A comprehensive study on diesel oil bioremediation under microcosm conditions using a combined microbiological, enzymatic, mass spectrometry, and metabarcoding approach.

This study aims at the application of a marine fungal consortium (Aspergillus sclerotiorum CRM 348 and Cryptococcus laurentii CRM 707) for the bioremediation of diesel oil-contaminated soil under microcosm conditions. The impact of biostimulation (BS) and/or bioaugmentation (BA) treatments on diesel-oil biodegradation, soil quality, and the structure of the microbial community were studied. The use of the fungal consortium together with nutrients (BA/BS) resulted in a TPH (Total Petroleum Hydrocarbon) degradation 42% higher than that obtained by natural attenuation (NA) within 120 days. For the same period, a 72 to 92% removal of short-chain alkanes (C12 to C19) was obtained by BA/BS, while only 3 to 65% removal was achieved by NA. BA/BS also showed high degradation efficiency of long-chain alkanes (C20 to C24) at 120 days, reaching 90 and 92% of degradation of icosane and heneicosane, respectively. In contrast, an increase in the levels of cyclosiloxanes (characterized as bacterial bioemulsifiers and biosurfactants) was observed in the soil treated by the consortium. Conversely, the NA presented a maximum of 37% of degradation of these alkane fractions. The 5-ringed PAH benzo(a)pyrene, was removed significantly better with the BA/BS treatment than with the NA (48 vs. 38 % of biodegradation, respectively). Metabarcoding analysis revealed that BA/BS caused a decrease in the soil microbial diversity with a concomitant increase in the abundance of specific microbial groups, including hydrocarbon-degrading (bacteria and fungi) and also an enhancement in soil microbial activity. Our results highlight the great potential of this consortium for soil treatment after diesel spills, as well as the relevance of the massive sequencing, enzymatic, microbiological and GC-HRMS analyses for a better understanding of diesel bioremediation.

Application of extracellular polymers on soil communities exposed to oil and nickel contamination.

The petrochemical industry is responsible for many accidental releases of pollutants in soil such as hydrocarbons and toxic metals. This co-contamination is responsible for a delay in the degradation of the organic pollution. Many successful technologies to remove these metals apply extracellular polymeric substances (EPS). In this study, we tested the application of an EPS from a Paenibacillus sp. to aid the bioremediation of soils contaminated with crude oil and nickel. We conducted a microcosm experiment to soils containing combinations of oil, nickel, and EPS. The final concentration of oil was evaluated with an infrared spectrometer. Also, we sequenced the metagenomes of the samples in an ion torrent sequencer. The application of EPS did not aid the removal of hydrocarbons with or without the presence of nickel. However, it led to a smaller decrease in the diversity indexes. EPS decreased the abundance of Actinobacteria and increased that of Proteobacteria. The EPS also decreased the connectivity among Actinobacteria in the network analysis. The results indicated that the addition of EPS had a higher effect on the community structure than nickel. Altogether, our results indicate that this approach did not aid the bioremediation of hydrocarbons likely due to its effect in the community structure that affected hydrocarbonoclastic microorganisms.

A Novel Multifunctional ß-N-Acetylhexosaminidase Revealed through Metagenomics of an Oil-Spilled Mangrove.

The use of culture-independent approaches, such as metagenomics, provides complementary access to environmental microbial diversity. Mangrove environments represent a highly complex system with plenty of opportunities for finding singular functions. In this study we performed a functional screening of fosmid libraries obtained from an oil contaminated mangrove site, with the purpose of identifying clones expressing hydrolytic activities. A novel gene coding for a ß-N-acetylhexosaminidase with 355 amino acids and 43KDa was retrieved and characterized. The translated sequence showed only 38% similarity to a ß-N-acetylhexosaminidase gene in the genome of Veillonella sp. CAG:933, suggesting that it might constitute a novel enzyme. The enzyme was expressed, purified, and characterized for its enzymatic activity on carboxymethyl cellulose, p-Nitrophenyl-2acetamide-2deoxy-ß-d-glucopyranoside, p-Nitrophenyl-2acetamide-2deoxy-ß-d-galactopyranoside, and 4-Nitrophenyl ß-d-glucopyranoside, presenting ß-N-acetylglucosaminidase, ß-glucosidase, and ß-1,4-endoglucanase activities. The enzyme showed optimum activity at 30 °C and pH 5.5. The characterization of the putative novel ß-N-acetylglucosaminidase enzyme reflects similarities to characteristics of the environment explored, which differs from milder conditions environments. This work exemplifies the application of cultivation-independent molecular techniques to the mangrove microbiome for obtaining a novel biotechnological product.

The influence of nickel on the bioremediation of multi-component contaminated tropical soil: microcosm and batch bioreactor studies.

Large petrochemical discharges are responsible for organic and inorganic pollutants in the environment. The purpose of this study was to evaluate the influence of nickel, one of the most abundant inorganic element in crude oil and the main component of hydrogen catalysts for oil refining, on the microbial community structure in artificially petroleum-contaminated microcosms and in solid phase bioreactor studies. In the presence of metals, the oil biodegradation in microcosms was significantly delayed during the first 7 days of operation. Also, increasing amounts of moisture generated a positive influence on the biodegradation processes. The oil concentration, exhibiting the most negative influence at the end of the treatment period. Molecular fingerprinting analyses (denaturing gradient gel electrophoresis--DGGE) indicated that the inclusion of nickel into the contaminated soil promoted direct changes to the microbial community structure. By the end of the experiments, the results of the total petroleum hydrocarbons removal in the bioreactor and the microcosm were similar, but reductions in the treatment times were observed with the bioreactor experiments. An analysis of the microbial community structure by DGGE using various markers showed distinct behaviors between two treatments containing high nickel concentrations. The main conclusion of this study was that Nickel promotes a significant delay in oil biodegradation, despite having only a minor effect over the microbial community.

