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- ItemAcesso aberto (Open Access)Atividade celulolítica e proteolítica de bactérias isoladas do trato gastrointestinal e do processo de compostagem dos restos orgânicos de hipopótamo(Universidade Federal de São Paulo, 2013-09-08) Ramos, Geomarcia Feitosa da Cruz [UNIFESP]; Vasconcellos, Suzan Pantaroto de [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)The bioprospection for cellulase and protease producers is a promiss strategy for the discovery of potential biocatalysts for use in hydrolysis of ligninocellulosic materials as well as proteic residues. These enzymes can increment and viabilize production of second generation ethanol from different and alternative sources. In this context, the goal of the present study was the investigation about the cellulolytic and proteolytic abilities of bacteria isolated from gastrointestinal tract of a hippopotamus as well as from the composting process of its organic remnants. The bacterial isolates were obtained through the platting of the samples in different culture media and incubation temperatures. After the bacterial isolation, the strains were submitted to qualitative enzymatic assays to investigate their cellulolytic ability according methodology described by Hankin & Angnostakis (1975). Subsequently they were quantitatively evaluated (FPase), following procedures described by Xiao et al. (2004). Proteolytic analyzes were also developed using high throughput screening approach, thorugh the evaluation of fluorescent peptide probes described by Oliveira et al. (2012). As results, in the qualitative assays for cellulolytic ability detection, it was obtained 70 positive hits from 159 isolated bacterial strains. After quantitative analyzes , 44% of these hits were selected, but 7% (5 strains) (FPZSP_CTT 685; FPZSP_CTT 651; FPZSP_CTT 662; FPZSP_CTT 621; FPZSP_CTT 636) showed FPase activity up to 12.8 FPU/mL, which is compatible to the adopted positive control (Trichoderma reesei). In relation to the proteolytic activities, 8 strains presented relative activity above 10%, which superpassed results described by the literature. Molecular analyzes based on the identification of 16S rDNA, together with mass spectrometry results from MALDI-TOF analyzes, could reveal that the majority of the selected bacterial isolates, about their enzymatic abilities, were affiliated to Bacillus genus. In summary, from the obtained results, it could be affirmed that the obtained microflora constituted potential source of interesting biocatalysts for future investigations about its industrial applications, looking for cellulolytic and proteolytic activities.
- ItemAcesso aberto (Open Access)Atividade enzimática de fungos filamentosos voltada à aplicação em processos de geração de bioetanol(Universidade Federal de São Paulo, 2015-10-06) Lima, Lidiane Maria dos Santos [UNIFESP]; Vasconcellos, Suzan Pantaroto de [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)The search for mesophilic and thermophilic filamentous fungi isolated from various environments, as well as waste treatment processes such as composting, represents an appropriate strategy to obtain microbial enzymes, due to the high temperatures at which such processes occur. Furthermore, habitats are promising as regards the recovery of micro-organisms and with potential metabolic capacities of interest. This study investigated the enzyme potential of fungi isolated from the filter cake compost (by-product of the processing of sugarcane) plus soil samples from the Atlantic Forest and / or pruning waste ground in the interest of detecting and evaluating the enzymatic activity of hydrolases and oxidoreductases, for future use in cellulosic ethanol production processes. Approximately 200 different fungal strains (macro / microscopically) were isolated and subjected to determination of cellulolytic activity using the kit AZO-CM-Cellulose S-ACMC 04/07 (Megazyme®) for screening tests of cellulolytic action. In this initial screening, 13 isolates were selected as positive hits: FPZ_SP3 01, SP3 05, SP3 47, SP3 72, SP3 74, SP3 75, SP3 91, SP2 121, SP1 129, SP1 