Diogo Jurelevicius

Metagenomics reveals sediment microbial community response to Deepwater Horizon oil spill

The Deepwater Horizon (DWH) oil spill in the spring of 2010 resulted in an input of ∼4.1 million barrels of oil to the Gulf of Mexico; >22% of this oil is unaccounted for, with unknown environmental consequences. Here we investigated the impact of oil deposition on microbial communities in surface sediments collected at 64 sites by targeted sequencing of 16S rRNA genes, shotgun metagenomic sequencing of 14 of these samples and mineralization experiments using 14C-labeled model substrates. The 16S rRNA gene data indicated that the most heavily oil-impacted sediments were enriched in an uncultured Gammaproteobacterium and a Colwellia species, both of which were highly similar to sequences in the DWH deep-sea hydrocarbon plume. The primary drivers in structuring the microbial community were nitrogen and hydrocarbons. Annotation of unassembled metagenomic data revealed the most abundant hydrocarbon degradation pathway encoded genes involved in degrading aliphatic and simple aromatics via butane monooxygenase. The activity of key hydrocarbon degradation pathways by sediment microbes was confirmed by determining the mineralization of 14C-labeled model substrates in the following order: propylene glycol, dodecane, toluene and phenanthrene. Further, analysis of metagenomic sequence data revealed an increase in abundance of genes involved in denitrification pathways in samples that exceeded the Environmental Protection Agency (EPA)’s benchmarks for polycyclic aromatic hydrocarbons (PAHs) compared with those that did not. Importantly, these data demonstrate that the indigenous sediment microbiota contributed an important ecosystem service for remediation of oil in the Gulf. However, PAHs were more recalcitrant to degradation, and their persistence could have deleterious impacts on the sediment ecosystem.

Bacterial Community Response to Petroleum Hydrocarbon Amendments in Freshwater, Marine, and Hypersaline Water-Containing Microcosms

ABSTRACT Hydrocarbon-degrading bacterial communities from freshwater, marine, and hypersaline Brazilian aquatic ecosystems (with water salinities corresponding to 0.2%, 4%, and 5%, respectively) were enriched with different hydrocarbons (heptadecane, naphthalene, or crude oil). Changes within the different microcosms of bacterial communities were analyzed using cultivation approaches and molecular methods (DNA and RNA extraction, followed by genetic fingerprinting and analyses of clone libraries based on the 16S rRNA-coding gene). A redundancy analysis (RDA) of the genetic fingerprint data and a principal component analysis (PCA) of the clone libraries revealed hydrocarbon-enriched bacterial communities specific for each ecosystem studied. However, within the same ecosystem, different bacterial communities were selected according to the petroleum hydrocarbon used. In general, the results demonstrated that Acinetobacter and Cloacibacterium were the dominant genera in freshwater microcosms; the Oceanospirillales order and the Marinobacter, Pseudomonas, and Cycloclasticus genera predominated in marine microcosms; and the Oceanospirillales order and the Marinobacter genus were selected in the different hydrocarbon-containing microcosms in hypersaline water. Determination of total petroleum hydrocarbons (TPHs) in all microcosms after 32 days of incubation showed a decrease in the hydrocarbon concentration compared to that for the controls. A total of 50 (41.3%) isolates from the different hydrocarbon-contaminated microcosms were associated with the dominant operational taxonomic units (OTUs) obtained from the clone libraries, and their growth in the hydrocarbon contaminating the microcosm from which they were isolated as the sole carbon source was observed. These data provide insight into the general response of bacterial communities from freshwater, marine, and hypersaline aquatic ecosystems to petroleum hydrocarbon contamination.

