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Actinomycetes and fungi were successfully isolated from both New Zealand and Malaysian marine invertebrates and classified as facultatively marine based on their ability to grow on both sea water and non-sea water media. Most of the extracts obtained from selected isolates were cytotoxic. A clear preference of the actinomycetes for solid-state fermentation was observed, however, for fungi no significant preference was seen. Three isolates of Streptomyces spp., four Penicillium spp. and two Paecilomyces spp. whose extracts showed good cytotoxicity were selected for further investigation. A small-scale extract obtained from a solid culture of Streptomyces sp. (LA3L2) showed good cytotoxicity and a new cytotoxic metabolite was isolated from a large-scale extract of Streptomyces sp. (LA3L2). This metabolite was characterized as S-methyl 2,4-dihydroxy-6-isopropyl-3,5-dimethylbenzothioate (5.15) and is only the third compound reported to contain the S-methyl benzothioate group. Two known compounds, montagnetol (5.16) and erythrin (5.18), were isolated from a further large-scale cultivation of Streptomyces sp. (LA3L2) and is the first reported actinomycete to produce these lichen-related compounds. In addition, two known inactive metabolites (bohemamine (5.1) and bohemamine B (5.2)) were identified from the small-scale extract. Streptomyces sp. (LA3L2) was also investigated for the effect of temperature and salinity on growth and cytotoxicity and shown to produce bohemamine only at 20 - 28℃ and 4% sea salt concentration on solid media. This isolate gave a low yield of active metabolite under all conditions. Small-scale extracts of two other Streptomyces spp. yielded three known cytotoxic metabolites. These were thiazostatin B (7.14) from Streptomyces sp. (LA5L4) and chromomycin A2 (7.1), chromomycin A3 (7.2) and chromomycin 02-3D (7.3) from Streptomyces sp. (LA3L1). All four Penicillium spp. produced known metabolites. Penicillium sp. (LY1L5) yielded two known metabolites, cycloaspeptide A (7.4) and α-cyclopiazonic acid (7.5). α-Cyclopiazonic acid (7.5) and three other known metabolites (roquefortine A (7.6), cyclopeptin (7.7) and viridicatin (7.8)) were isolated from Penicillum sp. (KK3T23). Penicillium sp. (KK3T8) produced brefeldin A (7.10), while mycophenolic acid (7.12) and brevianamide A (7.11) were produced by Penicillium sp. (KK4T14b). The effect of salinity on growth and cytotoxicity was investigated for the two Penicillium isolates producing the cytotoxic metabolite, α-cyclopiazonic acid (7.5). Saline conditions were not required for growth but metabolite production differed between the two isolates with respect to salinity. Isolate LY1L5 required saline conditions for α-cyclopiazonic production whereas isolate KK3T23 produced the metabolite under non-saline conditions and in concentrations of sea salt up to 6%. Three known compounds, indole-3-carboxylic acid (7.15), indole-3-carboxylate (7.17) and 5-carboxymellein (7.16) were identified from Paecilomyces sp. (PR5L9). Investigation of a small-scale extract obtained from a solid culture of another Paecilomyces sp. (PR10T2) resulted in the isolation and characterization of a unique structure of a symmetrical cyclic depsipeptide, epi-angolide (NAM 6-1). NAM 6-1 was considered as a new compound based on four homoisomeric configurations (A1, A2, A3 and A4). The value of dereplication procedures with respect to the rapid identification of metabolites and enhancement of in-house metabolite libraries is discussed. Structural elucidation of nine known metabolites (7.1, 7.2, 7.3, 7.5, 7.6, 7.7, 7.8, 7.10 and 7.11) was greatly aided by the in-house dereplication techniques using LC-MS-UV and AntiMarin database. A significant advantage was gained by the use of the CapNMR which enabled NMR characterization of very small quantities of metabolites (<20 µg). Approximately <5 µg of materials were required to perform 1D proton NMR experiments for the dereplication of seven known compounds; bohemamine (5.1), bohemamine B (5.2), thiazostatin B (7.14), indole-3-carboxylate (7.17) and 5-carboxymellein (7.16). Approximately 20 µg of materials were needed to acquire 1D and 2D (HSQC, HMBC and NOE) NMR spectra for structural elucidation of the new metabolite, S-methyl 2,4-dihydroxy-6-isopropyl-3,5-dimethylbenzothioate (5.15). Some 8 µg of materials were sufficient to perform 1D and 2D (COSY, HSQC and HMBC) NMR experiments for complete structural characterization of two known metabolites, montagnetol (5.16) and erythrin (5.18). Approximately 10 µg of materials were needed to acquire 1D and 2D NMR (COSY, HSQC and HMBC) experiments for structural elucidation of the new compound, epi-angolide NAM 6-1 (A1, A2, A3 and A4). Rapid identification of known fungal metabolites enabled the in-house HPLC-UV/Rt library to be enhanced by eight metabolites (7.5, 7.6, 7.7, 7.8, 7.10, 7.11, 7.17 and 7.16). An HPLC-UV/Rt library for actinomycete metabolites was successfully established with the insertion of eight known metabolites (5.1, 5.2, 5.16, 5.18, 7.1, 7.2, 7.3 and 7.14).