135, SP1 141, SP1 148 and SP1 151. Such fungi followed for new enzymatic assays aimed at the detection and quantification of the active cellulolytic complex (FPase test) as well as evaluation of ligninases activities, proteases, chitinases and mono-oxygenase. The selected isolates were also subjected to taxonomic characterization analyzes, using phenotypic and molecular techniques. The results obtained in this study suggested that the isolated SP3 01 (0.123 FPU /ml), SP3 75 (0.085 FPU /ml); SP3 74and SP2 121 (0.084 FPU /ml), when the pH conditions are 4.8 efficient producers of cellulolytic enzymes. With regard to the proteolytic activity, three isolates (SP3 01, SP3 72 and SP1 135) were selected, suggesting the presence of metallo proteases and serine proteases. As for chitinolytic activity, 12 isolates demonstrated promising enzyme activities for exoquitinase, quitobiosidase and endochitinase. Under-oxide reductase, it was possible to identify four isolates SP3 47, SP3 75, SP1 129 and SP1 141 with ligninolytic activity. The isolated SP3 05 was selected with mono-oxygenase activity induced by the presence of PAHs (polyaromatic hydrocarbons). Regarding the characterization of isolates, the phenotypic and molecular techniques allowed the taxonomic identification of Aspergillus niger (SP3 01), Mucor sp. (SP3 47), Aspergillus niger (SP3 74), Trichoderma koningii (SP3 75), Geotrichium candidum (SP3 91) Byssochlamys nivea (SP1 129), Paecilomyces saturatus (SP1 141), Aspergillus fumigatus (SP1 148) and Aspergillus fumigatus (SP1 151). Thus, isolated from the filter cake compost were efficient producers of hydrolytic enzymes and oxidative of interest to future enforcement in re-use of lignocellulosic waste processes to the production of second generation ethanol.
- ItemAcesso aberto (Open Access)Avaliação da degradação de hidrocarbonetos por bactérias provenientes de compostagem(Universidade Federal de São Paulo, 2016-02-24) Araujo e Oliveira, Aline Marcia Silva [UNIFESP]; Niero, Cristina Viana [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Composting is an alternative to convert organic waste into fertilizer which occurs predominantly by the action of microorganisms. Thus, it is expected that microorganisms capable of degrading various compounds inhabit compost bins. The aim of this study was to isolate hydrocarbon-degrading bacteria from composting. Three samples originated from a compost bin in the Fundação Parque Zoológico de São Paulo were studied using selective cultivation methods with poor nutrients media containing n-hexadecane as sole carbon source. Forty-four microorganisms were isolated and identified by mass spectrometry (MALDI-TOF) or sequencing of a 16S rRNA gene fragment. The isolates were identified as belonging to from the genus Acinetobacter, Pseudomonas, Stenotrophomonas, Gordonia, Elizabethkingia, Pandorea, Aquamicrobium, Bacillus, Klebsiella and Chryseobacterium. Three isolated from the genus Gordonia were submitted to growth curves and 2,6-DCPIP assays with different hydrocarbons as sole carbon source, and demonstrated ability to assimilate aliphatic hydrocarbons, and one of the isolates stood out from the others due to the increased growth in the media. The genomes of these 3 isolates were sequenced and bioinformatics analysis revealed the presence of CYP153 system, one of the systems responsible for starting the hydrocarbon degradation. qPCR experiments were performed in Gordonia isolates of the same species and through them it was verified that two genes from this system (ferredoxin and CYP153) have increased expression in both isolates in the presence of n-hexadecane in and dodecane when compared to the expression in the presence of glucose. However, a distinct profile in the growth curve as well as in the qPCR among isolates of the same species can be verified. The CYP153 system of the two isolates were cloned and expressed in E.coli BL21(DE3). Enzimatic activity of this system in E.coli could not be confirmed. The results demonstrate that the studied compost bins harbor microorganisms capable of expressing proteins responsible for hydrocarbon degradation.