The Use of a Combination of alkB Primers to Better Characterize the Distribution of Alkane-Degrading Bacteria

The alkane monooxygenase AlkB, which is encoded by the alkB gene, is a key enzyme involved in bacterial alkane degradation. To study the alkB gene within bacterial communities, researchers need to be aware of the variations in alkB nucleotide sequences; a failure to consider the sequence variations results in the low representation of the diversity and richness of alkane-degrading bacteria. To minimize this shortcoming, the use of a combination of three alkB-targeting primers to enhance the detection of the alkB gene in previously isolated alkane-degrading bacteria was proposed. Using this approach, alkB-related PCR products were detected in 79% of the strains tested. Furthermore, the chosen set of primers was used to study alkB richness and diversity in different soils sampled in Carmópolis, Brazil and King George Island, Antarctica. The DNA extracted from the different soils was PCR amplified with each set of alkB-targeting primers, and clone libraries were constructed, sequenced and analyzed. A total of 255 alkB phylotypes were detected. Venn diagram analyses revealed that only low numbers of alkB phylotypes were shared among the different libraries derived from each primer pair. Therefore, the combination of three alkB-targeting primers enhanced the richness of alkB phylotypes detected in the different soils by 45% to 139%, when compared to the use of a single alkB-targeting primer. In addition, a dendrogram analysis and beta diversity comparison of the alkB composition showed that each of the sampling sites studied had a particular set of alkane-degrading bacteria. The use of a combination of alkB primers was an efficient strategy for enhancing the detection of the alkB gene in cultivable bacteria and for better characterizing the distribution of alkane-degrading bacteria in different soil environments.

Bacillus amyloliquefaciens TSBSO 3.8, a biosurfactant-producing strain with biotechnological potential for microbial enhanced oil recovery

A screening for biosurfactant-producing bacteria was conducted with 217 strains that were isolated from environmental samples contaminated with crude oil and/or petroleum derivatives. Although 19 promising biosurfactant producers were detected, strain TSBSO 3.8, which was identified by molecular methods as Bacillus amyloliquefaciens, drew attention for its production of a high-activity compound that presented an emulsification activity of 63% and considerably decreased surface (28.5 mN/m) and interfacial (11.4 mN/m) tensions in Trypticase Soy Broth culture medium. TSBSO 3.8 growth and biosurfactant production were tested under different physical and chemical conditions to evaluate its biotechnological potential. Biosurfactant production occurred between 0.5% and 7% NaCl, at pH values varying from 6 to 9 and temperatures ranging from 28 to 50 °C. Moreover, biosurfactant properties remained the same after autoclaving at 121 °C for 15 min. The biosurfactant was also successful in a test to simulate microbial enhanced oil recovery (MEOR). Mass spectrometry analysis showed that the surface active compound was a surfactin, known as a powerful biosurfactant that is commonly produced by Bacillus species. The production of a high-efficiency biosurfactant, under some physical and chemical conditions that resemble those experienced in an oil production reservoir, such as high salinities and temperatures, makes TSBSO 3.8 an excellent candidate and creates good expectations for its application in MEOR.

Endophytic microbial community in two transgenic maize genotypes and in their near-isogenic non-transgenic maize genotype

BackgroundDespite all the benefits assigned to the genetically modified plants, there are still no sufficient data available in literature concerning the possible effects on the microbial communities associated with these plants. Therefore, this study was aimed at examining the effects of the genetic modifications of two transgenic maize genotypes (MON810 – expressing the insecticidal Bt-toxin and TC1507 – expressing the insecticidal Bt-toxin and the herbicide resistance PAT [phosphinothricin-N-acetyltransferase]) on their endophytic microbial communities, in comparison to the microbial community found in the near-isogenic non-transgenic maize (control).ResultsThe structure of the endophytic communities (Bacteria, Archaea and fungi) and their composition (Bacteria) were evaluated by denaturing gradient gel electrophoresis (DGGE) and the construction of clone libraries, respectively. DGGE analysis and the clone libraries of the bacterial community showed that genotype TC1507 slightly differed from the other two genotypes. Genotype TC1507 showed a higher diversity within its endophytic bacterial community when compared to the other genotypes. Although some bacterial genera were found in all genotypes, such as the genera Burkholderia, Achromobacer and Stenotrophomonas, some were unique to genotype TC1507. Moreover, OTUs associated with Enterobacter predominated only in TC1507 clone libraries.ConclusionThe endophytic bacterial community of the maize genotype TC1507 differed from the communities of the maize genotype MON810 and of their near-isogenic parental genotypes (non-Bt or control). The differences observed among the maize genotypes studied may be associated with insertion of the gene coding for the protein PAT present only in the transgenic genotype TC1507.

