Maíra Paula de Sousa

Phylogenetic and functional diversity of metagenomic libraries of phenol degrading sludge from petroleum refinery wastewater treatment system

In petrochemical refinery wastewater treatment plants (WWTP), different concentrations of pollutant compounds are received daily in the influent stream, including significant amounts of phenolic compounds, creating propitious conditions for the development of particular microorganisms that can rapidly adapt to such environment. In the present work, the microbial sludge from a refinery WWTP was enriched for phenol, cloned into fosmid vectors and pyrosequenced. The fosmid libraries yielded 13,200 clones and a comprehensive bioinformatic analysis of the sequence data set revealed a complex and diverse bacterial community in the phenol degrading sludge. The phylogenetic analyses using MEGAN in combination with RDP classifier showed a massive predominance of Proteobacteria, represented mostly by the genera Diaphorobacter, Pseudomonas, Thauera and Comamonas. The functional classification of phenol degrading sludge sequence data set generated by MG-RAST showed the wide metabolic diversity of the microbial sludge, with a high percentage of genes involved in the aerobic and anaerobic degradation of phenol and derivatives. In addition, genes related to the metabolism of many other organic and xenobiotic compounds, such as toluene, biphenyl, naphthalene and benzoate, were found. Results gathered herein demonstrated that the phenol degrading sludge has complex phylogenetic and functional diversities, showing the potential of such community to degrade several pollutant compounds. This microbiota is likely to represent a rich resource of versatile and unknown enzymes which may be exploited for biotechnological processes such as bioremediation.

Identification of Genes and Pathways Related to Phenol Degradation in Metagenomic Libraries from Petroleum Refinery Wastewater

Two fosmid libraries, totaling 13,200 clones, were obtained from bioreactor sludge of petroleum refinery wastewater treatment system. The library screening based on PCR and biological activity assays revealed more than 400 positive clones for phenol degradation. From these, 100 clones were randomly selected for pyrosequencing in order to evaluate the genetic potential of the microorganisms present in wastewater treatment plant for biodegradation, focusing mainly on novel genes and pathways of phenol and aromatic compound degradation. The sequence analysis of selected clones yielded 129,635 reads at an estimated 17-fold coverage. The phylogenetic analysis showed Burkholderiales and Rhodocyclales as the most abundant orders among the selected fosmid clones. The MG-RAST analysis revealed a broad metabolic profile with important functions for wastewater treatment, including metabolism of aromatic compounds, nitrogen, sulphur and phosphorus. The predicted 2,276 proteins included phenol hydroxylases and cathecol 2,3- dioxygenases, involved in the catabolism of aromatic compounds, such as phenol, byphenol, benzoate and phenylpropanoid. The sequencing of one fosmid insert of 33 kb unraveled the gene that permitted the host, Escherichia coli EPI300, to grow in the presence of aromatic compounds. Additionally, the comparison of the whole fosmid sequence against bacterial genomes deposited in GenBank showed that about 90% of sequence showed no identity to known sequences of Proteobacteria deposited in the NCBI database. This study surveyed the functional potential of fosmid clones for aromatic compound degradation and contributed to our knowledge of the biodegradative capacity and pathways of microbial assemblages present in refinery wastewater treatment system.

Níveis de fósforo total em dietas para alevinos de tilápia-do-nilo

Four hundred and thirty-two Nile tilapia (Oreochromis niloticus) fingerlings averaging initial weight of 0.60 ± 0.02 g were allotted to thirty-six 150 L-aquaria to determine total phosphorus requirement. The experiment was analyzed as a completely randomized design, with six treatments (0.55, 0.73, 0.94, 1.14, 1.37, and 1.59% of total phosphorus), six replicates and 12 fishes per experimental unit. The fishes were fed diets with 32% of CP and 3,000 kcal of DE/kg, during 40 days. Quadratic effect was observed for feed/gain ratio and protein efficiency rate and the best results were estimated with 1.10% of total phosphorus, for both variables. Linear effect of the dietary phosphorus levels on P retention efficiency rate and P efficiency rate was noticed. No treatment effect on the other variables was observed. The dietary total phosphorus requirement for Nile tilapia corresponds to 1.10%.

Níveis de lisina, com base no conceito de proteína ideal, em rações para alevinos de tilápia-do-nilo

