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.

Methanogenic communities and methane emissions from enrichments of Brazilian Amazonia soils under land-use change.

Amazonian forest conversion into agricultural and livestock areas is considered one of the activities that contribute most to the emission of greenhouse gases, including methane. Biogenic methane production is mainly performed by methanogenic Archaea, which underscores the importance of understanding the drivers shaping microbial communities involved in the methane cycling and changes in methane metabolism. Here, we aimed to investigate the composition and structure of bacterial and archaeal communities in tropical soils in response to land-use changes, emphasizing the methanogenic communities. We collected soil samples from primary forest, pasture, and secondary forest of the Amazonian region and used a strategy based on the enrichment of the methanogenic community with three different methanogenic substrates followed by measurements of methane emission, quantification of mcrA gene copies by qPCR, and total 16 S rRNA gene sequencing (metataxonomics). We observed variations in the structure of bacterial and archaeal communities of soils under different uses. The richness of methanogenic communities was higher in pasture than forest soils and this richness remained during the incubation period, and as a consequence, the enrichment induced earlier methane emission in pastures-derived samples. Furthermore, pastures enrichments exhibited methanogenic archaea networks more complex than primary and secondary forests. In conclusion, pastures harbor a richer and more responsive methanogenic community than forest samples, suggesting that conversion of forest areas to pasture may boost methane emission.

Genomic analysis reveals the potential for hydrocarbon degradation of Rhodopirellula sp. MGV isolated from a polluted Brazilian mangrove.

Planctomycetes are bacteria found in several environments, such as mangroves. In the coastline of the State of Sao Paulo (Brazilian Southeast), mangroves occur in different stages of environmental contamination, promoted by the proximity to the city and industrial activities. One of these mangroves (located in the city of Bertioga) is characterized by the high impact due to past petroleum and ongoing urban contamination. We isolated five bacteria affiliated to Planctomycetes from this mangrove and further subjected them to phenotypical and genetic analysis. The tolerance for salinity was demonstrated by the cultivation under distinct concentrations of NaCl. The ability of this bacterium to use diverse carbon sources was revealed by the use of 30 C-sources from a total of 31 tests. We found the isolate Rhodopirellula sp. MGV very closely affiliated to species of the genus Rhodopirellula, harboring a genome with 7.16 Mbp and 55.3% of GC. The annotation of the 77 contigs resulted in 6.284 CDS, with a remarkable occurrence of sequences associated with aromatic carbon metabolism. In conclusion, we present the isolation and characterization of a Planctomycetes from mangroves, suggesting its participation in the degradation of hydrocarbons present in the contaminated mangroves studied.

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.

Use of plant materials for the bioremediation of soil from an industrial site.

Bioremediation is one of the existing techniques applied for treating oil-contaminated soil, which can be improved by the incorporation of low-cost nutritional materials. This study aimed to assess the addition of two low-cost plant residues, sugarcane bagasse (SCB) and leaf litter (LL) of the forest leguminous Mimosa caesalpiniifolia plant (sabiá), either separately or combined, to a contaminated soil from a petroleum refinery area, analyzed after 90 days of treatment. Individually, both amounts of SCB (20 and 40 g kg-1) favored the growth of total heterotrophic bacteria and total fungi, while LL at 20 g kg-1 better stimulated the hydrocarbon-degrading microorganism's activity in the soil. However, no TPH removal was observed under any of these conditions. Higher microbial growth was detected by the application of both plant residues in multicontaminated soil. The maximum TPH removal of 30% was achieved in amended soil with 20 g kg-1 SCB and 20 kg-1 LL. All the experimental conditions revealed changes in the microbial community structure, related to the handling of the soil, with abundance of Alphaproteobacteria. This study demonstrates the effectiveness of the plant residues SCB and LL as low-cost nutritional materials for biodegradation of hydrocarbon in real oil contaminated soil by indigenous populations.

Draft Genome Sequence of Rhodopirellula baltica Strain BR-MGV, a Planctomycete Isolated from Brazilian Mangrove Soil.

Members of the phylum Planctomycetes, which are capable of surviving in a wide range of environments, are some of the least-explored bacteria. Here, we report the near-complete draft genome sequence and annotation of the planctomycete Rhodopirellula baltica BR-MGV, which was isolated from the soil of a contaminated Brazilian mangrove.

Higher phylogenetic diversity prevents loss of functional diversity caused by successive drying and rewetting cycles.

