Molecular characterization of Paenibacillus antarcticus IPAC21, a bioemulsifier producer isolated from Antarctic soil.

Paenibacillus antarcticus IPAC21, an endospore-forming and bioemulsifier-producing strain, was isolated from King George Island, Antarctica. As psychrotolerant/psychrophilic bacteria can be considered promising sources for novel products such as bioactive compounds and other industrially relevant substances/compounds, the IPAC21 genome was sequenced using Illumina Hi-seq, and a search for genes related to the production of bioemulsifiers and other metabolic pathways was performed. The IPAC21 strain has a genome of 5,505,124 bp and a G + C content of 40.5%. Genes related to the biosynthesis of exopolysaccharides, such as the gene that encodes the extracellular enzyme levansucrase responsible for the synthesis of levan, the 2,3-butanediol pathway, PTS sugar transporters, cold-shock proteins, and chaperones were found in its genome. IPAC21 cell-free supernatants obtained after cell growth in trypticase soy broth at different temperatures were evaluated for bioemulsifier production by the emulsification index (EI) using hexadecane, kerosene and diesel. EI values higher than 50% were obtained using the three oil derivatives when IPAC21 was grown at 28°C. The bioemulsifier produced by P. antarcticus IPAC21 was stable at different NaCl concentrations, low temperatures and pH values, suggesting its potential use in lower and moderate temperature processes in the petroleum industry.

Metagenomic analysis of microbial communities across a transect from low to highly hydrocarbon-contaminated soils in King George Island, Maritime Antarctica.

Soil samples from a transect from low to highly hydrocarbon-contaminated soils were collected around the Brazilian Antarctic Station Comandante Ferraz (EACF), located at King George Island, Antarctica. Quantitative PCR (qPCR) analysis of bacterial 16S rRNA genes, 16S rRNA gene (iTag), and shotgun metagenomic sequencing were used to characterize microbial community structure and the potential for petroleum degradation by indigenous microbes. Hydrocarbon contamination did not affect bacterial abundance in EACF soils (bacterial 16S rRNA gene qPCR). However, analysis of 16S rRNA gene sequences revealed a successive change in the microbial community along the pollution gradient. Microbial richness and diversity decreased with the increase of hydrocarbon concentration in EACF soils. The abundance of Cytophaga, Methyloversatilis, Polaromonas, and Williamsia was positively correlated (p-value = <.05) with the concentration of total petroleum hydrocarbons (TPH) and/or polycyclic aromatic hydrocarbons (PAH). Annotation of metagenomic data revealed that the most abundant hydrocarbon degradation pathway in EACF soils was related to alkyl derivative-PAH degradation (mainly methylnaphthalenes) via the CYP450 enzyme family. The abundance of genes related to nitrogen fixation increased in EACF soils as the concentration of hydrocarbons increased. The results obtained here are valuable for the future of bioremediation of petroleum hydrocarbon-contaminated soils in polar environments.

Enrichment of potential pathogens in marine microbiomes with different degrees of anthropogenic activity.

Anthropogenic activities in coastal marine ecosystems can lead to an increase in the abundance of potentially harmful microorganisms in the marine environment. To understand anthropogenic impacts on the marine microbiome, we first used publicly available microbial phylogenetic and functional data to establish a dataset of bacterial genera potentially related to pathogens that cause diseases (BGPRD) in marine organisms. Representatives of low-, medium- and highly impacted marine coastal environments were selected, and the abundance and composition of their microbial communities were determined by quantitative PCR and 16 S rRNA gene sequencing. In total, 72 BGPRD were cataloged, and 11, 36 and 37 BGPRD were found in low-, medium- and highly human-impacted ecosystems, respectively. The absolute abundance of BGPRD and the co-occurrence of antibiotic resistance genes (AGR) increased with the degree of anthropogenic perturbation in these ecosystems. Anthropogenically impacted coastal microbiomes were compositionally and functionally distinct from those of less impacted sites, presenting features that may contribute to adverse outcomes for marine macrobiota in the Anthropocene era.

