Evaluation of bacterial diversity recovered from petroleum samples using different physical matrices

Unraveling the microbial diversity and its complexity in petroleum reservoir environments has been a challenge throughout the years. Despite the techniques developed in order to improve methodologies involving DNA extraction from crude oil, microbial enrichments using different culture conditions can be applied as a way to increase the recovery of DNA from environments with low cellular density for further microbiological analyses. This work aimed at the evaluation of different matrices (arenite, shale and polyurethane foam) as support materials for microbial growth and biofilm formation in enrichments using a biodegraded petroleum sample as inoculum in sulfate reducing condition. Subsequent microbial diversity characterization was carried out using Scanning Electronic Microscopy (SEM), Denaturing Gradient Gel Electrophoresis (DGGE) and 16S rRNA gene libraries in order to compare the microbial biomass yield, DNA recovery efficiency and diversity among the enrichments. The DNA from microbial communities in petroleum enrichments was purified according to a protocol established in this work and used for 16S rRNA amplification with bacterial generic primers. The PCR products were cloned, and positive clones were screened by Amplified Ribosomal DNA Restriction Analysis (ARDRA). Sequencing and phylogenetic analyses revealed that the bacterial community was mostly represented by members of the genera Petrotoga, Bacillus, Pseudomonas, Geobacillus and Rahnella. The use of different support materials in the enrichments yielded an increase in microbial biomass and biofilm formation, indicating that these materials may be employed for efficient biomass recovery from petroleum reservoir samples. Nonetheless, the most diverse microbiota were recovered from the biodegraded petroleum sample using polyurethane foam cubes as support material.47371272

Screening of Marine-derived Fungi Isolated from the sponge Didemnun ligulum for Biodegradation of Pentachlorophenol

Contamination by pesticides employed in agriculture has caused serious environmental harm. Pentachlorophenol (PCP) is a phenolic organochlorine compound and a dangerous pollutant which was banned from Brazil since 1985; however, there are still many contaminated areas. This pesticide is a serious problem because it has high toxicity and persistence at the environment due to its resistance to biotic and abiotic degradation. The use of microorganisms as degrading agents is considered an efficient method to reduce the adverse effects of environmental contaminants. It is noteworthy that fungi from marine environment are adapted to extreme conditions, including high chlorine concentrations, and can produce unique enzymes with interesting properties. Therefore, marine-derived fungi have an excellent enzymatic potential for the biotransformation of xenobiotics such as organochlorine pesticides. In this work, fifteen fungi strains isolated from a marine invertebrate, the ascidian Didemnun ligulum, were evaluated according to their ability to grow in solid culture media (3% malt extract agar) in the presence of different concentrations (10, 25, 30, 40, and 50 mg L–1) of PCP. Among the tested strains, nine could grow in at least one concentration, and Trichoderma harzianum CBMAI 1677 showed optimal growth at the higher evaluated concentration (50 mg L–1), showing toxicity resistance and suggesting its potential for biodegradation of PCP. In a later work, it was observed that T. harzianum CBMAI 1677 was able to degrade PCP. These results confirmed the efficiency of marine-derived fungi to biodegrade persistent compounds and could enable the development of bioremediation methodologies using these microorganism

Cellulolytic and proteolytic ability of bacteria isolated from gastrointestinal tract and composting of a hippopotamus