Microbial communities regulate nutrient cycling in soil, thus the impact of climate change on the structure and function of these communities can cause an imbalance of nutrients in the environment. Structural and functional changes of soil bacterial communities in two contrasting biomes in Brazil, the Atlantic Forest and the Tropical Dry Forest (Caatinga), were studied by simulating, in microcosms, rainfall and drought events. Soil samples were collected in three Brazilian states: Bahia, Pernambuco and São Paulo, in a total of four sampling sites. Analysis of 16S rRNA amplicon libraries revealed changes in microbial communities after three drying-rewetting cycles (60-30% water holding capacity). Alpha diversity indexes were obtained for bacterial communities, as well as the functional diversity index (Shannon) based on the activity of the following enzymes: acid and alkaline phosphatase, arylsulfatase, dehydrogenase, cellulase, amylase, urease and phytase. In general, the soils of Caatinga showed a decrease in the diversity indexes studied, conversely, however, the soils of Atlantic Forest were found to be more resistant during the drying-rewetting cycles. Functional diversity was significantly different for the two biomes, with a decrease in Caatinga soils, while Atlantic Forest samples demonstrated a greater stability of enzymatic activity. Further, the Atlantic Forest samples showed more resistance when compared to samples from Caatinga. The results found in this study have confirmed the hypothesis that biomes, independent of climate, when subjected to successive events of drought and rewetting exhibit structural and metabolic changes.

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.

Draft Genome Sequence of Plant Growth-Promoting Drought-Tolerant Bacillus sp. Strain CMAA 1363 Isolated from the Brazilian Caatinga Biome.

The strain of Bacillus sp. CMAA 1363 was isolated from the Brazilian Caatinga biome and showed plant growth-promoting traits and ability to promote maize growth under drought stress. Sequencing revealed genes involved in stress response and plant growth promotion. These genomic features might aid in the protection of plants against the negative effects imposed by drought.

Dry Season Constrains Bacterial Phylogenetic Diversity in a Semi-Arid Rhizosphere System.

The rhizosphere is viewed as a deterministic environment led by the interaction between plants and microorganisms. In the case of semi-arid plants, this interaction is strengthened by the harshness of the environment. We tested the hypothesis that dry season represents a constraint on the bacterial diversity of the rhizosphere from semi-arid plants. To accomplish this, we sampled two leguminous species at five locations during the dry and rainy seasons in the Caatinga biome and characterised bacterial community structures using qPCR and 16S rRNA sequencing. We found that the main differences between seasons were due to reduced phylogenetic diversity caused by dryness. Variation partitioning indicated that environmental characteristics significant impacts in ß-diversity. Additionally, distance decay relationship and taxa area relationship indicate a higher spatial turnover at the rainy season. During the dry season, decreased bacterial abundance is likely due to the selection of resistant or resilient microorganisms; with the return of the rain, the sensitive populations start to colonise the rhizosphere by a process that is strongly influenced by environmental characteristics. Thus, we propose that the reduction of PD and strong influence of environmental parameters on the assemblage of these communities make them prone to functional losses caused by climatic disturbances.

Genome Sequence of Streptomyces caatingaensis CMAA 1322, a New Abiotic Stress-Tolerant Actinomycete Isolated from Dried Lake Bed Sediment in the Brazilian Caatinga Biome.

The genome sequence of the first Streptomyces species isolated from the Brazilian Caatinga is reported here. Genes related to environmental stress tolerance were prevalent and included many secondary metabolic gene clusters.

Draft Genome Sequence of Bacillus sp. Strain CMAA 1185, a Cellullolytic Bacterium Isolated from Stain House Lake, Antarctic Peninsula.

The aim of this study was to report the genome sequence of the cellulolytic Bacillus sp. strain CMAA 1185, isolated from Stain House Lake, Antarctica.

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.

Exploring the sheep rumen microbiome for carbohydrate-active enzymes.

The rumen is a complex ecosystem enriched for microorganisms able to degrade biomass during the animal's digestion process. The recovery of new enzymes from naturally evolved biomass-degrading microbial communities is a promising strategy to overcome the inefficient enzymatic plant destruction in industrial production of biofuels. In this context, this study aimed to describe the bacterial composition and functions in the sheep rumen microbiome, focusing on carbohydrate-active enzymes (CAE). Here, we used phylogenetic profiling analysis (inventory of 16S rRNA genes) combined with metagenomics to access the rumen microbiome of four sheep and explore its potential to identify fibrolytic enzymes. The bacterial community was dominated by Bacteroidetes and Firmicutes, followed by Proteobacteria. As observed for other ruminants, Prevotella was the dominant genus in the microbiome, comprising more than 30 % of the total bacterial community. Multivariate analysis of the phylogenetic profiling data and chemical parameters showed a positive correlation between the abundance of Prevotellaceae (Bacteroidetes phylum) and organic matter degradability. A negative correlation was observed between Succinivibrionaceae (Proteobacteria phylum) and methane production. An average of 2 % of the shotgun metagenomic reads was assigned to putative CAE when considering nine protein databases. In addition, assembled contigs allowed recognition of 67 putative partial CAE (NCBI-Refseq) representing 12 glycosyl hydrolase families (Pfam database). Overall, we identified a total of 28 lignocellulases, 22 amylases and 9 other putative CAE, showing the sheep rumen microbiome as a promising source of new fibrolytic enzymes.

