Ecogenômica de archaea e monitoramento de comunidades de procariotos redutores de sulfato: aplicações na indústria de petróleo e gás

The human activities responsible for the ambient degradation in the modern world are diverse. The industrial activities are preponderant in the question of the impact consequences for brazilian ecosystems. Amongst the human activities, the petroliferous industry in operation in Potiguar Petroliferous Basin (PPB) displays the constant risk of ambient impacts in the integrant cities, not only for the human populations and the environment, but also it reaches the native microorganisms of Caatinga ground and in the mangrove sediment. Not hindering, the elaboration of strategies of bioremediation for impacted areas pass through the knowledge of microbiota and its relations with the environment. Moreover, in the microorganism groups associated to oil, are emphasized the sulfate-reducing prokaryotes (SRP) that, in its anaerobic metabolism, these organisms participate of the sulfate reduction, discharging H2S, causing ambient risks and causing the corrosion of surfaces, as pipelines and tanks, resulting in damages for the industry. Some ancestries of PRS integrate the Archaea domain, group of microorganisms whose sequenced genomes present predominance of extremophilic adaptations, including surrounding with oil presence. This work has two correlated objectives: i) the detection and monitoring of the gene dsrB, gift in sulfate-reducing prokaryotes, through DGGE analysis in samples of mDNA of a mangrove sediment and semiarid soil, both in the BPP; ii) to relate genomic characteristics to the ecological aspects of Archaea through in silico studies, standing out the importance to the oil and gas industry. The results of the first work suggest that the petrodegraders communities of SRP persist after the contamination with oil in mangrove sediment and in semiarid soil. Comparing the populations of both sites, it reveals that there are variations in the size and composition during one year of experiments. In the second work, functional and structural factors are the probable cause to the pressure in maintenance of the conservation of the sequences in the multiple copies of the 16S rDNA gene. Is verified also the discrepancy established between total content GC and content GC of the same gene. Such results relating ribosomal genes and the ambient factors are important for metagenomic evaluations using PCR-DGGE. The knowledge of microbiota associated to the oil can contribute for a better destination of resources by the petroliferous industry and the development of bioremediation strategies. Likewise, search to lead to the best agreement of the performance of native microbiota in biogeochemical cycles in Potiguar Petroliferous Basin ecosystem

Metagenômica: busca de novos genes envolvidos com a biodegradação de hidrocarbonetos

Industrial activities, oil spills and its derivatives, as well as the incomplete combustion of fossil fuels have caused a great accumulation of hydrocarbons in the environment. The number of microorganisms on the planet is estimated at 1030 and prokaryotes the most abundant. They colonized diverse environments for thousands of years, including those considered extreme and represent an untapped source of metabolic and genetic diversity with a large biotechnological potential. It is also known that certain microorganisms have the enzymatic capacity to degrade petroleum hydrocarbons and, in many ecosystems, there is an indigenous community capable of performing this function. The metagenomic has revolutionized the microbiology allowing access uncultured microbial communities, being a powerful tool for elucidation of their ecological functions and metabolic profiles, as well as for identification of new biomolecules. Thus, this study applied metagenomic approaches not only for functional selection of genes involved in biodegradation and emulsification processes of the petroleum-derived hydrocarbons, but also to describe the taxonomic and metabolic composition of two metagenomes from aquatic microbiome. We analyzed 123.116 (365 ± 118 bp) and 127.563 sequences (352 ± 120 bp) of marine and estuarine metagenomes, respectively. Eight clones were found, four involved in the petroleum biodegradation and four were able to emulsify kerosene indicating their abilities in biosurfactants synthesis. Therefore, the metagenomic analyses performed were efficient not only in the search of bioproducts of biotechnological interest and in the analysis of the functional and taxonomic profile of the metagenomes studied as well

Potential microbiological contamination of effluents in poultry and swine abattoirs