Genetics and regulation of nitrogen fixation in Paenibacillus brasilensis PB24.

Biological nitrogen fixation (BNF), performed by diazotrophic prokaryotes, is responsible for reducing dinitrogen (N2) present in the biosphere into biologically available forms of nitrogen. Paenibacillus brasilensis PB24 is a diazotrophic Gram-positive bacterium and is considered ecologically and industrially important because it is able to produce antimicrobial substances and 2,3-butanediol. However, the genetics and regulation of its nitrogen fixing (nif) genes have never been assessed so far. Therefore, the present study aimed to (i) identify the structural and regulatory genes related to BNF in the PB24 genome, (ii) perform comparative genomics analysis of the nif operon among different Paenibacillus species and (iii) study the expression of these genes in the presence and absence of NH4. Strain PB24 showed a nif operon composed of nine genes (nifBHDKENXhesAV), with a conserved synteny (with small variations) among the Paenibacillus species evaluated. BNF regulatory genes, glnK and amtB (encoding GlnK signal transduction protein and AmtB transmembrane protein, respectively) and glnR and glnA genes (encoding the transcription factor GlnR and glutamine synthetase) were found in the PB24 genome. Primers were designed for qPCR amplification of the nitrogenase structural (nifH, nifD and nifK) and regulatory (glnA and amtB) BNF genes. The structural gene expression in PB24 was up- and downregulated in the absence and presence of NH4, respectively. The gene expression levels indicated a GlnR-mediated repression of genes associated with ammonium import (amtBglnK) and BNF (nif genes). Additionally, the regulatory mechanism of GlnR in P. brasilensis PB24 differed from the other Paenibacillus evaluated, considering the different distribution of binding sites recognized by GlnR.

Potential application of Pseudomonas stutzeri W228 for removal of copper and lead from marine environments.

High concentrations of metals in the environment alter bacterial diversity, selecting resistant and tolerant species. The study evaluated the selection of a potential bacterial strain from Sepetiba Bay-Rio de Janeiro, Brazil marine sediments to remove Cu and Pb. The bacterial strain isolated from the sediments was used in three different bioassays: (1) Cu at concentrations of 0 (control), 6 and 50 µg.mL-1; (2) Pb at concentrations of 0 (control), 6 and 50 µg.mL-1; (3) Cu + Pb in concentrations of 3 µg.mL-1 Cu + 3 µg.mL-1 Pb (6 µg.mL-1) and 25 µg.mL-1 Cu + 25 µg.mL-1 Pb (50 µg.mL-1). The number of cells and the enzymatic activities of dehydrogenases and esterases were quantified. Results of taxonomic identification indicated the selection of the Pseudomonas stutzeri W228 strain, showing a greater degree of similarity (±73%) with the database used. There was no significant variation in the number of cells, 108 cells.mL-1, which represents a high biomass production in the presence of stressors. However, we observed a reduction in dehydrogenase activity at all tested concentrations of Cu, Pb and Cu + Pb. The activity of esterase increased, indicating a higher energy demand to complete the bacterial life cycle. The study showed significant results for the absorption of Pb by the extracellular polymeric substances (EPS) and the efflux of Cu. The capacity of Pb absorption by EPS can be considered a resistance mechanism, as well as the efflux of Cu, so that the available EPS sites could be occupied by the most toxic ions demonstrating that Pseudomonas stutzeri is resistant to Pb and Cu.

Microbial community shift under exposure of dredged sediments from a eutrophic bay.