Actinomycetes and fungi were successfully isolated from both New Zealand and Malaysian marine invertebrates and classified as facultatively marine based on their ability to grow on both sea water and non-sea water media. Most of the extracts obtained from selected isolates were cytotoxic. A clear preference of the actinomycetes for solid-state fermentation was observed, however, for fungi no significant preference was seen. Three isolates of Streptomyces spp., four Penicillium spp. and two Paecilomyces spp. whose extracts showed good cytotoxicity were selected for further investigation. A small-scale extract obtained from a solid culture of Streptomyces sp. (LA3L2) showed good cytotoxicity and a new cytotoxic metabolite was isolated from a large-scale extract of Streptomyces sp. (LA3L2). This metabolite was characterized as S-methyl 2,4-dihydroxy-6-isopropyl-3,5-dimethylbenzothioate (5.15) and is only the third compound reported to contain the S-methyl benzothioate group. Two known compounds, montagnetol (5.16) and erythrin (5.18), were isolated from a further large-scale cultivation of Streptomyces sp. (LA3L2) and is the first reported actinomycete to produce these lichen-related compounds. In addition, two known inactive metabolites (bohemamine (5.1) and bohemamine B (5.2)) were identified from the small-scale extract. Streptomyces sp. (LA3L2) was also investigated for the effect of temperature and salinity on growth and cytotoxicity and shown to produce bohemamine only at 20 - 28℃ and 4% sea salt concentration on solid media. This isolate gave a low yield of active metabolite under all conditions. Small-scale extracts of two other Streptomyces spp. yielded three known cytotoxic metabolites. These were thiazostatin B (7.14) from Streptomyces sp. (LA5L4) and chromomycin A2 (7.1), chromomycin A3 (7.2) and chromomycin 02-3D (7.3) from Streptomyces sp. (LA3L1). All four Penicillium spp. produced known metabolites. Penicillium sp. (LY1L5) yielded two known metabolites, cycloaspeptide A (7.4) and α-cyclopiazonic acid (7.5). α-Cyclopiazonic acid (7.5) and three other known metabolites (roquefortine A (7.6), cyclopeptin (7.7) and viridicatin (7.8)) were isolated from Penicillum sp. (KK3T23). Penicillium sp. (KK3T8) produced brefeldin A (7.10), while mycophenolic acid (7.12) and brevianamide A (7.11) were produced by Penicillium sp. (KK4T14b). The effect of salinity on growth and cytotoxicity was investigated for the two Penicillium isolates producing the cytotoxic metabolite, α-cyclopiazonic acid (7.5). Saline conditions were not required for growth but metabolite production differed between the two isolates with respect to salinity. Isolate LY1L5 required saline conditions for α-cyclopiazonic production whereas isolate KK3T23 produced the metabolite under non-saline conditions and in concentrations of sea salt up to 6%. Three known compounds, indole-3-carboxylic acid (7.15), indole-3-carboxylate (7.17) and 5-carboxymellein (7.16) were identified from Paecilomyces sp. (PR5L9). Investigation of a small-scale extract obtained from a solid culture of another Paecilomyces sp. (PR10T2) resulted in the isolation and characterization of a unique structure of a symmetrical cyclic depsipeptide, epi-angolide (NAM 6-1). NAM 6-1 was considered as a new compound based on four homoisomeric configurations (A1, A2, A3 and A4). The value of dereplication procedures with respect to the rapid identification of metabolites and enhancement of in-house metabolite libraries is discussed. Structural elucidation of nine known metabolites (7.1, 7.2, 7.3, 7.5, 7.6, 7.7, 7.8, 7.10 and 7.11) was greatly aided by the in-house dereplication techniques using LC-MS-UV and AntiMarin database. A significant advantage was gained by the use of the CapNMR which enabled NMR characterization of very small quantities of metabolites (<20 µg). Approximately <5 µg of materials were required to perform 1D proton NMR experiments for the dereplication of seven known compounds; bohemamine (5.1), bohemamine B (5.2), thiazostatin B (7.14), indole-3-carboxylate (7.17) and 5-carboxymellein (7.16). Approximately 20 µg of materials were needed to acquire 1D and 2D (HSQC, HMBC and NOE) NMR spectra for structural elucidation of the new metabolite, S-methyl 2,4-dihydroxy-6-isopropyl-3,5-dimethylbenzothioate (5.15). Some 8 µg of materials were sufficient to perform 1D and 2D (COSY, HSQC and HMBC) NMR experiments for complete structural characterization of two known metabolites, montagnetol (5.16) and erythrin (5.18). Approximately 10 µg of materials were needed to acquire 1D and 2D NMR (COSY, HSQC and HMBC) experiments for structural elucidation of the new compound, epi-angolide NAM 6-1 (A1, A2, A3 and A4). Rapid identification of known fungal metabolites enabled the in-house HPLC-UV/Rt library to be enhanced by eight metabolites (7.5, 7.6, 7.7, 7.8, 7.10, 7.11, 7.17 and 7.16). An HPLC-UV/Rt library for actinomycete metabolites was successfully established with the insertion of eight known metabolites (5.1, 5.2, 5.16, 5.18, 7.1, 7.2, 7.3 and 7.14).