- ItemAcesso aberto (Open Access)Avaliação do grau de maturação de compostos orgânicos produzidos por compostagem tradicional, vermicompostagem e gongocompostagem.(Universidade Federal de São Paulo, 2023-01-03) Nascimento, Beatriz Carvalho do [UNIFESP]; Shinzato, Mirian Chieko [UNIFESP]; http://lattes.cnpq.br/3795345587829203A prática da compostagem pode ser uma importante ferramenta para diminuir o descarte incorreto dos resíduos sólidos orgânicos, que representa mais de 50% de todos os resíduos sólidos gerados no Brasil. A compostagem tem como produto compostos orgânicos com alto grau de maturação e concentração de nutrientes que pode ser utilizado como fertilizante natural. O presente trabalho teve como objetivo avaliar e comparar o grau de maturação dos compostos orgânicos gerados pelo método tradicional de compostagem (comum), vermicompostagem e gongocompostagem de resíduos orgânicos de origem vegetal. O grau de maturação dos compostos indica quando o produto está livre de substâncias fitotóxicas e, por isso, sua avaliação é de extrema importância para o uso seguro na agricultura. O grau de maturação foi analisado por meio da determinação dos índices de humificação do composto obtidas pelas frações de substâncias húmicas (ácidos húmicos e fúlvicos) e grupos funcionais (identificadas pela espectroscopia na região do infravermelho com transformada de Fourier, FTIR). Tendo em vista a quantificação das substâncias húmicas no extrato alcalino (EA) dos compostos, foi possível inferir que os ácidos húmicos representam 96,14% ± 1,01, 98,21% ± 0,46 e 95,67% ± 0,56 de todo o EA do composto comum, vermicomposto e gongocomposto, respectivamente. Os índices de humificação também indicaram uma maior quantidade de ácidos húmicos em relação aos ácidos fúlvicos (AH/AF > 1), apresentando grau de polimerização igual a 26,23 ± 5,72, 58,20 ± 14,48 e 22,51 ± 3,47 para a compostagem comum, vermicompostagem e gongocompostagem, respectivamente. A análise dos grupos funcionais de FTIR revelaram picos de absorção próximos a faixa de 1620 cm–¹, que caracterizam componentes de carbonos aromáticos, superiores àqueles encontrados na faixa de 2920 cm-¹, pertencente aos carbonos alifáticos. Os índices de aromaticidade também indicaram uma maior quantidade de carbonos aromáticos em relação aos carbonos alifáticos, indicando uma alta mineralização da matéria orgânica dos compostos. Dessa forma, os resultados apontam que todos os compostos apresentaram concentrações de moléculas mais complexas e estáveis superiores às moléculas mais simples, sendo estas primeiras características de compostos orgânicos com elevado grau de maturação. Portanto, é possível concluir que a análise do grau de maturação dos compostos deve ser realizada através da combinação de mais de um parâmetro, sendo a análise por FTIR a técnica mais simples e fácil de se obter estes resultados.
- ItemAcesso aberto (Open Access)Bioprospecção do potencial antimicrobiano, antiparasitário e antitumoral de metabólitos secundários produzidos por fungos do gênero Aspergillus Spp.(Universidade Federal de São Paulo (UNIFESP), 2018-06-28) Calado, Juliana Cristina Pereira [UNIFESP]; Batista, Wagner Luiz [UNIFESP]; http://lattes.cnpq.br/4373797404389169; http://lattes.cnpq.br/4353724670961832; Universidade Federal de São Paulo (UNIFESP)Filamentous fungi produce a wide variety of secondary metabolites that have a significant impact on society. Some metabolites are exploited by their activity as antibiotics and drugs; others are involved in diseases, in the interaction of fungi with plants and animals. In this context, the objective of this work was to obtain biologically active secondary metabolites produced by fungi isolated from the composting of the Fundação Zoológico de São Paulo (FPZSP). In this study, 71 organic extracts were used, 60 filamentous fungi and 11 yeasts. Of the 71 analyzed samples 25 presented antimicrobial activity in disc diffusion assays, and 23 samples showed activity against Staphylococcus aureus, 15 against Bacillus cereus and only one sample (I75) showed activity against Escherichia coli and Pseudomonas aeruginosa. The minimum inhibitory concentration (MIC) values were determined by microdilution in broth. Four of these samples had good antimicrobial activity (MIC 62.5 μg / mL) and 7 showed moderate activity (100500 μg / mL). Four samples showed antitumor activity with selectivity varying from 1.8 to 7.5 times. The best results were obtained for the activity against Leishmania (Leishmania) amazonensis. In the tests of leishmanicidal activity against the promastigote forms, 12 samples presented IC50 values equal to or less than 5 μg / mL, of which 3 showed good selectivity against the parasite