Health risks in the effluents of seven swine abattoirs and of seven poultry abattoirs were evaluated with regard to environment degradation and to dissemination of pathogenic microorganisms during the rainy and dry seasons. Supply-water samples from affluents and effluents of the treatment systems at different sites within the abattoir processing system were analysed. Similarly, water samples from the three recipient sites (emission point, 100 m upstream, 100 m downstream) were also analysed. Temperature, free residual chlorine (FRC), total coliform bacteria, Escherichia coli, enterococci, identification and serotyping of salmonellae were assessed. Scalding is the most significant stage in the slaughtering chain (P<0.05) when temperature is taken into account. Temperatures Lit effluents and at the sampled sites in the water bodies accorded to state and federal legislation standards. Supply waters did not meet the standards for FRC and microbial count standards according to the Ministry of Health and within limits imposed by the Industrial and Sanitary Inspection Regulations for Animal Products. Feather plucking and evisceration in Poultry slaughter and the cleansing of carcasses and facilities in Poultry and swine slaughtering had the highest contamination impact. The three loci at the water bodies were above the microbiological standards for classes II and III sites, in conformity with Law 8468 of the state of São Paulo, Brazil and Conama. Salmonella was found at several sites during slaughter, at both types of abattoirs, including in the effluent treatment system. This showed that these sites were the dissemination sources of the microorganism.

Avaliação da eficiência da desinfecção de teteiras e dos tetos no processo de ordenha mecânica de vacas

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Variação das características físico-químicas e microbiológicas das salmouras empregadas na salga de queijos tipo mussarela durante o período de sua utilização

Foram analisadas 40 amostras de salmouras empregadas na salga por submersão de queijos tipo mussarela, em uma indústria de laticínios do Estado de São Paulo, Brasil, com o objetivo de se conhecer a variação das características físico-químicas e microbiológicas durante o período de sua utilização. Os valores médios do pH, concentração de cloreto de sódio e de proteínas solúveis, desde o preparo da salmoura até o 21° dia de utilização, variaram de 7,21 a 5,76, 27,1 a 24,5 e de zero a 0,126 mg/ml, respectivamente. Por outro lado, os valores médios das contagens de microrganismos mesófilos e do número mais provável de coliformes totais e de origem fecal variaram de 5,8 x 10 UFC/ml a 6,9 x 10(4) UFC/ml, zero a 1,6 x 10(5)/100ml e zero a 1,1 x 10(5)/100ml, respectivamente. Além disso, os valores médios das contagens de bolores e leveduras e de Staphylococcus coagulase positiva, variaram de 0,4 x 10 UFC/ml a 2,0 x 10³ UFC/ml e de zero a 1,3 x 10 UFC/ml, respectivamente. Os resultados encontrados sugerem a existência de condições higiênicas inadequadas durante o preparo e utilização das salmouras, de modo a representar uma importante fonte de contaminação para os queijos. em decorrência deste fato, existe a possibilidade do comprometimento da qualidade dos queijos, de modo a representar risco potencial à população consumidora.

Risco à saúde representado pelo consumo de leite cru comercializado clandestinamente

Foram submetidas às contagens de microrganismos mesófilos, Staphylococccus coagulase positiva, e às determinações do número mais provável (NMP) de coliformes totais e de coliformes fecais, 60 amostras de leite cru comercializado clandestinamente nos Municípios de Botucatu e de São Manuel, Estado de São Paulo, Brasil. Os resultados obtidos evidenciaram a ocorrência de 41 (68,3%) e 50 (83,3%) amostras com contagens de microrganismos mesófilos e de coliformes totais, respectivamente, acima dos limites máximos estabelecidos pelo Ministério da Saúde para o leite pasteurizado tipo C. Evidenciaram, ainda, a presença de 30 (50,0%) e 11 (18,3%) amostras contaminadas por cepas de Staphylococcus coagulase positiva e por coliformes fecais, respectivamente. Apenas 5 (8,3%) amostras mostraram-se dentro dos referidos padrões legais.