Neste trabalho foram reativadas 51 linhagens fúngicas isoladas a partir da ascídia Didemnum ligulum. Após a reativação, as linhagens foram cultivadas em meio de cultura líquido (250 mL), a partir dos quais foram obtidos os respectivos extratos brutos. Os extratos foram avaliados em bioensaio de atividade citotóxica, em três linhagens de células tumorais. Observou-se que 14 extratos apresentaram atividade citotóxica. Os extratos foram avaliados por HPLC-UV-MS para determinação do perfil químico. Após esta análise, a linhagem DLM2-12, identificada como Penicillium citrinum, foi selecionada para crescimento em meio de cultura líquido em quantidade suficiente para se realizar o isolamento dos metabólitos secundários do extrato de seu meio de cultura. O extrato de 250 mL do meio de cultura de P. citrinum foi submetido a diferentes métodos de separação cromatográfica. Estas separações permitiram o isolamento e identificação da citrinina (47) como sendo majoritária. Para isolamento de outros compostos presentes no extrato, a linhagem foi cultivada em maior escala (8 L) e obtido o extrato de seu meio de cultura. O novo extrato foi submetido a separações cromatográficas em gel de Sephadex LH-20, sílica gel e sílica gel derivatizada com grupo cianopropila, bem como purificações por HPLC. Estas separações permitiram o isolamento e identificação de três quinolonas, as quinolactacinas C (50) e B (53) e a quinolactacina E (51), inédita na literatura, um ácido tetrâmico, o penicilenol A (54) e duas antraquinonas, a citreoroseína (57) e a emodina (61). As antraquinonas citreoroseína (57) e emodina (61) foram submetidas aos bioensaios de atividade antiviral, antimicrobiana, Leishmanicida e citotóxica. A citreoroseína (57) apresentou atividade contra as linhagens virais BVDV, HSV1 e aMPV e apresentou atividade citotóxica ativa frente à linhagem celular HCT-116 sendo inativa frente às linhagens celulares HL-60, SF295, OVCAR-8, K562, HCT-8, MDA-MB435 e MVF-7. A emodina (61) apresentou atividade contra as linhagens virais BVDV, HSV1, aMPV e hepatite C, atividade antimicrobiana, atividade Leishmanicida, atividade citotóxica frente às linhagens celulares HL-60, HCT-116, SF295, OVCAR-8, K562 e HCT-8 sendo inativa frente às linhagens celulares MDA-MB435 e MVF-7. / The present investigation describes the isolation of secondary metabolites from the culture medium of a marine-derived strain of the fungus Penicillium citrinum. Initially, 51 fungal strains isolated from the ascidian Didemnum ligulum and preserved in stereilized sea water were re-activated in Petri dishes. After reactivation, the strains were grown in 250 mL of liquid culture medium. All media were extracted with EtOAc and, after evaporation, aliquots of the extracts were evaluated in a cytotoxicity assay on three tumor cell lines. It was observed that 14 extracts showed cytotoxic activity. These 14 extracts were analyzed by HPLC-UV-MS in order to investigate their corresponding chemical profile. As a result, the DLM2-12 strain, identified as Penicillium citrinum, was selected for semi-preparative growth in liquid culture medium in sufficient quantity to accomplish the isolation of secondary metabolites. A first growth in 250 mL of culture medium yielded an extract which was subjected to various chromatographic separations, to give citrinin (42) as the major compound. An additional growth of P. citrinum DLM2-12 in a larger volume (8 L) yielded an extract which was subjected to chromatographic separations on Sephadex LH-20, cyanopropyl-bonded silica gel, and by reversed-phase HPLC. These separations gave, after the identification of pure compounds: a) three quinolones, quinolactacins C (51) and B (54) and a new quinolactacin (52), unreported in the literature; b) the tetramic acid penicilenol A (55), and; c) two anthraquinones, citreorosein (57) and emodin (61). The anthraquinones 57 and 61 were evaluated in antiviral, antimicrobial, antileishmanial and cytotoxic assays. Citreorosein (57) displayed activity against the viral strains BVDV, HSV1 and aMPV and showed cytotoxicity against the HCT-116 cell line, being inactive against HL-60, SF295, OVCAR-8, K562, HCT-8, MDA-MB435 and MVF-7 cell lines. Emodin (61) displayed activity against the viral strains BVDV, HSV1, AMPV and hepatitis C, as well as antimicrobial, leishmanicidal, as well as cytotoxic activity against HL-60, HCT-116, SF295, OVCAR-8, K562 and HCT-8 cancer cell lines, being inactive against MDA-MB435 and MVF-7 cell lines.

Recent paleolimnological research in the eastern Canadian high Arctic on the ecological impact of the Thule c.1000-1500 AD has documented the influence of prehistoric anthropogenic activities. Six lake and pond sites (three pairs) on the south-western coast of Baffin Island, Nunavut, were used to compare impacted and non-impacted sites in the southern-most region of Thule occupation, as well as to compare Thule occupation sites with sites occupied by another early Arctic population group, the Dorset. Tanfield 1 and Tanfield 2 (impacted and control, respectively) are adjacent to several multiple-occupation Dorset sites on Cape Tanfield; Juet 1 and 2 (impacted and control, respectively) are adjacent to a short-term occupation Dorset site on Juet Island; McKellar 1 is adjacent to a multiple-occupation Thule site near McKellar Bay. A nearby site (McKellar 2) was also studied, but it was clearly an anomalous, eutrophic site, rather than a control for McKellar 1. Diatom assemblages and sedimentary 15N profiles were analyzed in sediment cores from all study sites. Selected paired sediment intervals were AMS radiocarbon dated using both humic acids and terrestrial macrofossils in an attempt to establish basal dates for each core. Significant differences between several of the paired AMS radiocarbon dates serve as a cautionary note for dating Arctic sediments using either humic acids or terrestrial macrofossils. Paleolimnological analyses revealed that at both multiple-occupation sites (Tanfield 1 and McKellar 1), the activities of the Dorset and the Thule influenced lake ecology, while at the short-term occupation site (Juet 1), the Dorset occupation was not sufficiently large to have a discernible impact. McKellar 1 showed a greater impact compared to Tanfield 1, consistent with the intense marine mammal hunting by Thule at the former, compared to the moderate marine mammal hunting by Dorset at Tanfield 1. The origin of marine-derived nutrients at McKellar 2 could not be ascertained with certainty. The influence of early Arctic population groups remains obvious in present-day nutrient- and production-related water chemistry variables. This research points to the value of collaborations between paleolimnologists and archaeologists and may provide insight into the future implications of current anthropogenic activities in the Arctic. / Thesis (Master, Biology) -- Queen's University, 2011-10-04 15:52:18.29