Microbial diversity and hydrocarbon depletion in low and high diesel-polluted soil samples from Keller Peninsula, South Shetland Islands

Abstract The bioremediation of Antarctic soils is a challenge due to the harsh conditions found in this environment. To characterize better the effect of total petroleum hydrocarbon (TPH) concentrations on bacterial, archaeal and microeukaryotic communities in low (LC) and high (HC) hydrocarbon-contaminated soil samples from the Maritime Antarctic clone libraries (small-subunit rRNA genes) were constructed. The results showed that a high concentration of hydrocarbons resulted in a decrease in bacterial and eukaryotic diversity; however, no effect of the TPH concentration was observed for the archaeal community. The HC soil samples demonstrated a high relative abundance of bacterial operational taxonomic units (OTUs) affiliated with unclassified group TM7 and eukaryotic OTUs affiliated with unclassified fungi from Pezizomycotina subphyla. Chemical analyses of the LC and HC soil samples revealed the presence of negligible amounts of nitrogen, thereby justifying the use of biostimulation to remediate these Antarctic soils. Microcosm experiments showed that the application of fertilizers led to an increase of up to 27.8% in the TPH degradation values. The data presented here constitute the first step towards developing the best method to deploy bioremediation in Antarctic soils and provide information to indicate an appropriate action plan for immediate use in the case of new accidents.

Does the essential oil of Lippia sidoides Cham. (pepper-rosmarin) affect its endophytic microbial community?

BackgroundLippia sidoides Cham., also known as pepper-rosmarin, produces an essential oil in its leaves that is currently used by the pharmaceutical, perfumery and cosmetic industries for its antimicrobial and aromatic properties. Because of the antimicrobial compounds (mainly thymol and carvacrol) found in the essential oil, we believe that the endophytic microorganisms found in L. sidoides are selected to live in different parts of the plant.ResultsIn this study, the endophytic microbial communities from the stems and leaves of four L. sidoides genotypes were determined using cultivation-dependent and cultivation-independent approaches. In total, 145 endophytic bacterial strains were isolated and further grouped using either ERIC-PCR or BOX-PCR, resulting in 76 groups composed of different genera predominantly belonging to the Gammaproteobacteria. The endophytic microbial diversity was also analyzed by PCR-DGGE using 16S rRNA-based universal and group-specific primers for total bacteria, Alphaproteobacteria, Betaproteobacteria and Actinobacteria and 18S rRNA-based primers for fungi. PCR-DGGE profile analysis and principal component analysis showed that the total bacteria, Alphaproteobacteria, Betaproteobacteria and fungi were influenced not only by the location within the plant (leaf vs. stem) but also by the presence of the main components of the L. sidoides essential oil (thymol and/or carvacrol) in the leaves. However, the same could not be observed within the Actinobacteria.ConclusionThe data presented here are the first step to begin shedding light on the impact of the essential oil in the endophytic microorganisms in pepper-rosmarin.

Exploiting the aerobic endospore-forming bacterial diversity in saline and hypersaline environments for biosurfactant production

BackgroundBiosurfactants are surface-active biomolecules with great applicability in the food, pharmaceutical and oil industries. Endospore-forming bacteria, which survive for long periods in harsh environments, are described as biosurfactant producers. Although the ubiquity of endospore-forming bacteria in saline and hypersaline environments is well known, studies on the diversity of the endospore-forming and biosurfactant-producing bacterial genera/species in these habitats are underrepresented.MethodsIn this study, the structure of endospore-forming bacterial communities in sediment/mud samples from Vermelha Lagoon, Massambaba, Dois Rios and Abraão Beaches (saline environments), as well as the Praia Seca salterns (hypersaline environments) was determined via denaturing gradient gel electrophoresis. Bacterial strains were isolated from these environmental samples and further identified using 16S rRNA gene sequencing. Strains presenting emulsification values higher than 30 % were grouped via BOX-PCR, and the culture supernatants of representative strains were subjected to high temperatures and to the presence of up to 20 % NaCl to test their emulsifying activities in these extreme conditions. Mass spectrometry analysis was used to demonstrate the presence of surfactin.ResultsA diverse endospore-forming bacterial community was observed in all environments. The 110 bacterial strains isolated from these environmental samples were molecularly identified as belonging to the genera Bacillus, Thalassobacillus, Halobacillus, Paenibacillus, Fictibacillus and Paenisporosarcina. Fifty-two strains showed emulsification values of at least 30%, and they were grouped into18 BOX groups. The stability of the emulsification values varied when the culture supernatants of representative strains were subjected to high temperatures and to the presence of up to 20% NaCl. The presence of surfactin was demonstrated in one of the most promising strains.ConclusionThe environments studied can harbor endospore-forming bacteria capable of producing biosurfactants with biotechnological applications. Various endospore-forming bacterial genera/species are presented for the first time as biosurfactant producers.