Esporos de Clostridium botulinum em mel comercializado no Estado de São Paulo e em outros Estados brasileiros

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Survey on Chlamydophila psittaci in captive ramphastids in São Paulo State, Brazil

Chlamydophila psittaci (C. psittaci) has been detected in 460 avian species, among them the most frequent are the Psittaciformes, Columbiformes, Anseriformes and raptors. In Brazil, the main avian species recognized as healthy carriers belong to the order Psittaciformes and Columbiformes, but very few studies have been done in other bird families. Reports of the occurrence of this disease in the clinical form are rare in the Ramphastids; consequently, they are not commonly evaluated for this agent. The present study reports the investigation of C. psittaci in 25 captive ramphastids from a zoological park in São Paulo State, Brazil. Swabs samples from the cloaca were submitted to semi-nested polymerase chain reaction (semi-nested PCR) for direct detection of the microorganism. Additionally, blood samples obtained from these birds were submitted to the Complement Fixation Test (CFT) for detection of antibodies anti-C. psittaci. The presence of C. psittaci was not detected in the cloacal swab samples tested by the PCR. Nevertheless, 16% (4/25) of the bird's sera were positive by the CFT. Among the species with positive results, there are the saffron toucanet (Pteroglossus bailloni) and black-necked-aracari (Pteroglossus aracari), two species with no descriptions of the survey of C. psittaci published in the literature. Intermittent elimination of C. psittaci is a feature of chronically infected birds; however the absence of a positive-antigen sample did not guarantee that the bird is Chlamydophila-free. The serological results obtained show that the ramphastids tested were previously exposed to the pathogen and developed immune response, but showed no clinical signs of the disease and didn't eliminate regularly the organism in their feces in the moment of the sample collection.

Ocorrência de Megabacteriose em Periquitos Autralianos, Canários e Agapornis na Região de Ribeirão Preto- Estado de São Paulo - Brasil

Este trabalho tem como objetivo relatar a ocorrência de um agente etiológico, denominado na Europa, Australia e EUA como megabactéria, observado em estômago de pequenas aves (canários belgas, agapornis e periquitos australianos), provenientes da região de Ribeirão Preto, Estado de São Paulo/SP. As necropsias de 64 aves silvestres (4 periquitos australianos, 12 agapornis e 48 canários), realizadas no perído de 1994 a 1997, foram analisadas, constatando-se em 56% dos casos a presença de estruturas filiformes, acidofílicas sob coloração Giemsa, gram positivas, existentes no muco do proventrículo, descritas na literatura como megabactérias. Foram testados diversos tipos de meios de cultura para reprodução in vitro deste microrganismo. Foram ainda comparadas as dimensões (comprimento e largura) dessa bactéria obtida apartir do esfregaço fresco de muco proventricular e da megabactéria proveniente de cultivo in vitro. Também foram listados os principais achados macroscópicos do animais portadores desta bactéria.

Comparison of microbial numbers in soils by using various culture media and temperatures

The influence of different media and incubation temperatures on the quantification of microbial populations in sorghum, eucalyptus and forest soils was evaluated. Microbial growth was compared by using complex (tryptone soybean agar, TSA, casein-starch, CS, and Martin) and saline (Thorton, M3, Czapeck) media and incubation temperatures of 25 and 30&DEG; C. Higher numbers of total bacterial. and fungal colony-forming units (CFU) were observed in sorghum soils, and of spore-forming and Gram-negative bacteria in forest soils than other soils. Actinomycetes counts were highest in forest soil when using CS medium at 30&DEG; C and in sorghum soil at 25&DEG; C in M3 medium. Microorganism counts were dependent on the media and incubation temperatures. The counts at temperatures of 30&DEG; C were significantly higher than at 25&DEG; C. Microbial quantification was best when using TSA medium for total. and spore-forming bacteria, Thorton for Gram-negative bacteria, M3 for actinomycetes, and Martin for fungi. © 2005 Elsevier GmbH. All rights reserved.

