Thermo-resistant enzyme-producing microorganisms isolated from composting / Microrganismos produtores de enzimas termo-resistentes isolados de compostagem

Organo-mineral fertilizers supplemented with biological additives are an alternative to chemical fertilizers. In this study, thermoresistant microorganisms from composting mass were isolated by two-step procedures. First, samples taken at different time points and temperatures (33 days at 52 ºC, 60 days at 63 ºC, and over 365 days at 26 ºC) were pre-incubated at 80 oC for 30 minutes. Second, the microbial selection by in vitro culture-based methods and heat shock at 60 oC and 100 oC for 2h and 4h. Forty-one isolates were able to grow at 60 °C for 4h; twenty-seven at 100 °C for 2h, and two at 100 °C for 4h. The molecular identification by partial sequencing of the 16S ribosomal gene using universal primers revealed that thirty-five isolates were from eight Bacillus species, one Brevibacillus borstelensis, three Streptomyces thermogriseus, and two fungi (Thermomyces lanuginosus and T. dupontii). Data from amylase, phytase, and cellulase activity assays and the enzymatic index (EI) showed that 38 of 41 thermo-resistant isolates produce at least one enzyme. For amylase activity, the highest EI value was observed in Bacillus licheniformis (isolate 21C2, EI= 4.11), followed by Brevibacillus borstelensis (isolate 6C2, EI= 3.66), Bacillus cereus (isolate 18C2, EI= 3.52), and Bacillus paralicheniformis (isolate 20C2, EI= 3.34). For phytase, the highest EI values were observed for Bacillus cereus (isolate 18C2, EI= 2.30) and Bacillus licheniformis (isolate 3C1, EI= 2.15). Concerning cellulose production, B. altitudinis (isolate 6C1) was the most efficient (EI= 6.40), followed by three Bacillus subtilis (isolates 9C1, 16C2, and 19C2) with EI values of 5.66, 5.84, and 5.88, respectively, and one B. pumilus (isolate 27C2, EI= 5.78). The selected microorganisms are potentially useful as a biological additive in organo-mineral fertilizers and other biotechnological processes.

Os fertilizantes organo-minerais suplementados com aditivos biológicos são uma alternativa aos adubos químicos. Neste estudo, microrganismos termoresistentes foram isolados de compostagem por procedimentos de duas etapas. Inicialmente, as amostras tomadas em diferentes períodos e temperaturas (33 dias a 52 ºC, 60 dias a 63 ºC e mais de 365 dias a 26 ºC) foram pré-incubadas a 80 oC por 30 minutos. Posteriormente, a seleção microbiana foi conduzida por métodos baseados em cultura in vitro e choque térmico a 60 oC e 100 oC por 2h e 4h. Quarenta e um isolados foram capazes de crescer a 60 °C por 4h; vinte e sete a 100 °C por 2h e dois a 100 °C por 4h. A identificação molecular por sequenciamento parcial do gene ribossomal 16S usando primers universais revelou que trinta e cinco isolados eram de oito espécies de Bacillus, um Brevibacillus borstelensis, três Streptomyces thermogriseus e dois fungos (Thermomyces lanuginosus e T. dupontii). Os dados dos ensaios de atividade de amilase, fitase e celulase e o índice enzimático (IE) mostraram que 38 dos 41 isolados termorresistentes produziram pelo menos uma enzima. Para a atividade da amilase, o maior valor de IE foi observado em Bacillus licheniformis (isolado 21C2, IE = 4,11), seguido por Brevibacillus borstelensis (isolado 6C2, IE = 3,66), Bacillus cereus (isolado 18C2, IE = 3,52) e Bacillus paralicheniformis (isolado 20C2, IE = 3,34). Para a fitase, os maiores valores de IE foram observados para B. cereus (isolado 18C2, IE = 2,30) e B. licheniformis (isolado 3C1, IE = 2,15). Em relação à produção de celulose, B. altitudinis (isolado 6C1) foi o mais eficiente (IE = 6,40), seguido por três Bacillus subtilis (isolados 9C1, 16C2 e 19C2) com valores de IE de 5,66, 5,84 e 5,88, respectivamente, e um B. pumilus (isolado 27C2, IE = 5,78). Pode-se inferir que os microrganismos selecionados são potencialmente úteis como aditivos biológicos em fertilizantes organo-minerais e outros processos biotecnológicos.

Identification and control of wood-deteriorating fungi / Identificação e controle de fungos deterioradores de madeira

Wood is rich in organic compounds; thus, it is susceptible to attacks by several deteriorating agents because they acknowledge such attacks as a necessary energy supply. Fungi stand out among these agents since they can attack the wood in trees (before felling) and in several post-felling stages. Fungi are biological agents that use organic compounds as food sources, and that is the reason why they degrade most chemical components found in wood. Wood attacked by fungi suffers significant value loss, since these degrading agents affect their mechanical and aesthetic properties. Fungi ­ which are classified as staining, molding and rotting ­ are one of the main responsible for the biggest losses in the timber industry. Wood can be virtually used in a whole range of environments, a fact that makes it susceptible to attacks by different fungal species, since the ideal conditions for fungal development change from species to species. In other words, the fact that one or more fungal species are capable of deteriorating wood is directly related to conditions wood is subjected to. Thus, it is essential knowing how to evaluate fungal attacks, symptoms to be taken into consideration at the time to identify the type of attacking organism and the attack stage, as well as the control and prevention measures to be applied to these organisms.(AU)

