larvas dos mosquitos e nos carrapatos adultos. Foram analisadas alterações de movimentação e paralisação dos vetores em diferentes tempos de exposição entre zero e 20 minutos e três e 24 horas. Resultados: duas bactérias e um fungo promoveram uma desaceleração dos movimentos e/ou um aumento da movimentação dos ectoparasitas logo após a administração. Dois isolados bacterianos promoveram a paralisação dos movimentos de uma larva do mosquito Aedes aegypti em seu primeiro estágio de desenvolvimento, enquanto que um fungo provocou aumento da movimentação das larvas em seu estágio de desenvolvimento mais avançado. Conclusão: os microrganismos mostraram potencial uso no controle de vetores sanitários. Testes subsequentes de atividade dos possíveis metabólitos secundários produzidos e das formas de administração das culturas microbianas serão executados. Os resultados encontrados encorajam futuros estudos de otimização e caracterização dos extratos celulares, os quais poderão ser utilizados como ferramenta sustentável no controle biológico.(AU)

Background and Objectives: the use of microorganisms as biological control of health vectors can be considered a less aggressive practice to the environment, in comparison with the chemicals used. The present study evaluated the efficiency of cell suspensions of fungi and bacteria isolated from industrial textile effluents in the sanitary control of the natural vectors Aedes aegypti and Dermacentor nitens as a sustainable alternative for biological control. Methods: seven fungi and six bacteria strains were evaluated. The isolates were grown in nutrient broth and potato broth, for bacteria and fungi, respectively. 2 mL aliquots of each microbial suspension were added directly to mosquito larvae and adult ticks. Changes in movement and paralysis of vectors at different exposure times between zero and 20 minutes and three and 24 hours were analyzed. Results: two bacteria and a fungus promoted a slowdown in movement and / or an increase in the movement of ectoparasites shortly after administration. Two bacterial isolates caused the movement of a larva of the Aedes aegypti mosquito to stop in its first stage of development, while a fungus caused increased movement of the larvae in their most advanced stage of development. Conclusion: the microorganisms showed potential use in the control of health vectors. Subsequent activity tests of the possible secondary metabolites produced and the ways of administering the microbial cultures will be performed. The results found encourage future studies of optimization and characterization of cell extracts, which can be used as a sustainable tool in biological control.(AU)

Justificación y objetivos: el uso de microorganismos como control biológico de vectores sanitarios puede considerarse una práctica menos agresiva para el medio ambiente en comparación con los productos químicos utilizados. El presente estudio evaluó la eficiencia de las suspensiones de células fúngicas y bacterianas de efluentes industriales textiles en el control sanitario de los vectores Aedes aegypti y Dermacentor nitens como una alternativa sostenible para el control biológico. Métodos: evaluaron siete hongos y seis de bacterias. Los aislamientos se cultivaron en medio de cultivo, caldo nutrientes y de papa para bacterias y hongos, respectivamente. Se agregaron alícuotas de 2 mL de cada suspensión microbiana directamente a las larvas de mosquito y las garrapatas adultas. Analizaron los cambios en el movimiento y la parálisis de los vectores a diferentes tiempos de exposición entre cero y 20 minutos y tres y 24 horas. Resultados: dos bacterias y un hongo causaron una reducido el movimiento y/o aumentó el movimiento del ectoparásito poco después de la administración. Dos bacterias paralizaron los movimientos de las larvas de un mosquito en su primera etapa de desarrollo y un hongo causó un mayor movimiento de las larvas en su etapa posterior de desarrollo. Conclusión: los microorganismos mostraron uso potencial como control de vectores sanitarios. Se realizarán pruebas de actividad de los posibles metabolitos secundarios producidos y las formas de administración de los cultivos microbianos. Los resultados fomentan más estudios de optimización y caracterización de extractos celulares, que pueden utilizarse como herramienta sostenible en el control biológico.(AU)

Exploring the genetic potential of a fosmid metagenomic library from an oil-impacted mangrove sediment for metabolism of aromatic compounds.

Aromatic hydrocarbons (AH) are widely distributed in nature, and many of them have been reported as relevant environmental pollutants and valuable carbon sources for different microorganisms. In this work, high-throughput sequencing of a metagenomic fosmid library was carried out to evaluate the functional and taxonomic diversity of genes involved in aromatic compounds degradation in oil-impacted mangrove sediments. In addition, activity-based approach and gas chromatography were used to assess the degradation potential of fosmid clones. Results indicated that AH degradation genes, such as monooxygenases and dioxygenases, were grouped into the following categories: anaerobic degradation of aromatic compounds (20.34%), metabolism of central aromatic intermediates (35.40%) and peripheral pathways for catabolism of aromatic compounds (22.56%). Taxonomic affiliation of genes related to aromatic compounds metabolism revealed the prevalence of the classes Alphaproteobacteria, Actinobacteria, Betaproteobacteria, Gammaproteobacteria and Deltaproteobacteria. Aromatic hydrocarbons (phenol, naphthalene, phenanthrene, pyrene and benzopyrene) were used as the only carbon source to screen clones with degradation potential. Of the 2500 clones tested, 48 showed some respiratory activity in at least one of the five carbon sources used. The hydrocarbon degradation ability of the top ten fosmid clones was confirmed by GC-MS. Further, annotation of assembled metagenomic fragments revealed ORFs corresponding to proteins and functional domains directly or indirectly involved in the aromatic compound metabolism, such as catechol 2,3-dioxygenase and ferredoxin oxidoreductase. Finally, these data suggest that the indigenous mangrove sediment microbiota developed essential mechanisms towards ecosystem remediation of petroleum hydrocarbon impact.