Identificação e avaliação de rizobactérias isoladas de raízes de milho

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Avaliação de dois clones de Escherichia coli recombinante quanto ao crescimento e expressão de antígenos de Leishmania chagasi (kmp11 e P36)

Visceral leishmaniosis caused by Leishmania chagasi, also known as calazar, presented, in the period from 1990 to 2005, tax of incidence in Brazil varying between 1 and 3 cases for 100 000 inhabitants. The Northeast region that up to the year of 2000 contributed with almost 90% of the registered cases is reducing his participation in the current decade, reaching 56% in 2005. Conventional leishmaniasis treatment is costly and it shows high toxicity, demanding more research for alternative treatments, with special interest in development of vaccines and diagnosis kits which include production of recombinant antigens by host cells. Escherichia coli has been the microorganism most studied and used as a host for recombinant protein production. Therefore, the aim of this work was to study the influence of induction on cellular growth and to verify the type of Leishmania chagasi antigens expression (intra or extracellular) during two recombinant E. coli clones (kmp11 and P36) cultivation in rotary incubator (shaker) using three different media (2xTY, TB, FASS+EL). For that, tests were carried out using conditions established in the literature for E. coli (37°C and 200 rpm) and media supplemented with antibiotics to guarantee that only competent cells grows. First, tests were carried out without induction in order to verify the two microorganisms kinetic behavior (growth and substrate consumption) in different media. Next, the induction was carried out through the addition of IPTG (1mM as final concentration), at the first hour of cultivation. It was observed that protein expression were intracellular for all clones and media tested, however the highest level of expression was clearly observed by the electrophoresis band density (intensity) for 2xTY medium and kmp11 protein. Although it contains the lowest substrate concentration, consequently, a reduced cellular concentration when compared to other media, it appeared that this medium and clone combination is the most indicated for recombinant protein production. Therefore, the objective of this work was achieved, since the interested proteins were produced. Consequently, this result motivates new studies for production optimization using different cultivation strategies

Monitoramento em tempo real de processos fermentativos por espectroscopia no infravermelho próximo (NIRS)

A chemical process optimization and control is strongly correlated with the quantity of information can be obtained from the system. In biotechnological processes, where the transforming agent is a cell, many variables can interfere in the process, leading to changes in the microorganism metabolism and affecting the quantity and quality of final product. Therefore, the continuously monitoring of the variables that interfere in the bioprocess, is crucial to be able to act on certain variables of the system, keeping it under desirable operational conditions and control. In general, during a fermentation process, the analysis of important parameters such as substrate, product and cells concentration, is done off-line, requiring sampling, pretreatment and analytical procedures. Therefore, this steps require a significant run time and the use of high purity chemical reagents to be done. In order to implement a real time monitoring system for a benchtop bioreactor, these study was conducted in two steps: (i) The development of a software that presents a communication interface between bioreactor and computer based on data acquisition and process variables data recording, that are pH, temperature, dissolved oxygen, level, foam level, agitation frequency and the input setpoints of the operational parameters of the bioreactor control unit; (ii) The development of an analytical method using near-infrared spectroscopy (NIRS) in order to enable substrate, products and cells concentration monitoring during a fermentation process for ethanol production using the yeast Saccharomyces cerevisiae. Three fermentation runs were conducted (F1, F2 and F3) that were monitored by NIRS and subsequent sampling for analytical characterization. The data obtained were used for calibration and validation, where pre-treatments combined or not with smoothing filters were applied to spectrum data. The most satisfactory results were obtained when the calibration models were constructed from real samples of culture medium removed from the fermentation assays F1, F2 and F3, showing that the analytical method based on NIRS can be used as a fast and effective method to quantify cells, substrate and products concentration what enables the implementation of insitu real time monitoring of fermentation processes

Estudo da produção de pectinase por fermentação em estado sólido utilizando pedúnculo de caju como substrato