Rica em compostos orgânicos, a madeira é suscetível ao ataque de diversos agentes deterioradores por ser reconhecida como suprimento necessário de energia. Entre esses agentes, destacam-se os fungos, cujo início de ataque pode se dar ainda na árvore, antes do abate e nas diversas fases posteriores. Os fungos são agentes biológicos que necessitam de compostos orgânicos como fontes de alimento e, por essa razão, degradam grande parte dos componentes químicos da madeira. As madeiras, quando atacadas por fungos, sofrem grandes perdas de valor, afetando suas propriedades mecânicas e estéticas. Os fungos são considerados um dos principais responsáveis pelos maiores prejuízos na indústria madeireira e são classificados como manchadores, emboloradores e apodrecedores. Praticamente, a madeira pode estar em uso em uma variedade de ambientes; sabendo-se que as condições ideais de desenvolvimento de um fungo são diferentes para cada espécie, a madeira ficará suscetível ao ataque por diferentes espécies de fungos. Ou seja, o fato de uma ou mais espécies de fungos serem encontradas deteriorando a madeira está diretamente relacionado às condições em que ela se encontra. Dessa forma, torna-se importante saber como avaliar os ataques feitos por fungos, os sintomas a serem considerados para identificar o tipo de organismo e o estágio do ataque, bem como as medidas de controle e prevenção desses organismos.(AU)

Actividad antimicrobiana de cuatro variedades de plantas frente a patógenos de importancia clínica en Colombia / Antimicrobial activity of four varieties of plants against pathogens clinical significance in Colombia

Resumen Objetivo. Evaluar la actividad antimicrobiana de los extractos de las plantas Bauhinia sp., Sambucus nigra, Eichhornia crassipes y Taraxacum officinale frente a patógenos de importancia clínica. Método. La metodología incluyó la adquisición, secado, maceración, molienda, preparación de los extractos crudos etanólicos y concentración por rotaevaporación, análisis fitoquimico y se separaron las fracciones por cromatografía en capa fina. Las pruebas antimicrobianas se realizaron con diferentes concentraciones de los extractos según las indicaciones de Clinical and Laboratory Standars Institute. Los microorganismos utilizados fueron Enterococcus faecium resistente a vancomicina, Streptococcus pneumoniae, Klebsiella pneumoniae con presencia de KPC, Providencia rettgeri con presencia de ESBLs, Pseudomonas aeruginosa, Enterobacter cloacae, Escherichia coli, Staphylococcus aureus (3-lisina y Candida albicans. Resultados. Las cromatografías permitieron comprobar la presencia de flavonoides, terpenos, saponinas, fenoles, quinonas y alcaloides que han sido reportados con actividad antimicrobiana. En los ensayos de susceptibilidad antimicrobiana se encontró que los extractos presentaban diversos grados de inhibición frente a los microrganismos de estudio, siendo el más eficaz los tallos de T. officinale. Conclusión. Se puede concluir que los extractos vegetales podrían ser una alternativa de tratamiento para infecciones nosocomiales.

Objective. To evaluate the antimicrobial activity of extracts of plants Bauhinia sp., Sambucus nigra, Taraxacum officinale and Eichhornia crassipes against clinically important pathogens. Method. The methodology included the acquisition, drying, soaking, grinding, preparing ethanolic crude extracts and concentration by rotary evaporation; phytochemical analysis, visualization by thin layer chromatography. Antimicrobial tests were performed with different concentrations of extracts as indicated by the Clinical and Laboratory Standards Institute. The microorganisms used were Enterococcus faecium vancomycin-resistant, Streptococcus pneumoniae, Klebsiella pneumoniae with KPC, Providencia rettgeri presence of ESBLs, Pseudomonas aeruginosa, Enterobacter cloacae, Escherichia coli, Staphylococcus aureus (3-lysine and Candida albicans. Results. Chromatographies allowed checking the presence of flavonoids, terpenes, saponins, phenols, quinones and alkaloids have been reported having antimicrobial activity. In antimicrobial susceptibility tests it found that extracts showed varying degrees of inhibition against microorganisms study, the most effective stems T. officinale. Conclusion. It can be concluded that plant extracts may be alternative in the treatment of nosocomial infections.

Toxicidade equivalente da atmosfera por dioxinas, furanos e bifenilas policloradas,com uso de duas técnicas de coleta, passiva e ativa / Equivalent toxicity of the atmosphere for dioxins, furans and polychlorinated biphenyls, using two air sampling techniques, passive and active

Introdução: Dioxinas, furanos e bifenilas policloradas são poluentes tóxicos para a saúde humana incluindo riscos de incidência de cânceres, efeitos de neurodesenvolvimento, lesões dérmicas, cloroacne. Estes compostos são poluentes orgânicos persistentes (POPs) que podem ser transportados de longas distâncias da fonte de emissão e se bioacumular em ecossistemas. A atmosfera poluída foi recentemente classificada como carcinogênica para os seres humanos pela Organização Mundial da Saúde, mostrando a importância de sua caracterização, principalmente para compostos tóxicos. Entretanto, técnica de coleta ativa tem custo elevado para POPs, e existem poucos estudos de calibração que validem a substituição. Objetivos: Avaliar a toxicidade equivalente da atmosfera por dioxinas, furanos e bifenilas cloradas, utilizando técnicas de coleta ativa e passiva, e verificar gradiente de concentração nos ambientes urbano, urbano/industrial e de background. Método: Amostras de ar foram coletadas, utilizando coletores ativos e passivos, durante dois períodos consecutivos de quatro meses: de setembro a dezembro de 2014 (período 1) e de maio a agosto de 2015 (período 2) em três cidades de São Paulo, SP, em ambientes urbano, urbano/industrial e de background. Todas as amostras foram extraídas com solução de tolueno:acetona (9:1) em Soxhlet por 24 h e padrões marcados (13C12-PCDD/Fs e 13C12-PCBs) foram adicionados em cada amostra antes do processo de extração. Os extratos foram purificados em coluna de sílica mista (40 por cento H2SO4 e 10 por cento AgNO3) seguida por coluna de alumina. O procedimento analítico foi realizado utilizando HRGC/HRMS (High Resolution Gas Chromatograph/High Resolution Mass Spectrometer) operando em ionização de impacto de elétrons com energia de 35 eV no modo SIM (Select Ion Monitoring) e resolução de 10.000. Resultados mostraram que: (1) existe variação sazonal para concentrações de PCDD/Fs no ar entre os períodos 1 e 2 (p=0,03), enquanto as concentrações de dl-PCBs não foram estatisticamente diferentes nestes períodos (p=0,52); (2) existe gradiente de concentração de PCDD/Fs e dl- PCBs que aumenta na seguinte ordem: background