Orientadores: Lucia Regina Durrant, Lara Durães Sette / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-15T23:25:48Z (GMT). No. of bitstreams: 1 Santos_RafaellaCostaBonugli_D.pdf: 1864466 bytes, checksum: 5bb70197e101a9e0e85c0147626dbf2e (MD5) Previous issue date: 2010 / Resumo: Os ecossistemas marinhos representam uma fonte potencial de recursos genéticos para diversas aplicações biotecnológicas. Neste sentido, os fungos filamentosos derivados do ambiente marinho podem ser considerados estratégicos para a produção de compostos naturais bioativos e para aplicações em processos industriais que requerem tolerância às condições salinas. Neste contexto, o presente trabalho teve como objetivo geral avaliar a diversidade genética de fungos derivados de macro-organismos marinhos (cnidários e esponjas) e o potencial destes isolados para a biorremediação de poluentes ambientais. A caracterização da diversidade dos fungos isolados de cnidários marinhos demonstrou que a maioria dos isolados pertencem ao filo Ascomycota, sendo identificado apenas um único isolado do filo Zygomycota (gênero Mucor). Diversos fungos filamentosos isolados dos cnidários marinhos apresentaram potencial biotecnológico para produção das enzimas ligninolíticas. Entretanto, os fungos Aspergillus sclerotiorum CBMAI 849, Cladosporium cladosporioides CBMAI 857 (Ascomycota) e o Mucor racemosus CBMAI 847 (Zygomycota) foram selecionados devido à capacidade de produção de quantidades significativas de enzimas ligninolíticas na triagem inicial. Esses três isolados foram submetidos à avaliação de diferentes fatores (fonte de carbono, farelo de trigo e salinidade) envolvidos na atividade ligninolítica. A atividade da lacase e MnP foi ampliada quando a glicose foi utilizada como fonte de carbono, contudo LiP foi produzida apenas no meio contendo extrato de malte. A adição do farelo de trigo não influenciou a produção das enzimas, contudo, a salinidade foi o fator mais importante na atividade ligninolítica. Valores mais altos de MnP e lacase foram produzidos por M. racemosus CBMAI 847 em 12,5% e 23% de salinidade, sendo o primeiro relato da atividade ligninolítica para o gênero Mucor. Levando-se em consideração que os fungos basidiomicetos são os principais produtores de enzimas ligninolíticas, três basidiomicetos isolados de esponjas marinhas, identificados como Marasmiellus sp. CBMAI 1062, Tinctoporellus sp. CBMAI 1061 e Peniophora CBMAI 1063 foram investigados quanto à atividade de LiP, MnP e Lac, diversidade dos genes que codificam para a atividade extracelular da lacase e degradação do corante Remazol Brilhante Blue R (RBBR). A atividade enzimática foi altamente significativa para os três basidiomicetos derivados marinhos em meio contendo extrato de malte como fonte de carbono (condição não salina) e em meio formulado com água do mar artificial (condição salina). A atividade ligninolítica aumentou quando o farelo de trigo e CuSO4 foram adicionados ao meio de cultivo contendo glicose como fonte de carbono. Uma elevada diversidade de genes que codificam para a lacase foi encontrada nos três basidiomicetos estudados, sugerindo a detecção de novas lacases, que podem apresentar características diferentes das produzidas por micro-organimos terrestres. Em adição, os três basidiomicetos estudados apresentaram capacidade significativa de descoloração do corante RBBR. O fungo Tinctoporellus sp. CBMAI 1061 foi o mais eficiente na degradação do corante RBBR, com 100% de degradação após 3 dias de cultivo e a MnP foi a principal enzima produzida durante a descoloração do corante por este fungo. Os resultados obtidos no presente estudo demonstram o potencial biotecnológico dos fungos derivados de ambientes marinhos pertencentes a diferentes grupos taxonômicos (ascomicetos, zigomicetos e basidiomicetos), principalmente na degradação de poluentes por enzimas ligninolíticas em ambiente ou processos salinos, como na biorremediação de Hidrocarbonetos Policíclicos Aromáticos (HPAs) e vários compostos aromáticos derivados do derramamento de petróleo nos oceanos e mares ou no tratamento de efluentes têxteis, os quais contêm altas concentrações de sais / Abstract: Marine ecosystems represent potential genetic resources for various biotechnological applications. In this sense, the fungi derived from marine environments can be considered strategic for the production of natural bioactive compounds and for applications in industrial processes that require tolerance to saline conditions. In this context, this study aimed to evaluate the biotechnological potential of fungi derived from marine macroorganisms (cnidarians and sponges) on the degradation of environmental pollutants, as well as to characterize the diversity of isolates derived from such samples. Our results showed that most marine-derived fungal isolates belong to the phylum Ascomycota, and only one isolate was identified as representative of the genus Mucor, phylum Zigomycota. Several filamentous fungi isolated from marine cnidarians showed potential for industrial applications. However, Aspergillus sclerotiorum CBMA 849, Cladosporium cladosporioides CBMA 857 (Ascomycota) and Mucor racemosus CBMAI 847 (Zygomycota) were selected for their ability to produce significant amounts of ligninolytic enzymes in the initial screening. These three isolates were submitted to experiments related to the influence of different variable parameters (carbon source, wheat bran and salinity) on ligninolytic activity. Lac and MnP activities