Pectinolytic enzymes, or simply pectinases, are complex enzymes that degrade pectic polymers. They have many uses, such as fruit juice extraction and purification, textile fiber treatment and vegetal oil extraction. The aim of this work was to study the kinetics of pectinases production by solid-state fermentation, using dry cashew apple residue as substrate and the microorganism Aspergillus niger CCT 0916. The influence of the initial medium moisture and medium supplementation with a source of nitrogen and phosphorus was evaluated using the factorial experimental planning and response surface methodology. Ammonia sulphate and potassium phosphate were used as nitrogen and phosphorus source, respectively. The variables time of contact (T) and ratio volume solvent/fermented medium (RZ), in systems with and without agitation, were evaluated in order to study the best extraction condition of the produced enzyme. Washed and unwashed cashew apple residues were tested as the growth medium. The unwashed residue was obtained by drying the residue after the extraction of the juice, while the washed residue was obtained by water washing 5 times using the proportion of 1 kg pulp/2 liters of water. Samples were taken every 12 hours for moisture content, pH, protein, reducing sugars, polygalacturonase activity (PG) and viscosity reduction. The physical-chemical composition of the residues had different sugar and pectin levels. For the unwashed residue, the peak activity was reached with 40% of initial moisture content, 1% of nitrogen supplementation without phosphorus addition after 30 hours of process. These conditions led to 16 U/g of PG activity and 82% of viscosity reduction. The calculated models reached similar values to the experimental ones in the same process conditions: 15.55 U/g of PG and 79.57% of viscosity eduction. Similarly, the greatest enzyme production for washed residue was reached with 40% initial moisture content, 1% nitrogen supplementation without phosphorus addition after 22 hours of cultivation. In this condition it was obtained polygalacturonase activity of 9.84 U/g and viscosity reduction of 81.36%. These values are close to experimental values that were of 10.1 U/g and 81%, respectively. The conditions that led to the best PG activity results was the agitated one and the best extraction condition was obtained with 100 minutes of solvent/medium contact and RZ of 5 (mL/g)

Análise de fluxos metabólicos para otimização da síntese do antibiótico cosmomicina por Streptomyces olindensis ICB20

Metabolic flux analysis (MFA) is a powerful tool for analyzing cellular metabolism. In order to control the growth conditions of a specific organism, it is important to have a complete understanding of its MFA. This would allowed us to improve the processes for obtaining products of interest to human and also to understand how to manipulate the genome of a cell, allowing optimization process for genetic engineering. Streptomyces olindensis ICB20 is a promising producer of the antibiotic cosmomycin, a powerful antitumor drug. Several Brazilian researchers groups have been developing studies in order to optimize cosmomycin production in bioreactors. However, to the best of our knowledge, nothing has been done on metabolic fluxes analysis field. Therefore, the aim of this work is to identify several factors that can affect the metabolism of Streptomyces olindensis ICB20, through the metabolic flux analysis. As a result, the production of the secondary metabolite, cosmomycin, can be increased. To achieve this goal, a metabolic model was developed which simulates a distribution of internal cellular fluxes based on the knowledge of metabolic pathways, its interconnections, as well as the constraints of microorganism under study. The validity of the proposed model was verified by comparing the computational data obtained by the model with the experimental data obtained from the literature. Based on the analysis of intracellular fluxes, obtained by the model, an optimal culture medium was proposed. In addition, some key points of the metabolism of Streptomyces olindensis were identified, aiming to direct its metabolism to a greater cosmomycin production. In this sense it was found that by increasing the concentration of yeast extract, the culture medium could be optimized. Furthermore, the inhibition of the biosynthesis of fatty acids was found to be a interesting strategy for genetic manipulation. Based on the metabolic model, one of the optimized medium conditions was experimentally tested in order to demonstrate in vitro what was obtained in silico. It was found that by increasing the concentration of yeast extract in the culture medium would induce to an increase of the cosmomycin production