The effects of the digestible lysine level were assessed in feeds for Nile tilapia fingerlings (Oreochromis niloticus), based on the ideal protein concept. Four hundred and thirty two reverted fingerlings were used, average weight 1.12 ± 0.02 g, in a randomized complete design, consisting of 6 feeds, six replications and twelve fish per experimental unit. The diets consisted of a basal diet with 29.12% crude protein and 3,000 kcal/kg digestible energy, supplemented with synthetic amino acids, resulting in six diets with 0.95; 1.10; 1.25; 1.40; 1.55 and 1.70% digestible lysine and minimum ratios between methionine plus cystine, threonine, thryptophan, isoleucine, arginine with the lysine (66, 77, 23, 64 and 85%, respectively), based on digestible values. The fish were maintained in 130 liter aquaria equipped with individual water and controlled temperature and aeration. The fish were fed to apparent satiation, six times a day, for 30 days. Growth performance, body composition, body protein and fat deposition and nitrogen retention efficiency of the fish were evaluated. The increase in the dietary digestible lysine did not affect the survival rate and body fat level of the fishes. However, there was linear improvement in all the other parameters assessed, except lysine use efficiency and the body humidity, that showed quadratic and lineardecline, respectively. The levels of 1.80 (0.600% Mcal of DE) total lysine and 1.70% (0.567% Mcal of DE) digestible lysine, respectively, result in the best performance and carcass characteristics of Nile tilapia fingerlings, when the ideal protein concept is used to formulate the experimental diets.Avaliaram-se os efeitos dos niveis de lisina digestivel da racao, com base no conceito de proteina ideal, no desempenho de alevinos de de tilapia-do-nilo (Oreochromis niloticus). Utilizaram-se 432 alevinos revertidos, linhagem tailandesa, com peso inicial de 1,12 ± 0,02 g, em delineamento inteiramente ao acaso, composto de 6 dietas, 6 repeticoes e 12 peixes por unidade experimental. Como tratamentos, avaliaram-se uma dieta basal com 29,12% de proteina bruta e 3.000 kcal de energia digestivel/kg, suplementada com aminoacidos sinteticos, resultando em dietas com 0,95; 1,10; 1,25; 1,40; 1,55 e 1,70% de lisina digestivel e relacoes minimas entre metionina + cistina, treonina, triptofano, isoleucina e arginina com a lisina (66, 77, 23, 64 e 85%, respectivamente), com base em valores digestiveis. Os peixes foram mantidos em aquarios de 130 litros, dotados de abastecimento de agua, temperatura controlada e aeracao individual, e alimentados a vontade em seis refeicoes diarias durante 30 dias. Avaliaram-se os parâmetros de desempenho, a composicao corporal, a deposicao de proteina e gordura corporais e a eficiencia de retencao de nitrogenio dos peixes. A elevacao do teor de lisina digestivel na racao nao influenciou a taxa de sobrevivencia e o teor de gordura corporal dos peixes, mas melhorou de forma linear todos os demais parâmetros avaliados, com excecao da eficiencia de utilizacao de lisina e a umidade corporal, que pioraram de forma quadratica e linear, respectivamente. O nivel de 1,80% de lisina total (0,600% Mcal de ED) e o de 1,70% de lisina digestivel (0,567% Mcal de ED) proporcionam os melhores resultados de desempenho e caracteristicas de carcaca de alevinos de tilapia-do-nilo quando se utiliza o conceito de proteina ideal na formulacao das racoes experimentais.

Culturable bacterial diversity from a feed water of a reverse osmosis system, evaluation of biofilm formation and biocontrol using phages

Biofilm formation on reverse osmosis (RO) systems represents a drawback in the application of this technology by different industries, including oil refineries. In RO systems the feed water maybe a source of microbial contamination and thus contributes for the formation of biofilm and consequent biofouling. In this study the planktonic culturable bacterial community was characterized from a feed water of a RO system and their capacities were evaluated to form biofilm in vitro. Bacterial motility and biofilm control were also analysed using phages. As results, diverse Protobacteria, Actinobacteria and Bacteroidetes were identified. Alphaproteobacteria was the predominant group and Brevundimonas, Pseudomonas and Mycobacterium the most abundant genera. Among the 30 isolates, 11 showed at least one type of motility and 11 were classified as good biofilm formers. Additionally, the influence of non-specific bacteriophage in the bacterial biofilms formed in vitro was investigated by action of phages enzymes or phage infection. The vB_AspP-UFV1 (Podoviridae) interfered in biofilm formation of most tested bacteria and may represent a good alternative in biofilm control. These findings provide important information about the bacterial community from the feed water of a RO system that may be used for the development of strategies for biofilm prevention and control in such systems.

Níveis de lisina digestível em rações para alevinos de tilápia-do-nilo

The effect of digestible lysine levels was evaluated in diets for Nile tilapia fingerlings (Oreochromis niloticus), Thailand lineage, based on the ideal protein concept. Four hundred and thirty two Nile tilapia fingerlings with an initial body weight of 0.98 ± 0.03 g were allotted in a randomized complete design, with six treatments, six replications per treatment and twelve fish per experimental unit. The treatments consisted of six isoenergetic (3.000 kcal of DE/kg feed) and isoproteic (39.14% CP) diets with different levels of digestible lysine (1.50, 1.66, 1.82, 1.98, 2.14 and 2.30%). Feed intake, digestible lysine intake, average weight gain, specific growth rate, survival rate, feed conversion, daily protein deposition rate, daily fat deposition rate, chemical body composition (moisture, protein and fat content) and nitrogen retention efficiency were evaluated. The different levels of lysine improved linearly the parameters of digestible lysine intake, feed conversion, body protein content and body protein deposition. The Linear Response Plateau model best fitted the parameters of feed consumption, average weight gain, specific growth rate and nitrogen retention efficiency, estimating at 2.06%, 2.17, 2.14 and 2.12% respectively, the level of digestible lysine to maximize each of the parameters. The digestible and total digestible lysine requirement for Nile tilapia fingerlings was 2.17% (0.723%/Mcal of DE) and 2.32% (0.773%/Mcal of DE), respectively, to meet the main performance parameters and carcass traits, when the ideal protein concept was used in diet formulation.