Response of marine bacteria to oil contamination and to high pressure and low temperature deep sea conditions

The effect of pressure and temperature on microbial communities of marine environments contaminated with petroleum hydrocarbons is understudied. This study aims to reveal the responses of marine bacterial communities to low temperature, high pressure, and contamination with petroleum hydrocarbons using seawater samples collected near an offshore Brazilian platform. Microcosms containing only seawater and those containing seawater contaminated with 1% crude oil were subjected to three different treatments of temperature and pressure as follows: (1) 22°C/0.1 MPa; (2) 4°C/0.1 MPa; and (3) 4°C/22 MPa. The effect of depressurization followed by repressurization on bacterial communities was also evaluated (4°C/22 MPaD). The structure and composition of the bacterial communities in the different microcosms were analyzed by PCR‐DGGE and DNA sequencing, respectively. Contamination with oil influenced the structure of the bacterial communities in microcosms incubated either at 4°C or 22°C and at low pressure. Incubation at low temperature and high pressure greatly influenced the structure of bacterial communities even in the absence of oil contamination. The 4°C/22 MPa and 4°C/22 MPaD treatments resulted in similar DGGE profiles. DNA sequencing (after 40 days of incubation) revealed that the diversity and relative abundance of bacterial genera were related to the presence or absence of oil contamination in the nonpressurized treatments. In contrast, the variation in the relative abundances of bacterial genera in the 4°C/22 MPa‐microcosms either contaminated or not with crude oil was less evident. The highest relative abundance of the phylum Bacteroidetes was observed in the 4°C/22 MPa treatment.

Distribution of Anaerobic Hydrocarbon-Degrading Bacteria in Soils from King George Island, Maritime Antarctica

Anaerobic diesel fuel Arctic (DFA) degradation has already been demonstrated in Antarctic soils. However, studies comparing the distribution of anaerobic bacterial groups and of anaerobic hydrocarbon-degrading bacteria in Antarctic soils containing different concentrations of DFA are scarce. In this study, functional genes were used to study the diversity and distribution of anaerobic hydrocarbon-degrading bacteria (bamA, assA, and bssA) and of sulfate-reducing bacteria (SRB—apsR) in highly, intermediate, and non-DFA-contaminated soils collected during the summers of 2009, 2010, and 2011 from King George Island, Antarctica. Signatures of bamA genes were detected in all soils analyzed, whereas bssA and assA were found in only 4 of 10 soils. The concentration of DFA was the main factor influencing the distribution of bamA-containing bacteria and of SRB in the analyzed soils, as shown by PCR-DGGE results. bamA sequences related to genes previously described in Desulfuromonas, Lautropia, Magnetospirillum, Sulfuritalea, Rhodovolum, Rhodomicrobium, Azoarcus, Geobacter, Ramlibacter, and Gemmatimonas genera were dominant in King George Island soils. Although DFA modulated the distribution of bamA-hosting bacteria, DFA concentration was not related to bamA abundance in the soils studied here. This result suggests that King George Island soils show functional redundancy for aromatic hydrocarbon degradation. The results obtained in this study support the hypothesis that specialized anaerobic hydrocarbon-degrading bacteria have been selected by hydrocarbon concentrations present in King George Island soils.