Introduction: Dioxins, furans and polychlorinated biphenyls are toxic pollutants for human health including risks of cancer incidence, neurodevelopmental effects, dermal lesions, chloracne. These compounds are persistent organic pollutants (POPs) that can be transported to long distances from the emission source and they are bioaccumulated in ecosystems. Recently, the outdoor air pollution were classified as carcinogenic to humans by the World Health Organization, showing the importance of its characterization for toxic compounds. However, active air monitoring has a high cost for POPs, and there is a few calibration studies which support that substitution. Objective: To assess the equivalent toxicity of the atmosphere regarding the measurement of dioxins, furans and polychlorinated biphenyls, using active and passive air samplers, and to evatuate the contrasting concentrations at urban, urban/industrial and background sites. Method: Air samples were collected, using active and passive samplers, over two consecutive periods of four months: from September to December 2014 (period 1) and from May to August 2015 (period 2) at three cities in São Paulo, SP, covering urban, urban/industrial and background sites. All samples were extracted with toluene:acetone (9:1) in a Soxhlet apparatus for 24 hours and surrogate standards (13C12-PCDD/F and 13C12-PCBs) were spiked on each sample media prior to extraction procedure. The extracts were purified on an silica column (40 per cent H2SO4 and 10 per cent AgNO3) followed by an alumina column. The analytical procedure was carried out using HRGC/HRMS (High Resolution Gas Chromatograph/High Resolution Mass Spectrometer) operating in electron impact ionization with an energy of 35 eV in SIM (selected ion monitoring) mode and 10.000 resolution power. Results show that (1) there are seasonal variations for PCDD/F concentrations in air between period 1 and 2 (p=0.03), whereas dl-PCB levels were not statistically different (p=0.52) in those periods. (2) PCDD/F and dl-PCB air levels are in the following order: background

Busca por oxidantes quirais para a transformação enantiosseletiva de compostos orgânicos de boro / Search for chiral oxidants for the enantioselective transformation of organic boron compounds

Neste trabalho, avaliou-se o potencial do uso de oxidantes quirais em oxidações enantiosseletivas de compostos orgânicos de boro. É de conhecimento geral que compostos orgânicos de boro, especialmente ésteres e ácidos borônicos são facilmente oxidados por hidroperóxidos em meio básico. No entanto, são escassos na literatura exemplos destas reações de modo enantiosseletivo. A fim de realizar as reações mencionadas, sintetizou-se os hidroperóxidos quirais TADOOH ({(4R,5R)-5-[(hidroperoxidifenil)metil]-2,2-dimetil-1,3-dioxolan-4il}difenilmetanol) e o hidroperóxido quiral derivado de carboidrato, 2,3-dideoxi1-O-oxidanil-4,6-di-O-pivaloil-α-D-eritro-hex-2-enopiranose (di-O-PivOOH). Estes compostos apresentaram resultados interessantes na literatura em oxidações enantiosseletiva de sulfetos orgânicos, em epoxidações de alcenos e em oxidações de Baeyer-Villiger. Inicialmente o potencial oxidativo de ambos hidroperóxidos, bem como a seletividade destes, foi avaliado frente a diversos ésteres borônicos, sendo que somente o TADOOH apresentou resultados promissores. (Ver esquema no PDF) Observou-se uma melhor seletividade do TADOOH frente a ésteres borônicos que possuíam grupos carbonílicos em sua estrutura. Ao submeter o ß-boronil-éster, 3-fenil-3-(4,4,5,5-tetrametil-1,3,2-dioxaborolan-2-il)propanoato de etila, à oxidação com o TADOOH em THF utilizando NaOH como base, a -30°C por 1 hora, obteve-se o respectivo álcool com 40% de e.e. Cálculos de DFT para o estado de transição na oxidação dos ésteres borônicos com o TADOOH foram realizados em colaboração com o grupo do Prof. Dr. Ataualpa Albert Carmo Braga. Estes cálculos demonstraram que o estado de transição é estabilizado por uma ligação de hidrogênio não clássica entre o oxigênio da carbonila e umas das ligações C-H dos grupos fenila do TADOOH. Além dos estudos relatados, a reconhecida metodologia de Sharpless na epoxidação assimétrica de alcoóis alílicos foi adaptada para a oxidação enantiosseletiva de ésteres borônicos. Ao trocar o ligante derivado de éster tártarico, normalmente utilizado nas epoxidações de Sharpless, por (-)-efedrina observou-se uma moderada seletividade deste sistema frente ao pinacol l-fenietilboronato. Investigações mais detalhadas demonstraram que a presença do Ti(IV) não era necessária, sendo que a (-)efedrina era a responsável pela ativação e indução quiral nesta reação.