The bioprospection for cellulase and protease producers is a promise strategy for the discovery of potential biocatalysts for use in hydrolysis of lignocellulosic materials as well as proteic residues. These enzymes can increment and turn viable the production of second generation ethanol from different and alternative sources. In this context, the goal of this study was the investigation of cellulolytic and proteolytic abilities of bacteria isolated from the gastrointestinal tract of a hippopotamus as well as from its composting process. It is important to highlight that hippopotamus gastrointestinal samples were a non-typical sources of efficient hydrolytic bacteria with potential for application in biotechnological industries, like biofuel production. Looking for this, a total of 159 bacteria were isolated, which were submitted to qualitative and quantitative enzymatic assays. Proteolytic analyzes were conducted through the evaluation of fluorescent probes. Qualitative assays for cellulolytic abilities revealed 70 positive hits. After quantitative analyzes, 44 % of these positive hits were selected, but five (5) strains showed cellulolytic activity up to 11,8 FPU/mL. Regarding to proteolytic activities, six (6) strains showed activity above 10 %, which overpassed results described in the literature. Molecular analyzes based on the identification of 16S rDNA, revealed that all the selected bacterial isolates were affiliated to Bacillus genus. In summary, these results strongly indicate that the isolated bacteria from a hippopotamus can be a potential source of interesting biocatalysts with cellulolytic and proteolytic activities, with relevance for industrial applications.Brazilian research agency (FAPESP)Brazilian research agency (CAPES)Brazilian research agency (CNPq)Univ Fed Sao Paulo, Dept Biol Sci, Rua Sao Nicolau 210, BR-09913030 Sao Paulo, BrazilSao Paulo Zoo Pk Fdn, Av Miguel Estefano 4241, BR-04301905 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Biophys, Rua 3 Maio,100, BR-04044020 Sao Paulo, SP, BrazilFed Univ Latin Amer Integrat, Latin Amer Inst Life Sci & Nat, Av Tarquinio Joslin Santos 1000, BR-85870901 Foz Do Iguacu, Parana, BrazilUniv Fed Sao Paulo, Dept Biol Sci, Rua Sao Nicolau 210, BR-09913030 Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Biophys, Rua 3 Maio,100, BR-04044020 Sao Paulo, SP, BrazilFAPESP: 2010/51992-5CNPq: 475166/2013-2Web of Scienc

Enzymatic and biodegrader activities of petroleum microorganisms from Campos Basin (Pampo Sul)