in relation to the murine fibroblast lineage (values ranging from 5 to 50 times). Similar results for leishmanicidal activity against amastigote forms were observed in two of these extracts (I06 and I75). The I06 extract induced cell death by mitochondrial apoptosis and ROS production in the promastigote forms of L. (L.) amazonensis. The therapeutic potential of the organic extract of isolate I06 in cutaneous leishmaniasis model was also evaluated. BALB / c mice infected with L. (L.) amazonensis and treated with I06 extract showed a significant reduction in the lesion size in the paw as well as the parasite load. No signs of hepatic or renal toxicity were observed from histological analyzes and evaluation of serum levels of some biochemical markers. For the identification of the fungal species used in this study, partial sequencing of the intergenic region (STI) of ribosomal DNA and a region of the calmodulin gene was performed. All isolates with some biological activity belong to different strains of Aspergillus fumigatus. Thus, this dataset strongly suggests that a molecule or set of organic extract molecules from secondary metabolites of A. fumigatus (isolated I06) has leishmanicidal activity with high selectivity and low toxicity, thus presenting great potential for therapeutic use.
- ItemSomente MetadadadosCapillary electrophoresis coupled to contactless conductivity detection for analysis of amino acids of agricultural interest in composting(Wiley-Blackwell, 2016) Cappelini, Luciana Teresa Dias [UNIFESP]; Menegoci Eugenio, Patricia de Fatima; Gomes Carneiro Leao, Paulo Augusto; Alberice, Juliana Vieira; Urbaczek, Ana Carolina; Assuncao, Nilson Antonio [UNIFESP]; Juliano, Luiz [UNIFESP]; Carrilho, EmanuelComposting is a sustainable approach to manage animal and vegetal waste generated in the Fundacao Parque Zoologico de Sao Paulo. The resulting compost is often used in ZOO's premises as an organic fertilizer for the production of vegetables, which is further used to feed the animals. The composting product provides many forms of mineral and also amino acids (AA) that are absorbed by plants as nutrients. Since most amino acids absorb only slightly or not at all in the UV wavelengths, we developed a method for the determination of AA of agricultural interest in the composting samples. Due to the complexity of samples, we used ion exchange chromatography for the purification of AA prior to analysis. The proposed CZE-(CD)-D-4 method allowed a separation of the AA in a short analysis time (less than 3.0 min), with great linearity (with R-2 ranging from 0.993 to 0.998). Using a BGE of 10 mmol/L TEA, reduction of high-frequency noise and lower baseline fluctuations were obtained. The LOQ for the five AA were around 35 mu mol/L, and were adequate for our purpose. In addition, the method showed good precision (RSD of peak area and migration time less than 1.55 and 1.16%, respectively).
- ItemAcesso aberto (Open Access)Compostagem comunitária: experimentos coletivos pelo método termofílico da ONG Missão Ambiental, Ipiranga - São Paulo(Universidade Federal de São Paulo, 2023) Souza, Ana Maria [UNIFESP]; Santos, Thiago Luiz Salvadeo [UNIFESP]; Smith, Pedro Cardoso; Semensatto Junior, Décio Luis [UNIFESP]; http://lattes.cnpq.br/1202506419685089; http://lattes.cnpq.br/0510421593724951; http://lattes.cnpq.br/6925496891580912; Universidade Federal de São Paulo (UNIFESP)The article presents a tool that uses an educational and playful method on environmental education called Reflection, developed with face-to-face and online activities, with the chosen target audience. At the end of the process, the tool will provide results in the form of a survey, promoting interaction and the exchange of knowledge between users. This tool will have two parts: in the first, workshops, lectures and visits to places relevant to the topic will be held; the second stage will be dedicated to practice, with educational games, plays, polls and discussion forums. While using the tool, a survey will be carried out to evaluate the effectiveness of learning on the proposed topic in relation to participants' environmental awareness. In addition, there will be interaction between users, who will be able to share their experiences and knowledge on the topic. It is extremely important because it addresses a relevant and current topic, environmental education, and proposes an innovative method to promote awareness and participation of individuals in protecting the environment. The conclusion and presentation of the research will contribute to the evaluation of the effectiveness of the educational method and to the improvement of future educational actions in the environmental area.