In this work, it was investigated the potential use of chiral oxidants in organic boron compound oxidation. It is known in the literature, that organic boron compounds can be easily oxidized by hydroperoxides. However, an enantioselective approach in literature is scarce. In order to perform these reactions, hydroperoxide TADOOH ({(4R,5R)-5[(hydroperoxydiphenyl)methyl]-2,2-dimethyl-l,3-dioxolan-4-yl}diphenylmethanol) and carbohydrate derived hydroperoxide, 2,3-dideoxy-1-O-oxidanyl-4,6-di-O-pivaloyl-α-D-erythro-hex-2-enopyranose (di-O-PivOOH), have been synthesized. These compounds showed interesting results in several enantioselective oxidations, as like, organic sulfides oxidation, alkenes epoxidation and Baeyer-Villiger oxidations. The oxidative potential of both hydroperoxides, as well as their selectivity, were evaluated against several boronic esters. Only TADOOH has shown promissing results for further studies. (See Scheme on PDF). Boronic esters containing a carbonyl moiety showed better selectivities with TADOOH, for example, the reaction of ß-boronyl-ester, ethyl 3-phenyl-3-(4,4,5,5-tetramethyl-l,3,2-dioxaborolan-2-yl)propanoate, gave the correponding alcohol with 40% e.e. DFT calculations for the transition state in the oxidation of the boronic esters with TADOOH were carried out in collaboration with the group of Prof. Dr. Ataualpa Albert Carmo Braga. These calculations have shown that the transition state is stabilized by a non-classical hydrogen bond between the carbonyl oxygen and one of the C-H bonds of the TADOOH phenyl groups. In addition to the studies, the well-known Sharpless protocol for asymmetric epoxidation of allylic alcohols was adapted in the enantioselective oxidation of boronic esters. By replacing the tartaric ester-derived, commonly used in the Sharpless experiments, for (-)-ephedrine moderate selectivity was observed with pinacol 1-phenylethyl boronate. Further investigations showed that the presence of Ti (IV) was not necessary, and (-)-ephedrine was responsible for the activation and chiral induction in this reaction.

Chemical and physical properties of an anthropogenic dark earth soil from Bragança, Para, Eastern Amazon / Propriedades físicas e químicas de Terra Preta Antropogênica em Bragança, Pará, Amazônia Oriental

Although anthropogenic dark earth (ADE) is generally found in non-floodable land, it also occurs on floodplains but, there is no information about the chemical and physical characteristics of ADE in this environment. In this study, we propose to check the hypothesis that a Gleysol, classified as ADE, presents improved chemical and physical conditions than an adjacent soil, no anthropogenic. Thus, the objective of this study was to characterize the chemical and physical properties of the top layer of two ADE profiles in a Gleysol and compare them with an adjacent soil. Samples were taken from two areas classified as ADE in Bragança, Pará State, Brazil, at the "Jabuti" archaeological site, and from an adjacent non-anthropogenic site. Disturbed and undisturbed soil samples were collected at the soil depth of 0.05-0.10 m for chemical (pH, potential acidity, exchangeable cations, and soil organic carbon) and physical (soil particle size distribution, particles density, water retention curve, total porosity, microporosity, macroporosity, and bulk density) analysis. The two areas of ADE in a Gleysol, showed improved soil chemical properties compared to the adjacent soil, particularly in relation to phosphorus and calcium levels that contributed to higher cation exchange capacity which, in turn, was positively related to organic carbon content. Changes in soil physical properties were less noticeable but both areas of ADE presented higher water retention capacity, particularly at low tension. The improved conditions of the ADE soil under Gleysols shows that these areas are adequate for soil cultivation, especially with plants adapted to floodplain.

Embora as terras pretas antropogênicas (TPA) geralmente são encontradas em áreas de terra firme, elas também ocorrem em áreas e várzeas, mas, não há informações sobre as características químicas e físicas de TPA neste ambiente. Neste estudo, nós propomos verificar a hipótese de que um Gleissolo, classificado como TPA, apresenta melhores condições químicas e físicas do que o solo adjacente, não antropogênico. Assim, o objetivo do estudo foi caracterizar as propriedades químicas e físicas da camada superficial de dois perfis de TPA em um Gleissolo e compará-los com um solo adjacente. Amostras de solo foram coletadas em duas áreas classificadas como TPA em Bragança, Estado do Pará, Brasil, no sítio arqueológico "Jabuti" e em uma área adjacente não antropogênica. Amostras de solo com estrutura deformada e indeformada foram coletadas na profundidade de 0,05-0,10 m para análises químicas (pH, acidez potencial, cátions trocáveis, e carbono orgânico do solo) e físicas (distribuição do tamanho das partículas do solo, densidade de partículas, curva de retenção de água, porosidade total, microporosidade, macroporosidade e densidade do solo). As duas áreas de TPA em Gleissolo, apresentaram melhores propriedades químicas comparadas com o solo adjacente, principalmente em relação aos níveis de fósforo e cálcio que contribuíram para a maior capacidade de troca catiônica que, por sua vez, foi positivamente relacionada com o conteúdo de carbono orgânico. Mudanças nas propriedades físicas do solo foram menos pronunciadas, mas as duas áreas de TPA exibiram maior capacidade de retenção de água, sobretudo em baixa tensão.

Tomato yield and quality under various combinations of organic compost / Produção e qualidade de tomate cultivado sob oito tipos de composto orgânico

The number of growers and planted area dedicated to organic farming have increased considerably. Consequently, demand has increased for alternative organic composts in terms of quantity and new options for direct use with crops. Making use of local industrial byproducts is also important throughout Brazil and the rest of the world. Therefore, it was evaluated the effect of eight types of organic composts, produced with different percentages of poultry litter and crushed sugarcane on tomato yield and quality. The experiment was carried out during the 2013 growing season at the experimental farm of the Agricultural Research Company of Minas Gerais state (Empresa de Pesquisa Agropecuária do Estado de Minas Gerais) located in Prudente de Morais, MG, Brazil. It was evaluated different formulations of poultry litter and crushed sugarcane (0, 10, 20, 30, 40, 50, 50, 60 and 70%) yielding eight types of alternative Bokashi used as organic compost in the production of two tomato cultivars (Santa Clara and the Verano hybrid). It was found that the various formulations of alternative Bokashi, except the alternative Bokashi B1, strengthened components of tomato yield and quality. We found that composts of poultry litter and crushed sugarcane increase tomato yield and quality and can be and excellent alternative, especially for organic farmers.