Orientadores: Anita Jocelyne Marsaioli, Valeria Maia de OliveiraTese (doutorado) - Universidade Estadual de Campinas, Instituto de QuimicaResumo: Amostras de águas de formação de petróleo e óleos foram coletadas na Formação Pampo, situada na Bacia de Campos, através das quais, aplicando-se diferentes condições de cultivo, foram recuperadas 92 linhagens de bactérias aeróbias. Dentre os isolados, obtidos, 29 foram identificados por técnicas moleculares baseadas em DNAr 16S, observando-se a predominância de bactérias do gênero Bacillus.. De forma a constatar sobre a produção microbiana de metabólitos secundários com atividade inibitória ao desenvolvimento de outras espécies no reservatório de petróleo investigado, realizou-se análises de antibiogramas e Concentrações Inibitórias Mínimas (MIC), constatando Bacillus possuíam tal atividade. Sob condições anaeróbias foram obtidos consórcios microbianos, os quais foram caracterizados por técnicas independentes-de-cultivo, identificando-se bactérias do gênero Petrotoga como constituintes majoritários das comunidades presentes nos poços de petróleo amostrados. Duas espécies distintas de arquéias metanogênicas, Methanohalophilus sp. e Methanomethylovorans sp., também foram identificadas. Foram realizadas análises para a detecção da atividade biodegradadora da microbiota anaeróbia sobre biomarcadores do petróleo, onde foi observada forte tendência da comunidade microbiana investigada à biotransformação de compostos oxigenados (ácido octadecanóico; a e b-amirina). A biodegradação de biomarcadores do petróleo sob condições aeróbias revelou que a maioria das 29 bactérias avaliadas demonstrou tendência à biotransformação de ácidos carboxílicos de cadeia longa (ácido nonadecanóico). A linhagem de Micrococcus sp., entretanto, foi exceção a esta tendência, biotransformando preferencialmente hidrocarbonetos (fitano ediidrofenantreno), mesmo em presença de compostos oxigenados. A existência de interações simbióticas entre as comunidades microbianas aeróbias e anaeróbias nos processos de biodegradação de biomarcadores do petróleo, foi avaliada simulando atuação de ambas as microbiotas sobre uma mistura de hidrocarbonetos e compostos oxigenados. Os resultados obtidos sugeriram que a microbiota anaeróbia poderia sobreviver às custas dos metabólitos produzidos pela microbiota aeróbia durante a biodegradação do petróleo. A produção de exopolímeros microbianos com atividade bioemulsificante de hidrocarbonetos também foi determinada, verificando-se que microrganismos tidos com excelentes produtores de EPS não corresponderam às linhagens que apresentaram atividade biodegradadora de hidrocarbonetos do petróleo. Análises de triagens enzimáticas de alto desempenho (HTS) foram realizadas, e Dietzia sp. sobressaiu-se quanto às excelentes atividades de lipases, esterases e monooxigenases, enquanto Halomonas sp. apresentou o melhor desempenho quanto à atividade de epóxido hidrolaseAbstract: Samples of petroleum formation water and oils were collected from Pampo Formation (Campos Basin). Applying different conditions of microbial cultivation it was recuperated 92 bacterial strains from among 29 were identified by molecular techniques based in rDNA 16S. The predominance of Bacillus genus was observed in this recuperated bacterial community. The production of microbial secondary metabolites with antibiotic activity was investigated through analysis of antibiograms and Microbial Inibitory Concentrations (MIC) assays. Strains of Bacillus produced metabolites with microbial growth inhibition that can actuate in the petroleum reservoir in the selection of microbial species. Under anaerobic conditions microbial consortia were characterized by independent of cultivation techniques, identifying bacteria of Petrotoga genus as main constituent from the sampled reservoir. Two distinct species of methanogenic archaea, Methanohalophilus sp and Methanomethylovorans sp, were identified too. Analysis for the detection of the biodegrader activities of anaerobic microbiota under petroleum biomarkers were realized. Strong tendency of this microbial community to oxygenated compounds biotransformation was observed. The biodegradation of petroleum biomarkers under aerobic conditions was investigated as a form to determinate the microbial preferences for the petroleum compounds, in mixture or isolate, evaluating the formed products. The results presented the preference for the biotransformation of long chain carboxylic acids before the metabolism of hydrocarbons. Although, the strain of Micrococcus sp. preferred the biotransformation of hydrocarbons (phytane and dihydrophenanthrene), not biodegrading the fatty acid in strong levels. To investigate about the existence of symbiotic interactions between aerobic and anaerobic microbial communities in petroleum biodegradation processes, the actuation of the both microbiota under a mixture of hydrocarbons and oxygenated compounds was simulated. The obtained results suggest that the anaerobic microbiota can survive from the aerobic metabolites produced by the aerobic community. The production of microbial exopolymers with bioemulsifier activity of hydrocarbons was determined too. It was verified that strains presented as excellent producers of bioemulsifiers are not efficient petroleum biodegraders. Analysis of High Throughput Screening (HTS) were realized and Dietzia sp. presented strong activities of lipases, esterases and monooxygenases, while Halomonas sp. was the best result how the epoxide hydrolase activityDoutoradoQuimica OrganicaDoutor em Ciência

The Support of Early Interaction in a Separation Situation After Birth

Tämä opinnäytetyö on osa Näyttöön perustuva kätilötyö -hanketta, jonka tavoitteena on löytää keinoja kuinka kätilö voi tukea varhaista vuorovaikutusta tilanteessa, jossa äiti ja vastasyntynyt hoidetaan erillään. Aihe on noussut tarpeesta parantaa äitien kokemuksia ja ohjausta lapsivuode- sekä vastasyntyneiden tarkkailuosastoilla. Opinnäytetyömme on kirjallisuuskatsaus. Tutkimusaineisto kerättiin hoitotieteellisistä tietokannoista, Ovidista, Cinahlista, PubMedista ja Kurresta. Aineiston kokonaismäärä oli 26 tutkimusta tai artikkelia, joista 8 on suomenkielisiä tutkimuksia ja 18 englanninkielisiä tutkimusartikkeleita. Aineisto analysoitiin induktiivisesti laadullisen sisällön analyysin avulla. Kirjallisuuskatsauksemme tulokset tuovat esiin mahdollisia hoitotyön toimintoja, joilla kätilö voi tukea äidin ja vastasyntyneen varhaista vuorovaikutusta erotilanteessa. Keskeisiksi tuloksiksi nousi esille koko perheen huomioiminen hoidon toteuttamisessa. Perhekeskeisyys on periaate, joka ohjaa kätilön toimintaa. Kätilön tulee tukea koko perheen varhaista vuorovaikutusta ja tarjota mahdollisimman hyvät lähtökohdat perheen tulevaisuudelle. Tuloksina saimme myös konkreettisia keinoja, kuten vanhempien kannustaminen osallistumaan vauvan hoitoon ja imetysohjauksen antaminen äidille ja vanhempien rohkaiseminen kenguruhoitoon, joilla kätilö voi tukea varhaista vuorovaikutusta tilanteessa, jossa äiti ja vastasyntynyt hoidetaan erillään. Kirjallisuuskatsaukseemme käytimme tutkimuksia, jotka oli tehty äidin tai vanhempien kokemuksien pohjalta. Äidin tai vanhempien näkökulmasta tehdyt tutkimukset antoivat arvokasta tietoa äitien ja isien kokemuksista, kun he ovat joutuneet erilleen vastasyntyneestä vauvastaan. Tämän tiedon pohjalta saimme erilaisia keinoja, joilla kätilö voi tukea varhaista vuorovaikutusta erotilanteessa. Aihetta on tutkittu paljon äidin ja vanhempien kokemusten kautta. Kehitys ehdotuksena voisi olla hoitajien kokemuksiin pohjautuvia tutkimuksia, jotka kartoittavat esimerkiksi sosiaalisen tuen antamista rajoittavia tekijöitä. Mielenkiintoista olisi tutkia myös isien kokemuksia varhaisesta vuorovaikutuksesta erotilanteessa, sekä vuorovaikutuksen kehittymistä vauvan kotiin pääsyn jälkeen