Evaluation of the efficiency of deterioration of aromatic hydrocarbons by bacteria from wastewater treatment plant of oil refinery

Three bacterial strains were isolated from the activated sludge system of petroleum refinery wastewater, identified by partial sequencing of 16S rDNA, and classified as Acinetobacter genomospecies 3, Bacillus pumilus, and Bacillus flexus. The degradation efficiency of aromatic hydrocarbons was evaluated by gas chromatography with a flame ionization detector. In a mineral medium containing anthracene and phenanthrene and the consortium of microorganisms, the removal efficiency was 96% and 99%, respectively, after 30 days. The good rate of hydrocarbon degradation proves the operational efficiency of the microbial consortium in treating effluents containing these compounds.

Growth inhibition of sulfate-reducing bacteria in produced water from the petroleum industry using essential oils

Strategies for the control of sulfate-reducing bacteria (SRB) in the oil industry involve the use of high concentrations of biocides, but these may induce bacterial resistance and/or be harmful to public health and the environment. Essential oils (EO) produced by plants inhibit the growth of different microorganisms and are a possible alternative for controlling SRB. We aimed to characterize the bacterial community of produced water obtained from a Brazilian petroleum facility using molecular methods, as well as to evaluate the antimicrobial activity of EO from different plants and their major components against Desulfovibrio alaskensis NCIMB 13491 and against SRB growth directly in the produced water. Denaturing gradient gel electrophoresis revealed the presence of the genera Pelobacter and Marinobacterium, Geotoga petraea, and the SRB Desulfoplanes formicivorans in our produced water samples. Sequencing of dsrA insert-containing clones confirmed the presence of sequences related to D. formicivorans. EO obtained from Citrus aurantifolia, Lippia alba LA44 and Cymbopogon citratus, as well as citral, linalool, eugenol and geraniol, greatly inhibited (minimum inhibitory concentration (MIC) = 78 µg/mL) the growth of D. alaskensis in a liquid medium. The same MIC was obtained directly in the produced water with EO from L. alba LA44 (containing 82% citral) and with pure citral. These findings may help to control detrimental bacteria in the oil industry.

Integrated diversity analysis of the microbial community in a reverse osmosis system from a Brazilian oil refinery

Oil refineries are known for the large volume of water used in their processes, as well as the amount of wastewater generated at the end of the production chain. Due to strict environmental regulations, the recycling of water has now become a viable alternative for refineries. Among the many methods available to treat wastewater for reuse, the use of membranes in reverse osmosis systems stands out due to several economic and environmental benefits. However, these systems are vulnerable to contamination and deposition of microorganisms, mainly because of the feedwater quality. In this study, the microbial diversity of feedwater and reverse osmosis membranes was investigated using a combination of culture-dependent and culture-independent methods in order to characterize the microorganisms colonizing and deteriorating the membranes. In total, 37 bacterial isolates, 17 filamentous fungi and approximately 400 clones were obtained and analyzed. Among the bacterial genera identified, the most represented were Sphingobium, Acidovorax, Microbacterium, Rhizobium and Shinella. The results revealed genera that acted as candidate key players in initial biofilm formation in membrane systems, and provided important information concerning the microbial ecology of oligotrophic aquatic systems.

Screening and characterization of prophages in Desulfovibrio genomes

Bacteria of the genus Desulfovibrio belong to the group of Sulphate Reducing Bacteria (SRB). SRB generate significant liabilities in the petroleum industry, mainly due to their ability to microbiologically induce corrosion, biofilm formation and H2S production. Bacteriophages are an alternative control method for SRB, whose information for this group of bacteria however, is scarce. The present study developed a workflow for the identification of complete prophages in Desulfovibrio. Poly-lysogenesis was shown to be common in Desulfovibrio. In the 47 genomes analyzed 53 complete prophages were identified. These were classified within the order Caudovirales, with 69.82% belonging to the Myoviridade family. More than half the prophages identified have genes coding for lysozyme or holin. Four of the analyzed bacterial genomes present prophages with identity above 50% in the same strain, whose comparative analysis demonstrated the existence of colinearity between the sequences. Of the 17 closed bacterial genomes analyzed, 6 have the CRISPR-Cas system classified as inactive. The identification of bacterial poly-lysogeny, the proximity between the complete prophages and the possible inactivity of the CRISPR-Cas in closed bacterial genomes analyzed allowed the choice of poly-lysogenic strains with prophages belonging to the Myoviridae family for the isolation of prophages and testing of related strains for subsequent studies.