O cenário atual da agricultura orgânica demonstra aumento considerável do número de produtores e área plantada. Consequentemente, há maior demanda por novas alternativas de compostos orgânicos, não só em volume, mas também disponibilizar novas opções para uso direto no sistema de cultivo. A busca pelo aproveitamento de subprodutos gerados pelas indústrias locais é pauta no Brasil e no mundo. O objetivo do trabalho foi avaliar o efeito de oito tipos de compostos orgânicos produzidos com diferentes porcentagens de cama de frango e cana de açúcar triturada na produção e qualidade do tomate.O experimento foi instalado na fazenda experimental da Empresa de Pesquisa Agropecuária do Estado de Minas Gerais localizada no município de Prudente de Morais, MG no ano agrícola de 2013. Foram avaliadas diferentes formulações entre cama de frango e cana de açúcar triturada(0, 10, 20, 30, 40, 50, 50, 60 e 70%) resultando em oito tipos de Bokashi alternativo utilizado como composto orgânico na produção de duas cultivares de tomate (Santa Clara e Híbrido Verano). Pode-se verificar que as diferentes formulações de Bokashi Alternativo, com exceção da B1, potencializaram os componentes de produção e qualidade do tomate. O uso de cama de frango associado com cana de açúcar triturada promove incrementos na produção e qualidade de tomate demonstrando ser uma excelente alternativa, especialmente para produtores orgânicos.

Process optimization for production and purification of a thermostable, organic solvent tolerant lipase from Acinetobacter sp. AU07

ABSTRACT The purpose of this study was to isolate, purify and optimize the production conditions of an organic solvent tolerant and thermostable lipase from Acinetobacter sp. AU07 isolated from distillery waste. The lipase production was optimized by response surface methodology, and a maximum production of 14.5 U/mL was observed at 30 ºC and pH 7, using a 0.5% (v/v) inoculum, 2% (v/v) castor oil (inducer), and agitation 150 rpm. The optimized conditions from the shake flask experiments were validated in a 3 L lab scale bioreactor, and the lipase production increased to 48 U/mL. The enzyme was purified by ammonium sulfate precipitation and ion exchange chromatography and the overall yield was 36%. SDS-PAGE indicated a molecular weight of 45 kDa for the purified protein, and Matrix assisted laser desorption/ionization time of flight analysis of the purified lipase showed sequence similarity with GDSL family of lipases. The optimum temperature and pH for activity of the enzyme was found to be 50 ºC and 8.0, respectively. The lipase was completely inhibited by phenylmethylsulfonyl fluoride but minimal inhibition was observed when incubated with ethylenediaminetetraacetic acid and dithiothreitol. The enzyme was stable in the presence of non-polar hydrophobic solvents. Detergents like SDS inhibited enzyme activity; however, there was minimal loss of enzyme activity when incubated with hydrogen peroxide, Tween 80 and Triton X-100. The kinetic constants (Km and Vmax) revealed that the hydrolytic activity of the lipase was specific to moderate chain fatty acid esters. The Vmax, Km and Vmax/Km ratio of the enzyme were 16.98 U/mg, 0.51 mM, and 33.29, respectively when 4-nitrophenyl palmitate was used as a substrate.

Evaluation and optimization of SYBR Green real-time reverse transcription polymerase chain reaction as a tool for diagnosis of the Flavivirus genus in Brazil

Abstract: INTRODUCTION: The genus Flavivirus includes several pathogenic species that cause severe illness in humans. Therefore, a rapid and accurate molecular method for diagnosis and surveillance of these viruses would be of great importance. Here, we evaluate and optimize a quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) method for the diagnosis of the Flavivirus genus. METHODS: We evaluated different commercial kits that use the SYBR Green system for real-time RT-PCR with a primer set that amplifies a fragment of the NS5 flavivirus gene. The specificity and sensitivity of the assay were tested using twelve flaviviruses and ribonucleic acid (RNA) transcribed from the yellow fever virus. Additionally, this assay was evaluated using the sera of 410 patients from different regions of Brazil with acute febrile illness and a negative diagnosis for the dengue virus. RESULTS: The real-time RT-PCR amplified all flaviviruses tested at a melting temperature of 79.92 to 83.49°C. A detection limit of 100 copies per ml was determined for this assay. Surprisingly, we detected dengue virus in 4.1% (17/410) of samples from patients with febrile illness and a supposedly negative dengue infection diagnosis. The viral load in patients ranged from 2.1×107to 3.4×103copies per ml. CONCLUSIONS: The real-time RT-PCR method may be very useful for preliminary diagnoses in screenings, outbreaks, and other surveillance studies. Moreover, this assay can be easily applied to monitor viral activity and to measure viral load in pathogenesis studies.

Fluorescent membrane markers elucidate the association of Borrelia burgdorferi with tick cell lines

This study aimed to describe the association of Borrelia burgdorferi s.s. with ixodid tick cell lines by flow cytometry and fluorescence and confocal microscopy. Spirochetes were stained with a fluorescent membrane marker (PKH67 or PKH26), inoculated into 8 different tick cell lines and incubated at 30°C for 24 h. PKH efficiently stained B. burgdorferi without affecting bacterial viability or motility. Among the tick cell lines tested, the Rhipicephalus appendiculatus cell line RA243 achieved the highest percentage of association/internalization, with both high (90%) and low (10%) concentrations of BSK-H medium in tick cell culture medium. Treatment with cytochalasin D dramatically reduced the average percentage of cells with internalized spirochetes, which passed through a dramatic morphological change during their internalization by the host cell as observed in time-lapse photography. Almost all of the fluorescent bacteria were seen to be inside the tick cells. PKH labeling of borreliae proved to be a reliable and valuable tool to analyze the association of spirochetes with host cells by flow cytometry, confocal and fluorescence microscopy.

Enhanced production and organic solvent stability of a protease fromBrevibacillus laterosporus strain PAP04

A bacterial strain (PAP04) isolated from cattle farm soil was shown to produce an extracellular, solvent-stable protease. Sequence analysis using 16S rRNA showed that this strain was highly homologous (99%) to Brevibacillus laterosporus. Growth conditions that optimize protease production in this strain were determined as maltose (carbon source), skim milk (nitrogen source), pH 7.0, 40°C temperature, and 48 h incubation. Overall, conditions were optimized to yield a 5.91-fold higher production of protease compared to standard conditions. Furthermore, the stability of the enzyme in organic solvents was assessed by incubation for 2 weeks in solutions containing 50% concentration of various organic solvents. The enzyme retained activity in all tested solvents except ethanol; however, the protease activity was stimulated in benzene (74%) followed by acetone (63%) and chloroform (54.8%). In addition, the plate assay and zymography results also confirmed the stability of the PAP04 protease in various organic solvents. The organic solvent stability of this protease at high (50%) concentrations of solvents makes it an alternative catalyst for peptide synthesis in non-aqueous media.