Draft Genome Sequence of Bacillus thuringiensis Strain BrMgv02-JM63, a Chitinolytic Bacterium Isolated from Oil-Contaminated Mangrove Soil in Brazil.

Here, we report the draft genome sequence and the automatic annotation of Bacillus thuringiensis strain BrMgv02-JM63. This genome comprises a set of genes involved in the metabolism of chitin and N-acetylglucosamine utilization, thus suggesting the possible role of this strain in the cycling of organic matter in mangrove soils.

Endo-and exoglucanase activities in bacteria from mangrove sediment

The mangrove ecosystem is an unexplored source for biotechnological applications. In this unique environment, endemic bacteria have the ability to thrive in the harsh environmental conditions (salinity and anaerobiosis), and act in the degradation of organic matter, promoting nutrient cycles. Thus, this study aimed to assess the cellulolytic activities of bacterial groups present in the sediment from a mangrove located in Ilha do Cardoso (SP, Brazil). To optimize the isolation of cellulolytic bacteria, enrichments in two types of culture media (tryptone broth and minimum salt medium), both supplemented with 5% NaCl and 1% of cellulose, were performed. Tests conducted with the obtained colonies showed a higher occurrence of endoglycolytic activity (33 isolates) than exoglycolytic (19 isolates), and the degradation activity was shown to be modulated by the presence of NaCl. The isolated bacteria were clustered by BOX-PCR and further classified on the basis of partial 16S rRNA sequences as Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, Firmicutes or Bacteroidetes. Therefore, this study highlights the importance of studies focusing on the endemic species found in mangroves to exploit them as novel biotechnological tools for the degradation of cellulose.

Streptomyces araujoniae sp. nov.: an actinomycete isolated from a potato tubercle.

The taxonomic position of a streptomycete isolated from a potato tubercle was determined by using a polyphasic approach. The organism had chemotaxonomic and morphological properties consistent with its classification in the genus Streptomyces and formed a distinct phyletic line in the Streptomyces 16S rRNA gene tree. It was found to be closely related to Streptomyces celluloflavus NRRL B-2493(T) (99.4 % 16S rRNA gene similarity) and shared a 99.0 % 16S rRNA gene similarity value with Streptomyces albolongus NRRL B-3604(T) and Streptomyces cavourensis subsp. cavourensis NBRC 13026(T); low levels of DNA-DNA relatedness with these organisms showed that the isolate belonged to a distinct genomic species. The isolate was distinguished readily from the type strains of these species using a combination of morphological and other phenotypic properties. On the basis of these results, it is proposed that isolate ASBV-1(T) (= CBMAI 1465(T) = CCMA 894(T) = NRRL B-24922(T)) be classified as the type strain of Streptomyces araujoniae sp. nov.

Use of molecular approach to verify the influence of a eutrophic lagoon in the nearby ocean's bacterioplankton communities

A lagoa Rodrigo de Freitas é um ambiente aquático eutrofizado, cujas águas são lançadas ao mar nas praias de Ipanema e Leblon através do canal do Jardim de Alah. Nesse trabalho, foi estudada a influência desse aporte na comunidade bacteriana dessas praias. Para isso coletou-se água de onze estações distribuídas entre a lagoa e as praias. Essas amostras foram analisadas quanto a parâmetros moleculares e microbiológicos. Foi realizado também PCR-DGGE utilizando-se iniciadores para o gene rpoB, a partir de DNA extraído das amostras de água coletadas. Resultados preliminares mostram que a influência da lagoa na comunidade bacteriana das praias pode ser verificada por todas as abordagens.

Use of molecular approach to verify the influence of a eutrophic lagoon in the nearby ocean's bacterioplankton communities

Rodrigo de Freitas lagoon is an eutrophic aquatic environment. The waters from the lagoon are released to the sea at Ipanema and Leblon beaches, through Jardim de Alah channel. In this work, the influence of these waters on the bacterial communities of these beaches was investigated. Eleven sampling stations were set between the lagoon and the beaches, and the samples were analyzed by molecular and microbiological parameters. PCR-DGGE of the DNA extracted from the samples was performed using rpoB primers. Preliminary results indicate that all used approaches could reveal the influence of the lagoon on the beaches bacterial communities.

A lagoa Rodrigo de Freitas é um ambiente aquático eutrofizado, cujas águas são lançadas ao mar nas praias de Ipanema e Leblon através do canal do Jardim de Alah. Nesse trabalho, foi estudada a influência desse aporte na comunidade bacteriana dessas praias. Para isso coletou-se água de onze estações distribuídas entre a lagoa e as praias. Essas amostras foram analisadas quanto a parâmetros moleculares e microbiológicos. Foi realizado também PCR-DGGE utilizando-se iniciadores para o gene rpoB, a partir de DNA extraído das amostras de água coletadas. Resultados preliminares mostram que a influência da lagoa na comunidade bacteriana das praias pode ser verificada por todas as abordagens.