A Novel Multifunctional ß-N-Acetylhexosaminidase Revealed through Metagenomics of an Oil-Spilled Mangrove.

The use of culture-independent approaches, such as metagenomics, provides complementary access to environmental microbial diversity. Mangrove environments represent a highly complex system with plenty of opportunities for finding singular functions. In this study we performed a functional screening of fosmid libraries obtained from an oil contaminated mangrove site, with the purpose of identifying clones expressing hydrolytic activities. A novel gene coding for a ß-N-acetylhexosaminidase with 355 amino acids and 43KDa was retrieved and characterized. The translated sequence showed only 38% similarity to a ß-N-acetylhexosaminidase gene in the genome of Veillonella sp. CAG:933, suggesting that it might constitute a novel enzyme. The enzyme was expressed, purified, and characterized for its enzymatic activity on carboxymethyl cellulose, p-Nitrophenyl-2acetamide-2deoxy-ß-d-glucopyranoside, p-Nitrophenyl-2acetamide-2deoxy-ß-d-galactopyranoside, and 4-Nitrophenyl ß-d-glucopyranoside, presenting ß-N-acetylglucosaminidase, ß-glucosidase, and ß-1,4-endoglucanase activities. The enzyme showed optimum activity at 30 °C and pH 5.5. The characterization of the putative novel ß-N-acetylglucosaminidase enzyme reflects similarities to characteristics of the environment explored, which differs from milder conditions environments. This work exemplifies the application of cultivation-independent molecular techniques to the mangrove microbiome for obtaining a novel biotechnological product.

Functional metagenomics of oil-impacted mangrove sediments reveals high abundance of hydrolases of biotechnological interest.

Mangroves are located in coastal wetlands and are susceptible to the consequences of oil spills, what may threaten the diversity of microorganisms responsible for the nutrient cycling and the consequent ecosystem functioning. Previous reports show that high concentration of oil favors the incidence of epoxide hydrolases and haloalkane dehalogenases in mangroves. This finding has guided the goals of this study in an attempt to broaden the analysis to other hydrolases and thereby verify whether oil contamination interferes with the prevalence of particular hydrolases and their assigned microorganisms. For this, an in-depth survey of the taxonomic and functional microbial diversity recovered in a fosmid library (Library_Oil Mgv) constructed from oil-impacted Brazilian mangrove sediment was carried out. Fosmid DNA of the whole library was extracted and submitted to Illumina HiSeq sequencing. The resulting Library Oil_Mgv dataset was further compared with those obtained by direct sequencing of environmental DNA from Brazilian mangroves (from distinct regions and affected by distinct sources of contamination), focusing on hydrolases with potential use in biotechnological processes. The most abundant hydrolases found were proteases, esterases and amylases, with similar occurrence profile in all datasets. The main microbial groups harboring such hydrolase-encoding genes were distinct in each mangrove, and in the fosmid library these enzymes were mainly assigned to Chloroflexaceae (for amylases), Planctomycetaceae (for esterases) and Bradyrhizobiaceae (for proteases). Assembly and analysis of Library_Oil Mgv reads revealed three potentially novel enzymes, one epoxide hydrolase, one xylanase and one amylase, to be further investigated via heterologous expression assays.

Compositional profile of α / ß-hydrolase fold proteins in mangrove soil metagenomes: prevalence of epoxide hydrolases and haloalkane dehalogenases in oil-contaminated sites.

The occurrence of genes encoding biotechnologically relevant α/ß-hydrolases in mangrove soil microbial communities was assessed using data obtained by whole-metagenome sequencing of four mangroves areas, denoted BrMgv01 to BrMgv04, in São Paulo, Brazil. The sequences (215 Mb in total) were filtered based on local amino acid alignments against the Lipase Engineering Database. In total, 5923 unassembled sequences were affiliated with 30 different α/ß-hydrolase fold superfamilies. The most abundant predicted proteins encompassed cytosolic hydrolases (abH08; ∼ 23%), microsomal hydrolases (abH09; ∼ 12%) and Moraxella lipase-like proteins (abH04 and abH01; < 5%). Detailed analysis of the genes predicted to encode proteins of the abH08 superfamily revealed a high proportion related to epoxide hydrolases and haloalkane dehalogenases in polluted mangroves BrMgv01-02-03. This suggested selection and putative involvement in local degradation/detoxification of the pollutants. Seven sequences that were annotated as genes for putative epoxide hydrolases and five for putative haloalkane dehalogenases were found in a fosmid library generated from BrMgv02 DNA. The latter enzymes were predicted to belong to Actinobacteria, Deinococcus-Thermus, Planctomycetes and Proteobacteria. Our integrated approach thus identified 12 genes (complete and/or partial) that may encode hitherto undescribed enzymes. The low amino acid identity (< 60%) with already-described genes opens perspectives for both production in an expression host and genetic screening of metagenomes.