Alteraciones orgánicas y funcionalesocasionadas por el uso excesivo depantallas de visualización de datos / Organic and functional alterations caused by the excessive use of data display screens

Los avances logrados por las Pantallas de Visualización de Datos (PVD) y su empleoen los computadores digitales y la telefonía móvil, entre muchas de sus aplicaciones,son extraordinarios tanto en el perfeccionamiento técnico como en la optimizaciónde sus funciones. Estos alcances se deben utilizar con lógica y raciocinio. Suutilización incorrecta puede ser la causa de muchas alteraciones o deformidadesorgánicas y subsecuentemente incapacidades laborales. Las más investigadas sonlas deformidades de la columna vertebral: hipercifosis; hiperlordosis, y la llamadaespalda plana en la cual hay disminución de la curvatura lumbar y trastorno de susfunciones. Todas estas alteraciones, provocadas por las posturas defectuosas queasumen los usuarios, se manifiestan con cervicalgia (dolor en el cuello) y a medianoo largo plazo con artrosis (degeneración del cartílago). Otra de las alteracionesdocumentadas y estudiadas es el síndrome del túnel carpiano, que es provocadopor el aumento de la presión en el interior del canal en el curso de una inflamaciónde los tendones de los músculos flexores que comprimen el nervio mediano en supaso por el canal y que se manifiesta con dolor difuso, parestesias, hipoestesia oanestesia de la mano. La aplicación inadecuada del teclado del computador, el empleoinapropiado y excesivo de herramientas (Internet, PVD, computadores digitalesy telefonía celular) y los movimientos manuales repetitivos pueden causar síndromedel túnel carpiano. La educación bien orientada y la prevención primaria podríandisminuir las alteraciones orgánicas y funcionales provocadas por el uso excesivode las pantallas de visualización de datos...

Response of Rhodococcus erythropolis strain IBBPo1 to toxic organic solvents

Abstract Recently, there has been a lot of interest in the utilization of rhodococci in the bioremediation of petroleum contaminated environments. This study investigates the response of Rhodococcus erythropolis IBBPo1 cells to 1% organic solvents (alkanes, aromatics). A combination of microbiology, biochemical, and molecular approaches were used to examine cell adaptation mechanisms likely to be pursued by this strain after 1% organic solvent exposure. R. erythropolis IBBPo1 was found to utilize 1% alkanes (cyclohexane, n-hexane, n-decane) and aromatics (toluene, styrene, ethylbenzene) as the sole carbon source. Modifications in cell viability, cell morphology, membrane permeability, lipid profile, carotenoid pigments profile and 16S rRNA gene were revealed in R. erythropolis IBBPo1 cells grown 1 and 24 h on minimal medium in the presence of 1% alkanes (cyclohexane, n-hexane, n-decane) and aromatics (toluene, styrene, ethylbenzene). Due to its environmental origin and its metabolic potential, R. erythropolis IBBPo1 is an excellent candidate for the bioremediation of soils contaminated with crude oils and other toxic compounds. Moreover, the carotenoid pigments produced by this nonpathogenic Gram-positive bacterium have a variety of other potential applications.

Valoración económica en salud y medio ambiente del control de contaminantes orgánicos persistentes en Colombia / Economic assessment in health and environment from control of persistent organic pollutants in Colombia

Objetivo Estimar los beneficios económicos en salud y ambientales relacionados con la implementación del Convenio de Estocolmo para el control de Compuestos Orgánicos Persistentes (COP) en el país. Método La estimación se realizó teniendo en cuenta dos escenarios: cumplimiento del convenio y no cumplimiento del Convenio y el beneficio bruto se derivó de la diferencia en valor presente entre los costos en salud y ambientales que se asumen en cada escenario. Resultados Los beneficios brutos por la disminución de gastos en salud derivados de la implementación del Convenio se estiman entre USD $511 y USD $501 millones. Al introducir variables como los costos de gestión del convenio y los posibles beneficios por acceso a mercados internacionales, los beneficios para el país se estiman entre USD $1631 y USD $3118 millones. Discusión A pesar de los beneficios económicos generados por menores gastos en salud con la aplicación del Convenio, los costos asociados a la reducción de estos contaminantes, generan un balance negativo, que solo es compensado al cruzar estas cifras con la expectativa de mayores ingresos por acceso a mercados internacionales. Se considera que esta valoración económica como un primer ejercicio es un aporte importante, pero que debe ser actualizado a la luz de metodologías de valoración que involucren otras variables de rentabilidad social y diferentes escenarios por tecnologías emergentes, nuevo conocimiento sobre estos contaminantes, cambios en la legislación y/o cambios en las condiciones de tratados internacionales, entre otros.(AU)

Objective To estimate the economic benefits related to environment and health in the context of the implementation of the Stockholm Convention for the control of Persistent Organic Pollutants in the country. Method: The estimation was conducted based on two scenarios: non-compliance with the agreement and compliance with the Convention. Gross profit was derived from the difference in present value between the health and environmental costs that are assumed in each scenario. Results Gross profit by decreasing health costs arising from the implementation of the Convention was estimated at USD $ 511 and USD $ 501 million. By introducing variables such as management costs and agreement on potential benefits for access to international markets, the benefits to the country were estimated at between USD $1631 and USD $ 3118 million. Discussion Despite the economic benefits generated by lower expenditure on health for the Convention implementation, the costs associated with reducing pollutant emissions generated a negative balance, compensated only by the expectation of higher revenues for international market access. We consider this initial economic assessment an important contribution, but it should be reviewed to include valuation methodologies involving other social profitability variables and different scenarios for emerging technologies, new scientific knowledge about these pollutants, changes in legislation and / or changes in trade agreement conditions, among others.(AU)