- ItemAcesso aberto (Open Access)Identificação e verificação de atividade celulolítica de microrganismos isolados de compostagem(Universidade Federal de São Paulo, 2016-02-29) Paganini, Thais Pires Martins [UNIFESP]; Vallim, Marcelo Afonso [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)The composting process is an effective and economical way of treating organic waste, mainly by microbial decomposition. The Fundação Parque Zoológico de São Paulo (FPZSP) has a compost production unit (UPCO) that takes organic matter from various sources, and one of the waste more used in the process are vegetable scraps, and consequently cellulose is the largest source of carbon present. This study explored the diversity in order to obtain cellulose-degrading microorganisms. Thus, a total of 54 microorganisms were isolated in medium supplemented with carboxymethyl cellulose as sole carbon source. Among them, a specie never described before as cellulose degrading, identified as Gordonia paraffinivorans, showed a high specific activity in enzymatic assays. Further, this microorganism had their genomes sequenced and the analysis of the genome identified sequences encoding two families (GH1 and GH3) of β-glucosidases.
- ItemAcesso aberto (Open Access)Isolamento, identificação e caracterização de bactérias cultiváveis presentes na compostagem de resíduos orgânicos do zoológico de São Paulo e produtoras de amilases e proteases(Universidade Federal de São Paulo, 2014-03-18) Magron, Claudio Fernando [UNIFESP]; Oliveira, Julio Cezar Franco de [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)A Fundação Parque Zoológico de São Paulo (FPZSP) possui uma Unidade de Produção de Composto Orgânico (UPCO) que recicla material orgânico, como: excremento de aproximadamente 3.500 animais (cerca de 400 espécies) oriundos de várias partes do mundo, cama de animais, resíduos de poda dos jardins do Parque Zoológico, restos vegetais da Mata Atlântica e resíduos alimentares. O composto final, cerca de 600 toneladas/ano, é utilizado como fertilizante orgânico na fazenda do Zoológico, para produção de cerca de 70% dos alimentos consumidos pelos animais do Parque, fechando assim um ciclo virtuoso de sustentabilidade. Além disso, a compostagem evita que a matéria orgânica seja depositada em aterros sanitários, o que causaria problemas ambientais. A riqueza microbiológica desse material é incalculável, visto que ali poderão ser encontradas espécies que ainda não foram descritas e/ou cultivadas, bem como espécies produtoras de enzimas de interesse industrial. Dentro de um acordo de parceria estabelecida entre a UNIFESP e a FPZSP, e dentro de colaboração com grupos de pesquisa do IQ-USP, este projeto de mestrado envolveu o isolamento de bactérias em meios de cultura ricos não seletivos provenientes de diferentes etapas de um processo de compostagem efetuado numa célula de compostagem da UPCO do Zoológico de São Paulo. Uma vez purificados e estocados no banco de microrganismos do LIMic (Laboratório de Interações Microbianas, Unifesp ? Diadema), os isolados microbianos foram triados para aqueles secretores de amilase ou protease, seguido de caracterização taxonômica por espectrometria de massa (MALDI/TOF-MS). Nove isolados bacterianos secretores de amilases foram selecionados para estudo quanto à atividade da atividade das amilases secretadas em meio de cultura líquido, e tiveram respectivos fragmentos do 16SrDNA amplificados por PCR, sequenciados e submetidos a análise de sequências para caracterização taxonômica.