were enhanced when glucose was used as carbon source. However LiP was produced only in medium containing malt extract. According to the statistical analysis, the addition of wheat bran did not influence the enzyme productions, but the salinity was the most important parameter on the ligninolytic activities. The highest amounts of MnP and laccase were produced by M. racemosus CBMAI 847 in 12.5% and 23% salinity and this was the first report related to ligninolytic activity for the genus Mucor. Taking into account that basidiomycetes are the major producers of ligninolytic enzymes, three basidiomycetes isolates from marine sponges identified as Marasmiellus sp. CBMAI 1062, Tinctoporellus sp. CBMAI 1061 and Peniophora sp. CBMAI 1063 were investigated in relation to LiP, MnP and Lac activities, as well as the diversity of extracellular laccase genes and the decolorization of Remazol Brilliant Blue R (RBBR) dye. The enzyme activity was highly significant for the three marine-derived basidiomycetes in medium containing malt as carbon source (non-saline condition) and in medium formulated with artificial seawater (saline condition). Ligninolytic activity was enhanced when wheat bran and CuSO4 were added to the culture medium containing glucose as carbon source. A high diversity of Lac genes was found in the three basidiomycetes studied, suggesting the detection of new laccase, which may present different characteristics from those produced by terrestrial microorganisms. In addition, the three basidiomycetes studied showed significant capacity to decolorize the RBBR dye. Tinctoporellus sp. CBMAI 1061 was the most efficient fungus in the dye decolorization, presenting 100% degradation after 3 days of cultivation in liquid medium, and MnP was the main enzyme produced during RBBR decolorization by the three marine-derived fungi. Results from our study revealed the biotechnology potential of marine-derived fungi belonging to different taxonomic groups (ascomycetes, zygomycetes and basidiomycetes), in the degradation of pollutants by ligninolytic enzymes in saline environments and/or processes, such as the bioremediation of Polycyclic Aromatic Hydrocarbons (PAHs) derived from oil spills in the ocean or in the treatment of industrial (textile) colored effluents, which contain high concentrations of salts. Os ecossistemas marinhos representam uma fonte potencial de recursos genéticos para diversas aplicações biotecnológicas. Neste sentido, os fungos filamentosos derivados do ambiente marinho podem ser considerados estratégicos para a produção de compostos naturais bioativos e para aplicações em processos industriais que requerem tolerância às condições salinas. Neste contexto, o presente trabalho teve como objetivo geral avaliar a diversidade genética de fungos derivados de macro-organismos marinhos (cnidários e esponjas) e o potencial destes isolados para a biorremediação de poluentes ambientais. A caracterização da diversidade dos fungos isolados de cnidários marinhos demonstrou que a maioria dos isolados pertencem ao filo Ascomycota, sendo identificado apenas um único isolado do filo Zygomycota (gênero Mucor). Diversos fungos filamentosos isolados dos cnidários marinhos apresentaram potencial biotecnológico para produção das enzimas ligninolíticas. Entretanto, os fungos Aspergillus sclerotiorum CBMAI 849, Cladosporium cladosporioides CBMAI 857 (Ascomycota) e o Mucor racemosus CBMAI 847 (Zygomycota) foram selecionados devido à capacidade de produção de quantidades significativas de enzimas ligninolíticas na triagem inicial. Esses três isolados foram submetidos à avaliação de diferentes fatores (fonte de carbono, farelo de trigo e salinidade) envolvidos na atividade ligninolítica. A atividade da lacase e MnP foi ampliada quando a glicose foi utilizada como fonte de carbono, contudo LiP foi produzida apenas no meio contendo extrato de malte. A adição do farelo de trigo não influenciou a produção das enzimas, contudo, a salinidade foi o fator mais importante na atividade ligninolítica. Valores mais altos de MnP e lacase foram produzidos por M. racemosus CBMAI 847 em 12,5% e 23% de salinidade, sendo o primeiro relato da atividade ligninolítica para o gênero Mucor. Levando-se em consideração que os fungos basidiomicetos são os principais produtores de enzimas ligninolíticas, três basidiomicetos isolados de esponjas marinhas, identificados como Marasmiellus sp. CBMAI 1062, Tinctoporellus sp. CBMAI 1061 e Peniophora CBMAI 1063 foram investigados quanto à atividade de LiP, MnP e Lac, diversidade dos genes que codificam para a atividade extracelular da lacase e degradação do corante Remazol Brilhante Blue R (RBBR). A atividade enzimática foi altamente significativa para os três basidiomicetos derivados marinhos em meio contendo extrato de malte como fonte de carbono (condição não salina) e em meio formulado com água do mar artificial (condição salina). A atividade ligninolítica aumentou quando o farelo de trigo e CuSO4 foram adicionados ao meio de cultivo contendo glicose como fonte de carbono. Uma elevada diversidade de genes que codificam para a lacase foi encontrada nos três basidiomicetos estudados, sugerindo a detecção de novas lacases, que podem apresentar características diferentes das produzidas por micro-organimos terrestres. Em adição, os três basidiomicetos estudados apresentaram capacidade significativa de descoloração do corante RBBR. O fungo Tinctoporellus sp. CBMAI 1061 foi o mais eficiente na degradação do corante RBBR, com 100% de degradação após 3 dias de cultivo e a MnP foi a principal enzima produzida durante a descoloração do corante por este fungo. Os resultados obtidos no presente estudo demonstram o potencial biotecnológico dos fungos derivados de ambientes marinhos pertencentes a diferentes grupos taxonômicos (ascomicetos, zigomicetos e basidiomicetos), principalmente na degradação de poluentes por enzimas ligninolíticas em ambiente ou processos salinos, como na biorremediação de Hidrocarbonetos Policíclicos Aromáticos (HPAs) e vários compostos aromáticos derivados do derramamento de petróleo nos oceanos e mares ou no tratamento de efluentes têxteis, os quais contêm altas concentrações de sais / Doutorado / Doutor em Ciência de Alimentos