Occurrence of culturable soil fungi in a tropical moist deciduous forest Similipal Biosphere Reserve, Odisha, India

Similipal Biosphere Reserve (SBR) is a tropical moist deciduous forest dominated by the species Shorea robusta. To the best of our knowledge their rich biodiversity has not been explored in term of its microbial wealth. In the present investigation, soil samples were collected from ten selected sites inside SBR and studied for their physicochemical parameters and culturable soil fungal diversity. The soil samples were found to be acidic in nature with a pH ranging from of 5.1–6.0. Highest percentage of organic carbon and moisture content were observed in the samples collected from the sites, Chahala-1 and Chahala-2. The plate count revealed that fungal population ranged from 3.6 × 104–2.1 × 105 and 5.1 × 104–4.7 × 105 cfu/gm of soil in summer and winter seasons respectively. The soil fungus, Aspergillus niger was found to be the most dominant species and Species Important Values Index (SIVI) was 43.4 and 28.6 in summer and winter seasons respectively. Among the sites studied, highest fungal diversity indices were observed during summer in the sites, Natto-2 and Natto-1. The Shannon-Wiener and Simpson indices in these two sites were found to be 3.12 and 3.022 and 0.9425 and 0.9373 respectively. However, the highest Fisher’s alpha was observed during winter in the sites Joranda, Natto-2, Chahala-1 and Natto-1 and the values were 3.780, 3.683, 3.575 and 3.418 respectively. Our investigation revealed that, fungal population was dependent on moisture and organic carbon (%) of the soil but its diversity was found to be regulated by sporulating species like Aspergillus and Penicillium.

Monitoring of metals, organic compounds and coliforms in water catchment points from the Sinos River basin / Monitoramento de metais, compostos orgânicos e coliforms em pontos de captação de água da bacia hidrográfica do Rio dos Sinos

Unplanned use and occupation of the land without respecting its capacity of assimilation and environmental purification leads to the degradation of the environment and of water used for human consumption. Agricultural areas, industrial plants and urban centres developed without planning and the control of effluent discharges are the main causes of water pollution in river basins that receive all the liquid effluents produced in those places. Over the last decades, environmental management has become part of governmental agendas in search of solutions for the preservation of water quality and the restoration of already degraded resources. This study evaluated the conditions of the main watercourse of the Sinos River basin by monitoring the main physical, chemical and microbiological parameters described in the CONAMA Resolution no. 357/2005.The set of parameters evaluated at five catchment points of water human consumption revealed a river that has different characteristics in each reach, as the upper reach was class 1, whereas the middle and lower reaches of the basin were class 4. Monitoring pointed to households as the main sources of pollutants in those reaches, although metals used in the industrial production of the region were found in the samples analyzed.

O uso e ocupação do solo não planejados, sem respeitar sua capacidade de assimilação e de purificação ambiental, levam à degradação do ambiente e da água utilizada para consumo humano. Áreas agrícolas, plantas industriais e centros urbanos desenvolvidos sem planejamento e controle de descargas de efluentes são as principais causas de poluição da água em bacias hidrográficas que recebem todos os efluentes líquidos produzidos naqueles lugares. Nas últimas décadas, gestão ambiental tornou-se parte das agendas governamentais em busca de soluções para a preservação da qualidade da água e a restauração de recursos já degradadas. Este estudo avaliou as condições do curso de água principal da bacia do Rio dos Sinos, monitorando o principal físico, químico e microbiológicos parâmetros descritos na Resolução CONAMA n.º 357/2005. O conjunto de parâmetros avaliados em cinco pontos de captação de água para consumo humano revelou um rio que tem características diferentes em cada alcance, como o superior chegar era classe 1, Considerando que o atinge médio e inferior da bacia foram classe 4. monitoramento apontado famílias como as principais fontes de poluentes naqueles atinge, apesar de metais utilizados na produção industrial da região foram encontrados nas amostras analisadas.

Optimization of the production of mycorrhizal inoculum on substrate with organic fertilizer

The system for production of inoculum of arbuscular mycorrhizal fungi (AMF) using sand and vermiculite irrigated with nutrient solution is promising. However, organic amendments added to the substrate can stimulate sporulation of AMF and replace the nutrient solution. The aim of this study was to maximize the production of AMF (Acaulospora longula, Claroideoglomus etunicatum, Dentiscutata heterogama and Gigaspora albida) using selected organic substrates (vermicompost, coir dust and Tropstrato) together with sand and vermiculite. The production of spores varied among the tested AMF and according to the organic source added to the substrate. The vermicompost promoted higher sporulation of A. longula in relation to the other AMF and substrates. The Tropstrato® inhibited the sporulation of D. heterogama while the reproduction of C. etunicatum was not affected by the organic compounds. The inoculum of A. longula also showed a high number of infective propagules and promoted biomass accumulation in maize plants. The system of inoculum production using sand and vermiculite + 10% vermicompost favors the production of infective inoculum of A. longula with the fungus benefiting growth of corn plants.

Metabolite profiling of Schizochytrium sp. by GC-MS, an oleaginous microbial source of biodiesel

The chemical screening carried out on Schizochytrium sp. biomass led the identification of 24 types of organic compounds belonging to n-alkanes, 1-alkenes, 1-alkanols, free fatty acids, methyl and ethyl esters of saturated and unsaturated fatty acids, saturated tri- and diglycerides, unsaturated monoglycerides, wax esters, sterols, triterpenes, and mono- and sesquiterpenes. Moreover, a sample containing fully saturated ethyl biodiesel was obtained experimentally with a yield of 28.72% w/w of the crude extract, and an average chain length of 15.52 carbons. This strain produced no toxins, but showed important nutrients, making it potentially applicable to the field of functional food, and biodiesel production.