- ItemSomente MetadadadosSeleção de fungos filamentosos como eficientes fontes de lignina-peroxidases(Universidade Federal de São Paulo (UNIFESP), 2020-08-21) Lima, Lidiane Maria dos Santos [UNIFESP]; Vasconcellos, Suzan Pantaroto de [UNIFESP]; Universidade Federal de São PauloSelection of Filamentous Fungi as Efficient Sources of Lignin-Peroxidases Lignin peroxidases (LiP) are ligninolytic enzymes normally produced by biodegradable fungi of lignocellulose in nature. Currently, interest in such enzymes has increased, due to their high potential for biotechnological application. In this sense, the search for microbial cultures, of different habitats, with different environmental conditions, exponentially increases the acquisition of notable enzymes for the most diverse purposes. An example is the application of biorefineries of cellulosic ethanol, which aims at the efficient transformation of lignocellulosic biomass into bioproducts, such as biofuels and energy, thus being considered a sustainable alternative to petroleum derivatives. However, one of the obstacles to the lignocellulose valorization on an industrial scale is the complexity and recalcitrance of biomass, especially lignin. It is in this context that the development and optimization of enzymatic preparations based on lignin-peroxidases are required, in order to enable the cleavage and digestibility of lignocellulosic fiber. Thus, the present study was initiated by the screening of environmental filamentous fungi with respect to the effectiveness of lignin-peroxidases, using dye degradation tests of aromatic molecular structures, such as remazol brilliant blue R (RBBR) and methylene blue (AM), as compounds for detecting fungal ligninolytic activity. Thus, six (6) filamentous fungi FPZSP3_01, FPZSP3_05, FPZSP3_47, FPZSP3_91, FPZSP1_129 and FPZSP1_141 isolated from soil increased by filter cake from sugar and alcohol refineries, were selected. Thus, such microorganisms were subjected to in-depth analysis about the enzymatic kinetics of their lignin-peroxidases, analyzing the crude enzymatic extract (microbial supernatant) from the following strains: FPZSP3_47 (Mucor sp.), FPZSP1_129 (Byssochlamys nivea) and FPZSP1 (Paecilomyces saturatus). The apparent kinetic constants KM, Vmax and kcat were determined in two conditions, pH 3.0 at room temperature (20 ºC±2), and pH 9.0, NaCl 4M, at 65 ºC, in addition to the influence of different temperatures, pH ranges and salinities in the activities enzyme detected. The filamentous fungus Mucor sp. (FPZSP3_47) belonging to the phylum Mucoromycota was selected for protein characterization studies. The apparent kinetic constants KM (mM) 55.65 ± 6.56, Vmax (mmol min-1) 414.75 ± 6.37 and kcat (min-1) 7.45 were higher at pH 3.0 at room temperature for Mucor sp. FPZSP3_47. The lignin-peroxidases of Mucor sp., Byssochlamys nivea and Paecilomyces saturatus exhibited activity and stability for 30 hours at all temperatures evaluated (4 ºC, ambient, 30 ºC and 65 ºC), with two optimal temperatures (30 ºC and 65 ºC) being selected. ºC). Such conditions were selected to analyze the activity dependence and enzymatic stability at pH. All ligninases of Mucor sp., Byssochlamys nivea and Paecilomyces saturatus were active and stable over a wide pH range (5.0 to 9.0). The pH 9.0 and the temperature of 65 ºC were selected to investigate the effect of NaCl (0.1 to 4 M) on the stability of LiP, and the results revealed wide tolerance to NaCl, for 24 hours. The LiP of the isolate Mucor sp. was purified by ion-exchange chromatography and analyzed on SDS-PAGE 12%, featuring a protein with a molecular mass estimated at 60 kDa and specific activity of 0.013 U/mg. It is worth mentioning that this is the first description of ligninases of a fungus of the genus Mucor sp. in the literature, therefore, a potential candidate for the optimization of processes associated with the production of ethanol from vegetable biomass.