Functional congruence of rhizosphere microbial communities associated to leguminous tree from Brazilian semiarid region.

Semiarid environments are characterized by the uneven spread of rain throughout the year. This leads to the establishment of a biota that can go through long periods without rain. In order to understand the dynamics of rhizosphere microbial communities across these contrasting seasons in Caatinga, we used the Ion Torrent platform to sequence the metagenome of the rhizosphere of a native leguminous plant (Mimosa tenuiflora). The annotation indicated that most abundant groups detected were the Actinobacteria and Proteobacteria, and the dominant functional groups were carbohydrate and protein metabolisms, and that in the wet season, the communities carried carbohydrate and amino acid metabolisms.The major differences observed between seasons were higher abundance of genes related to carbohydrate and amino acid metabolisms in the rainy season, indicating that the populations present might be better adapted to a higher abundance of organic matter. Besides, no clear separation of samples was detected based on their taxonomic composition whereas the functional composition indicates that samples from the rain season are more related. Altogether, our results indicate that there is al arge functional stability in these communities mostly due to the selection of features that aid the biota to endure the dry season and blossom during rain.

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.

Water regime influences bulk soil and Rhizosphere of Cereus jamacaru bacterial communities in the Brazilian Caatinga biome.

We used the T-RFLP technique combined with Ion Torrent (PGM) sequencing of 16S rRNA and multivariate analysis to study the structure of bulk soil and rhizosphere bacterial communities of a cactus, Cereus jamacaru, from the Brazilian Caatinga biome, which is unique to Brazil. The availability of water shapes the rhizosphere communities, resulting in different patterns during the rainy and dry seasons. Taxonomic approaches and statistical analysis revealed that the phylum Actinobacteria strongly correlated with the dry season, while samples from the rainy season exhibited a strong correlation with the phylum Proteobacteria for rhizosphere samples and with the phyla Bacteroidetes, Firmicutes, Lentisphaerae, and Tenericutes for bulk soil samples. The STAMP software also indicated that the phylum Bacteroidetes, as well as two classes in the Proteobacteria phylum (γ and δ), were the most significant ones during the rainy season. The average abundance of the phylum Actinobacteria and the genus Bacillus was significantly greater during the dry season. Some significant genera found during the dry season might reflect their tolerance to the extreme conditions found in the Caatinga biome. They may also indicate the ecological function that microorganisms play in providing plants with some degree of tolerance to water stress or in assisting in their development through mechanisms of growth promotion. Alterations in microbial communities can be due to the different abilities of native microorganisms to resist and adapt to environmental changes.

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.

Bacterial community composition of anthropogenic biochar and Amazonian anthrosols assessed by 16S rRNA gene 454 pyrosequencing.

Biochar (BC) is a common minor constituent of soils and is usually derived from the burning of wood materials. In the case of Amazonian dark earth (ADE) soils, the increased amount of this material is believed to be due to anthropogenic action by ancient indigenous populations. In this study, we use 16S rRNA gene pyrosequencing to assess the bacterial diversity observed in the BC found in ADEs as well as in the dark earth itself and the adjacent Acrisol. Samples were taken from two sites, one cultivated with manioc and one with secondary forest cover. Analyses revealed that the community structure found in each sample had unique features. At a coarse phylogenetic resolution, the most abundant phyla in all sequence libraries were Actinobacteria, Acidobacteria, Verrucomicrobia and Proteobacteria that were present in similar relative abundance across all samples. However, the class composition varied between them highlighting the difference between the Acrisol and the remaining samples. This result was also corroborated by the comparison of the OTU composition (at 97 % identity). Also, soil coverage has shown an effect over the community structure observed in all samples. This pattern was found to be significant through unweighted UniFrac as well as P tests. These results indicate that, although the ADEs are found in patches within the Acrisols, the contrasting characteristics found between them led to the development of significantly different communities.

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.