Neste trabalho foram reativadas 51 linhagens fúngicas isoladas a partir da ascídia Didemnum ligulum. Após a reativação, as linhagens foram cultivadas em meio de cultura líquido (250 mL), a partir dos quais foram obtidos os respectivos extratos brutos. Os extratos foram avaliados em bioensaio de atividade citotóxica, em três linhagens de células tumorais. Observou-se que 14 extratos apresentaram atividade citotóxica. Os extratos foram avaliados por HPLC-UV-MS para determinação do perfil químico. Após esta análise, a linhagem DLM2-12, identificada como Penicillium citrinum, foi selecionada para crescimento em meio de cultura líquido em quantidade suficiente para se realizar o isolamento dos metabólitos secundários do extrato de seu meio de cultura. O extrato de 250 mL do meio de cultura de P. citrinum foi submetido a diferentes métodos de separação cromatográfica. Estas separações permitiram o isolamento e identificação da citrinina (47) como sendo majoritária. Para isolamento de outros compostos presentes no extrato, a linhagem foi cultivada em maior escala (8 L) e obtido o extrato de seu meio de cultura. O novo extrato foi submetido a separações cromatográficas em gel de Sephadex LH-20, sílica gel e sílica gel derivatizada com grupo cianopropila, bem como purificações por HPLC. Estas separações permitiram o isolamento e identificação de três quinolonas, as quinolactacinas C (50) e B (53) e a quinolactacina E (51), inédita na literatura, um ácido tetrâmico, o penicilenol A (54) e duas antraquinonas, a citreoroseína (57) e a emodina (61). As antraquinonas citreoroseína (57) e emodina (61) foram submetidas aos bioensaios de atividade antiviral, antimicrobiana, Leishmanicida e citotóxica. A citreoroseína (57) apresentou atividade contra as linhagens virais BVDV, HSV1 e aMPV e apresentou atividade citotóxica ativa frente à linhagem celular HCT-116 sendo inativa frente às linhagens celulares HL-60, SF295, OVCAR-8, K562, HCT-8, MDA-MB435 e MVF-7. A emodina (61) apresentou atividade contra as linhagens virais BVDV, HSV1, aMPV e hepatite C, atividade antimicrobiana, atividade Leishmanicida, atividade citotóxica frente às linhagens celulares HL-60, HCT-116, SF295, OVCAR-8, K562 e HCT-8 sendo inativa frente às linhagens celulares MDA-MB435 e MVF-7. / The present investigation describes the isolation of secondary metabolites from the culture medium of a marine-derived strain of the fungus Penicillium citrinum. Initially, 51 fungal strains isolated from the ascidian Didemnum ligulum and preserved in stereilized sea water were re-activated in Petri dishes. After reactivation, the strains were grown in 250 mL of liquid culture medium. All media were extracted with EtOAc and, after evaporation, aliquots of the extracts were evaluated in a cytotoxicity assay on three tumor cell lines. It was observed that 14 extracts showed cytotoxic activity. These 14 extracts were analyzed by HPLC-UV-MS in order to investigate their corresponding chemical profile. As a result, the DLM2-12 strain, identified as Penicillium citrinum, was selected for semi-preparative growth in liquid culture medium in sufficient quantity to accomplish the isolation of secondary metabolites. A first growth in 250 mL of culture medium yielded an extract which was subjected to various chromatographic separations, to give citrinin (42) as the major compound. An additional growth of P. citrinum DLM2-12 in a larger volume (8 L) yielded an extract which was subjected to chromatographic separations on Sephadex LH-20, cyanopropyl-bonded silica gel, and by reversed-phase HPLC. These separations gave, after the identification of pure compounds: a) three quinolones, quinolactacins C (51) and B (54) and a new quinolactacin (52), unreported in the literature; b) the tetramic acid penicilenol A (55), and; c) two anthraquinones, citreorosein (57) and emodin (61). The anthraquinones 57 and 61 were evaluated in antiviral, antimicrobial, antileishmanial and cytotoxic assays. Citreorosein (57) displayed activity against the viral strains BVDV, HSV1 and aMPV and showed cytotoxicity against the HCT-116 cell line, being inactive against HL-60, SF295, OVCAR-8, K562, HCT-8, MDA-MB435 and MVF-7 cell lines. Emodin (61) displayed activity against the viral strains BVDV, HSV1, AMPV and hepatitis C, as well as antimicrobial, leishmanicidal, as well as cytotoxic activity against HL-60, HCT-116, SF295, OVCAR-8, K562 and HCT-8 cancer cell lines, being inactive against MDA-MB435 and MVF-7 cell lines.

O Capítulo 1 descreve a produção de celulases por 4 linhagens fúngicas de ambiente marinho (Aspergillus sydowii CBMAI 934, A. sydowii CBMAI 935, Penicillium citrinum CBMAI 1186 e Mucor racemosus CBMAI 847) e uma linhagem de ambiente terrestre (Aspergillus sp. CBMAI 1198) cultivados em meio sólido composto por farelo de trigo (5 g) e solução de peptona (0,75 g.L-1) enriquecida com sais inorgânicos. Foram realizadas otimizações da temperatura, pH inicial e umidade do meio de cultura das linhagens obtendo-se maiores atividades celulolíticas na faixa de temperatura entre 25-35 °C, com exceção do fungo A. sydowii CBMAI 935 que foi de 40 °C, e valores diferentes de pH ótimo, desde condições acídicas até alcalinas, bem como valores diferentes de teor de umidade ótima. Quando avaliou-se a influência do pH, da temperatura e do volume de extrato enzimático durante a hidrólise do papel de filtro cada conjunto de celulases produzidas apresentou pontos ótimos diferentes entre elas, e em alguns casos, dois valores ótimos de pH e temperatura. As celulases produzidas nas condições ótimas determinadas foram aplicadas na hidrólise da celulose do bagaço da cana-de-açúcar pré-tratado usando-se 10 U FPU/g de bagaço de cana-de-açúcar. As celulases dos fungos Aspergillus sp. CBMAI 1198 e A. sydowii CBMAI 934 apresentaram a maior capacidade para hidrolisar o bagaço da cana-de-açúcar pré-tratado, 75% e 78% de degradação do material lignocelulósico, respectivamente. No Capítulo 2 foi avaliada a capacidade de 6 bactérias isoladas de turfeira (Bacillus subtilis LQOB-SE1, B. coagulans LQOB-SE2, B. pumilus LQOB-SE3, Brevibacillus brevis LQOB-SE4, Lysinibacillus sp. LQOB-SE5 e Serratia marcescens