Validación de sybr-14 y 6-cfda para evaluar la viabilidad e integridad de la membrana plasmática en espermatozoides caninos de raza chihuahua / Validation of sybr-14 and 6-cfda to evaluate the viability and plasma membrane integrity in sperm of chihuahua canine breed

En la aplicación de técnicas reproductivas es importante determinar in vitro la capacidad fecundante de los espermatozoides, para ello se utilizan combinaciones de tinciones para evaluar los diferentes parámetros de función espermática, aumentando así la precisión de la estimación de la muestra. El objetivo del estudio fue comparar la efectividad de la utilización de los fluorocromos 6-CFDA y SYBR-14 combinados con PI para determinar la viabilidad e integridad de la membrana plasmática por citometría de flujo. Se utilizó semen fresco de caninos (n=5) de raza Chihuahua, con una concentración espermática >150x106 esp/ml y motilidad progresiva >80%. Tres protocolos fueron ensayados: grupo 1: SYBR-14/PI, grupo 2: 6-CFDA/PI y grupo 3: PI. La integridad de la membrana plasmática de los espermatozoides fue similar entre grupos 1 y 2, independiente del fluorocromo utilizado (37,26±13,9 y 33,8±14,6, respectivamente; p=0,4601). Asimismo, la viabilidad espermática entre los grupos 1, 2 y 3 (62,7±13,9, 66,1±14,6 y 66,4±13,3, respectivamente; p=0,8987). En conclusión, no se evidenció diferencias en la efectividad para determinar la viabilidad e integridad de la membrana plasmática mediante la utilización de SYBR-14 y 6-CFDA, ambas tinciones pueden ser incorporadas al análisis de rutina de semen canino de raza Chihuahua.

In applying reproductive techniques in vitro it is important to determine the fertilizing capacity of the sperm, for this a combination of dyes were used to assess different parameters of sperm function, thereby increasing the accuracy of the estimation of the sample. In dogs (Canis lupus familiaris) Chihuahua breed there is no precedent for evaluating sperm function parameters. The aim was to assess the viability and plasmatic membrane integrity, basic parameters of sperm function. Propidium iodide (PI) was used, a fluorescent dye-specific DNA, which combined with fluorochromes permeable acts as marker of the sperm membrane integrity. The effectiveness of the use of 6-CFDA and SYBR-14 fluorochromes combined with PI was also compared to determine viability and sperm membrane integrity using flow cytometry. Fresh semen of dogs (n=5) Chihuahua breed was used with a concentration of >200x106 sp/ml and progressive motility >80%. Three protocols were performed: group 1: SYBR-14/PI, group 2: 6-CFDA/PI and group 3: PI. The plasma membrane integrity of sperm was similar, independent of the fluorophore used between groups 1 and 2 (13.9±37.26 and 33.8±14.6, respectively, p=0.4601). This also applied to sperm viability between groups 1, 2 and 3 (62.7±13.9, 66.1±14.6 and 66.4±13.3, respectively, p=0.8987). No difference was demonstrated in effectiveness to determine the viability and integrity of the sperm membrane using SYBR-14 and 6-CFDA, both dyes can be incorporated in to routine analysis of semen in canine Chihuahua breed.

Efeito de compostos orgânicos sobre podridões radiculares no feijoeiro comum / Effect of organic compounds on root rots of common beans

Os fungos habitantes do solo, Rhizoctonia solani e Fusarium solani f.sp. phaseoli, constituem um complexo etiológico com interação sinérgica, responsável por perdas de produtividade de até 50% em feijoeiro. A matéria orgânica de diversas origens é utilizada na agricultura visando, entre outros motivos, estimular a atividade microbiana para limitar a atividade dos fitopatógenos habitantes do solo. O presente trabalho teve como objetivo avaliar o efeito de doses de compostos orgânicos sobre podridões radiculares causadas por R. solani e F. solani f.sp. phaseoli, em feijoeiro cultivar Pérola, em condições de campo. O delineamento experimental foi de blocos casualizados, em esquema fatorial de 2 x 6. Os compostos orgânicos à base de crotalária (Crotalaria juncea) e grama batatais (Paspalum notatum), associados com cama de frango, foram aplicados no sulco de plantio nas doses de 0, 20, 40, 80, 160 e 320 kg.ha-1. As avaliações de severidade foram realizadas, utilizando-se escala de notas. A maior severidade de F. solani f.sp. phaseoli em plantas de feijoeiro foi observada sob efeito da dose de 320 kg.ha-1 de crotalária + cama de frango e 80 kg.ha-1 para o composto à base de grama batatais + cama de frango. Para R. solani, os compostos orgânicos e doses não influenciaram a severidade de R. solani. A dose de 80 kg.ha-1, independente do composto orgânico, proporcionou a melhor produtividade.

The soil inhabiting fungi, Rhizoctonia solani and Fusarium solani f.sp. phaseoli, constitute an etiological complex with synergic interaction, responsible for up to 50% losses in common beans. Organic matter of diverse sources is used in agriculture to stimulate microbial activity limiting the activity of soil inhabiting plant pathogens, among other uses. This study evaluated the effect of organic compound doses on root rots caused by R. solani and F. solani f.sp. phaseoli, in common beans, cultivar Pérola, in field conditions. The experimental design was randomized blocks, as a 2 x 6 factorial. The organic compounds of Crotalaria juncea or Paspalum notatum, associated with poultry bedding, were applied in the planting furrow at the doses de 0, 20, 40, 80, 160 or 320 kg.ha-1. Evaluations of severity were done using a rating scale. Greater severity of F. solani f.sp. phaseoli in common bean plants were observed at the dose of 320 kg.ha-1 crotalaria + poultry bedding e 80 kg.ha-1 with paspalum compound + poultry bedding. The organic compounds and doses did not affect R. solani severity. The dose of 80 kg.ha-1, regardless of the organic compound, resulted in greater yield.