Structure-based engineering of Phanerochaete chrysosporium alcohol oxidase for enhanced oxidative power towards glycerol

Glycerol is a major byproduct of biodiesel production and enzymes that oxidize this compound have been long sought after. The recently described alcohol oxidase from the white-rot basidiomycete Phanerochaete chrysosporium (PcAOX) was reported to feature very mild activity on glycerol. Here we describe the comprehensive structural and biochemical characterization of this enzyme. PcAOX was expressed in Escherichia coli in high yields and displayed high thermostability. Steady-state kinetics revealed that PcAOX is highly active towards methanol, ethanol, and 1-propanol (kcat = 18, 19, and 11 s-1, respectively), but showed very limited activity towards glycerol (kcat = 0.2 s-1 at 2 M substrate). The crystal structure of the homo-octameric PcAOX was determined at 2.6 Å resolution. The catalytic center is a remarkable solvent-inaccessible cavity located at the re side of the flavin cofactor. Its small size explains the observed preference for methanol and ethanol as best substrates. These findings led us to design several cavity-enlarging mutants with significantly improved activity towards glycerol. Among them, the F101S variant had a high kcat value of 3 s-1 retaining a high degree of thermostability. The crystal structure of F101S PcAOX was solved, confirming the site of mutation and the larger substrate-binding pocket. Our data demonstrate that PcAOX is a very promising enzyme for glycerol biotransformation

Sustainable Synthesis of Novel Arylamide L-Cysteine Methyl Esters Peptidomimetic Derivatives: Inhibitors of Serine and Cysteine-like Proteases

The development of new protease inhibitors is always expanding for active therapies against diseases caused by pathogenic microorganisms that rely on proteases for replication and vital functions. This work aimed to synthesize new peptidomimetics arylamides, endeavoring to provide these compounds with the capacity to inhibit mainly cysteine and serine-like proteases.  The effectiveness of COMU as coupling reagent, under classical and sustainable approaches, were evaluated. The results confirmed that the use of dichloromethane and the classical methodology is efficient for new amide bond formation in terms of yield. Although, the use of dimethyl carbonate and the microwave-assisted methodology proved competitive performance and can be used as an alternative route due to its environmentally friendly approach (green solvent and energy efficiency).  In vitro screening assays attested that the proposed compounds have inhibitory activity for papain (IC50 between 5.56±0.21 μM and 18.50±0.69 μM) and β-trypsin (IC50 between 250±12 μM and 1410±30 μM). The compounds 1a and 1e were noteworthy with IC50 values of 5.56±0.21 μM and 7.06±0.33 μM for papain and 540±10 μM and 250±12 μM for β-trypsin, respectively. DOI: http://dx.doi.org/10.17807/orbital.v12i4.1575 </p