LQOB-SE6) em produzir 2,3-butanodiol a partir da fermentação de glicerol e a bactéria que apresentou tal capacidade (S. marcescens LQOB-SE6) foi usada para produzir 2,3-butanodiol também a partir da fermentação de glicose visando o reaproveitamento dos resíduos gerados na produção de biodiesel e de etanol. As melhores condições para o uso do glicerol foram: pH inicial 7, Caldo nutriente 8 g.L-1, concentração inicial de glicerol 50 g.L-1 e tempo de cultivo de 7 dias. Foram obtidos bons rendimento (0,30 g.g-1), produtividade (0,13 g.L-1.h-1) e concentração máxima de 2,3-butanodiol (22,4 g.L-1). As melhores condições para a fermentação da glicose foram: pH inicial 7, Caldo nutriente 8 g.L-1, concentração inicial de glicose 75 g.L-1 e tempo de cultivo de 5 dias. Obteve-se um rendimento de 0,42 g.g-1 em 5 dias de fermentação, produtividade de 0,45 g.L-1.h-1 após 2 dias e concentração máxima de 2,3-butanodiol de 31,2 g.L-1. A produção de 2,3-butanodiol a partir do hidrolisado gerado na hidrólise do bagaço de cana-de-açúcar pelas celulases do fungo de ambiente marinho A. sydowii CBMAI 934 não foi observada devido à baixa concentração de açúcares no hidrolisado. Os resultados obtidos nesta tese mostram o potencial biotecnológico da microbiota fúngica e bacteriana isoladas de diferentes biomas brasileiros. / In Chapter 1 it is reported the cellulase production by 4 marine-derived fungi strains (Aspergillus sydowii CBMAI 934, A. sydowii CBMAI 935, Penicillium citrinum CBMAI 1186 and Mucor racemosus CBMAI 847) and 1 terrestrial fungi strain (Aspergillus sp. CBMAI 1198). They were grown in solid state fermentation using wheat straw as substrate (5 g) and with addition of peptone solution (0,75 g.L-1) enriched with inorganic salts. It was performed the enhancement of the growth conditions by changing the temperature, initial pH and moisture. The optimum temperature for all strains varied between 25-35 °C but A. sydowii CBMAI 935 with 40 °C. The optimum pH was different for each strain, varying from acidic to alkaline conditions. The optimum moisture content also varied accordingly the studied strain. In order enhance the cellulose hydrolysis performed by the produced cellulases, it was varied the pH, temperature and amount of the crude cellulase extract during the filter paper hydrolysis reaction. The obtained optimum values were different among strains and, in some cases, there were two optimum pH and temperature for the hydrolysis of the filter paper. Then, the obtained cellulases, using the best conditions for hydrolysis, were used in the sugarcane bagasse hydrolysis (10 FPU/g of sugarcane bagasse). The cellulases from the strains Aspergillus sp. CBMAI 1198 and A. sydowii CBMAI 934 were capable of degrading 75% and 78% of the sugarcane bagasse, respectively, generating reducing sugars. In Chapter 2, the capability of 6 strains (Bacillus subtilis LQOB-SE1, B. coagulans LQOB-SE2, B. pumillus LQOB-SE3, Brevibacillus brevis LQOB-SE4, Lysinibacillus sp. LQOB-SE5 and Serratia marcescens LQOB-SE6), isolated from peat soil, of producing 2,3-butanediol from glycerol fermentation. The only strain that produced 2,3-butanediol was S. marcescens LQOB-SE6, which was also applied in 2,3-butanediol production from glucose fermentation. Therefore, wastes from biodiesel and bioethanol production can be reused in industrial scale. The best conditions for glycerol fermentation: initial pH 7, Nutrient Broth (8 g.L-1), initial glycerol concentration (50 g.L-1) and fermentation time of 7 days. It were obtained good yield (0.30 g.g-1), productivity (0.13 g.L-1.h-1) and 2,3-butanodiol concentration (22.4 g.L-1). The best conditions for glucose fermentation: initial pH 7, Nutrient Broth (8 g.L-1), initial glucose concentration (75 g.L-1) and fermentation time of 5 days. It were also obtained good yield (0.42 g.g-1) and 2,3-butanodiol concentration (31.2 g.L-1) after 5 days and productivity (0.45 g.L-1.h-1) after 2 days. The 2,3-butanediol production from the hydrolysate of sugarcane bagasse, obtained by using cellulases from A. sydowii CBMAI 934, was not observed due the low sugar concentration in the hydrolysate.

Este trabalho objetivou o estudo de rotas de formação de metabólitos secundários utilizando precursores isotopicamente marcados com 13C. Os experimentos de crescimento com adição de [1-13C]acetato, [1,2-13C2]acetato e [U-13C315N1]-L-cisteína para o fungo do ambiente marinho Penicillium sp. DRF2 mostrou que as ciclotiocurvularinas são provenientes da rota de formação de policetídeos e pela incorporação de L-cisteína, depois da transformação desta em 3-mercaptopiruvato. Os resultados sugerem que a formação das ciclotiocurvularinas provém de um processo de detoxificação da &alpha;,&beta;-desidrocurvularina. O estudo do metabolismo secundário de Aspergillus sp. DLM3-8, também do ambiente marinho, mostrou que o seu perfil metabólico produzido em experimentos de crescimento sob diferentes condições é constante. Os experimentos de incorporação de precursores isotopicamente marcados com 13C na naftoquinonaimina, produzida por Aspergillus sp. DLM3-8 foram inconclusivos, indicando que outras abordagens experimentais devem ser realizadas para se investigar a biossíntese deste metabólito. / This investigation aimed investigated the formation routes of secondary metabolites using 13C-labelled precursors. Feeding experiments with [1-13C]acetate, [1,2-13C2]acetate and [U-13C315N1]-L-cysteine within the growth medium of the marine-derived fungi Penicillium sp. DRF2 showed that cyclothiocurvularins are derived from polyketides and from the incorporation of a L-cysteine residue, after its transformation into 3-mercaptopyruvate. The results suggest that the formation of cyclothiocurvularins is derived from a detoxification process of&alpha;,&beta;-dehydrocurvularin. Investigation of the secondary metabolism of a marine-derived Aspergillus sp. DLM3-8 indicated a stable metabolic profile under a variety of growth conditions. Feeding experiments with 13C-labelled precursors for the biosynthesis investigation of naphthoquinoneimine were inconclusive, indicating that other methodologies should be envisaged in order to investigate the biosynthesis of this metabolite.

O Capítulo 1 descreve a produção de celulases por 4 linhagens fúngicas de ambiente marinho (Aspergillus sydowii CBMAI 934, A. sydowii CBMAI 935, Penicillium citrinum CBMAI 1186 e Mucor racemosus CBMAI 847) e uma linhagem de ambiente terrestre (Aspergillus sp. CBMAI 1198) cultivados em meio sólido composto por farelo de trigo (5 g) e solução de peptona (0,75 g.L-1) enriquecida com sais inorgânicos. Foram realizadas otimizações da temperatura, pH inicial e umidade do meio de cultura das linhagens obtendo-se maiores atividades celulolíticas na faixa de temperatura entre 25-35 °C, com exceção do fungo A. sydowii CBMAI 935 que foi de 40 °C, e valores diferentes de pH ótimo, desde condições acídicas até alcalinas, bem como valores diferentes de teor de umidade ótima. Quando avaliou-se a influência do pH, da temperatura e do volume de extrato enzimático durante a hidrólise do papel de filtro cada conjunto de celulases produzidas apresentou pontos ótimos diferentes entre elas, e em alguns casos, dois valores ótimos de pH e temperatura. As celulases produzidas nas condições ótimas determinadas foram aplicadas na hidrólise da celulose do bagaço da cana-de-açúcar pré-tratado usando-se 10 U FPU/g de bagaço de cana-de-açúcar. As celulases dos fungos Aspergillus sp. CBMAI 1198 e A. sydowii CBMAI 934 apresentaram a maior capacidade para hidrolisar o bagaço da cana-de-açúcar pré-tratado, 75% e 78% de degradação do material lignocelulósico, respectivamente. No Capítulo 2 foi avaliada a capacidade de 6 bactérias isoladas de turfeira (Bacillus subtilis LQOB-SE1, B. coagulans LQOB-SE2, B. pumilus LQOB-SE3, Brevibacillus brevis LQOB-SE4, Lysinibacillus sp. LQOB-SE5 e Serratia marcescens LQOB-SE6) em produzir 2,3-butanodiol a partir da fermentação de glicerol e a bactéria que apresentou tal capacidade (S. marcescens LQOB-SE6) foi usada para produzir 2,3-butanodiol também a partir da fermentação de glicose visando o reaproveitamento dos resíduos gerados na produção de biodiesel e de etanol. As melhores condições para o uso do glicerol foram: pH inicial 7, Caldo nutriente 8 g.L-1, concentração inicial de glicerol 50 g.L-1 e tempo de cultivo de 7 dias. Foram obtidos bons rendimento (0,30 g.g-1), produtividade (0,13 g.L-1.h-1) e concentração máxima de 2,3-butanodiol (22,4 g.L-1). As melhores condições para a fermentação da glicose foram: pH inicial 7, Caldo nutriente 8 g.L-1, concentração inicial de glicose 75 g.L-1 e tempo de cultivo de 5 dias. Obteve-se um rendimento de 0,42 g.g-1 em 5 dias de fermentação, produtividade de 0,45 g.L-1.h-1 após 2 dias e concentração máxima de 2,3-butanodiol de 31,2 g.L-1. A produção de 2,3-butanodiol a partir do hidrolisado gerado na hidrólise do bagaço de cana-de-açúcar pelas celulases do fungo de ambiente marinho A. sydowii CBMAI 934 não foi observada devido à baixa concentração de açúcares no hidrolisado. Os resultados obtidos nesta tese mostram o potencial biotecnológico da microbiota fúngica e bacteriana isoladas de diferentes biomas brasileiros. / In Chapter 1 it is reported the cellulase production by 4 marine-derived fungi strains (Aspergillus sydowii CBMAI 934, A. sydowii CBMAI 935, Penicillium citrinum CBMAI 1186 and Mucor racemosus CBMAI 847) and 1 terrestrial fungi strain (Aspergillus sp. CBMAI 1198). They were grown in solid state fermentation using wheat straw as substrate (5 g) and with addition of peptone solution (0,75 g.L-1) enriched with inorganic salts. It was performed the enhancement of the growth conditions by changing the temperature, initial pH and moisture. The optimum temperature for all strains varied between 25-35 °C but A. sydowii CBMAI 935 with 40 °C. The optimum pH was different for each strain, varying from acidic to alkaline conditions. The optimum moisture content also varied accordingly the studied strain. In order enhance the cellulose hydrolysis performed by the produced cellulases, it was varied the pH, temperature and amount of the crude cellulase extract during the filter paper hydrolysis reaction. The obtained optimum values were different among strains and, in some cases, there were two optimum pH and temperature for the hydrolysis of the filter paper. Then, the obtained cellulases, using the best conditions for hydrolysis, were used in the sugarcane bagasse hydrolysis (10 FPU/g of sugarcane bagasse). The cellulases from the strains Aspergillus sp. CBMAI 1198 and A. sydowii CBMAI 934 were capable of degrading 75% and 78% of the sugarcane bagasse, respectively, generating reducing sugars. In Chapter 2, the capability of 6 strains (Bacillus subtilis LQOB-SE1, B. coagulans LQOB-SE2, B. pumillus LQOB-SE3, Brevibacillus brevis LQOB-SE4, Lysinibacillus sp. LQOB-SE5 and Serratia marcescens LQOB-SE6), isolated from peat soil, of producing 2,3-butanediol from glycerol fermentation. The only strain that produced 2,3-butanediol was S. marcescens LQOB-SE6, which was also applied in 2,3-butanediol production from glucose fermentation. Therefore, wastes from biodiesel and bioethanol production can be reused in industrial scale. The best conditions for glycerol fermentation: initial pH 7, Nutrient Broth (8 g.L-1), initial glycerol concentration (50 g.L-1) and fermentation time of 7 days. It were obtained good yield (0.30 g.g-1), productivity (0.13 g.L-1.h-1) and 2,3-butanodiol concentration (22.4 g.L-1). The best conditions for glucose fermentation: initial pH 7, Nutrient Broth (8 g.L-1), initial glucose concentration (75 g.L-1) and fermentation time of 5 days. It were also obtained good yield (0.42 g.g-1) and 2,3-butanodiol concentration (31.2 g.L-1) after 5 days and productivity (0.45 g.L-1.h-1) after 2 days. The 2,3-butanediol production from the hydrolysate of sugarcane bagasse, obtained by using cellulases from A. sydowii CBMAI 934, was not observed due the low sugar concentration in the hydrolysate.