Microbial communities occur in almost every habitat. To evaluate the homeostasis disruption of in situ microbiomes, dredged sediments from Guanabara Bay-Brazil (GB) were mixed with sediments from outside of the bay (D) in three different proportions (25%, 50%, and 75%) which we called GBD25, GBD50, and GBD75. Grain size, TOC, and metals-as indicators of complex contamination-dehydrogenase (DHA) and esterase enzymes (EST)-as indicators of microbial community availability-were determined. Microbial community composition was addressed by amplifying the 16S rRNA gene for DGGE analysis and sequencing using MiSeq platform (Illumina).We applied the quality ratio index (QR) to the GB, D, and every GBD mixture to integrate geochemical parameters with our microbiome data. QR indicated high environmental risk for GB and every GBD mixture, and low risk for D. The community shifted from aerobic to anaerobic profile, consistent with the characteristics of GB. Sample D was dominated by JTB255 marine benthic group, related to low impacted areas. Milano-WF1B-44 was the most representative of GB, often found in anaerobic and sulfur enriched environments. In GBD, the denitrifying sulfur-oxidizing bacteria, Sulfurovum, was the most representative, typically found in suboxic or anoxic niches. The canonical correspondence analysis was able to explain 60% of the community composition variation and exhibit the decrease of environmental quality as the contamination increases. Physiological and taxonomic shifts of the microbial assemblage in sediments were inferred by QR, which was suitable to determine sediment risk. The study produced sufficient information to improve the dredging plan and management.

Chemical and biological dispersants differently affect the bacterial communities of uncontaminated and oil-contaminated marine water.

The use of dispersants in marine environments is a common practice worldwide for oil spill remediation. While the effects of chemical dispersants have been extensively studied, those of biosurfactants, mainly surfactin that is considered one of the most effective surfactants produced by bacteria, have been less considered. We constructed microcosms containing marine water collected from Grumari beach (W_GB, Brazil) and from Schiermonnikoog beach (W_SI, The Netherlands) with the addition of oil (WO), Ultrasperse II plus oil (WOS), surfactin plus oil (WOB), and both dispersants (WS or WB) individually. In these treatments, the composition of bacterial communities and their predictive biodegradation potential were determined over time. High-throughput sequencing of the rrs gene encoding bacterial 16S rRNA revealed that Bacteroidetes (Flavobacteria class) and Proteobacteria (mainly Gammaproteobacteria and Alphaproteobacteria classes) were the most abundant phyla found among the W_GB and W_SI microbiomes, and the relative abundance of the bacterial types in the different microcosms varied based on the treatment applied. Non-metrical multidimensional scaling (NMDS) revealed a clear clustering based on the addition of oil and on the dispersant type added to the GB or SI microcosms, i.e., WB and WOB were separated from WS and WOS in both marine ecosystems studied. The potential presence of diverse enzymes involved in oil degradation was indicated by predictive bacterial metagenome reconstruction. The abundance of predicted genes for degradation of petroleum hydrocarbons increased more in surfactin-treated microcosms than those treated with Ultrasperse II, mainly in the marine water samples from Grumari beach.

Chemical characterization and potential application of exopolysaccharides produced by Ensifer adhaerens JHT2 as a bioemulsifier of edible oils.

Bioemulsifiers are able to stabilize oil-in-water emulsions and are very important in several industrial processes, including food processing. In this study, a bioemulsifier produced by Ensifer adhaerens JHT2 was tested for its ability to emulsify edible oils (canola, corn, palm, olive and soy). Emulsification of soy and canola oils was detected, but the highest emulsification index (EI) was obtained when JHT2 culture supernatant was used for the emulsification of palm oil (EI=100%). Bioemulsifier production was evaluated using nine culture media and different NaCl concentrations (0.5 to 10%), pH (4 to 10) and temperatures (28 to 42°C). The highest emulsification activity was detected in the supernatants of JHT2 grown in trypticase soy broth containing 0.5-1.0% NaCl, pH6-7 and temperatures of 28-37°C. Characterization of the bioemulsifier produced by JHT2 revealed typical characteristics of exopolysaccharides (EPS), constituting a backbone of (1→4)-ß-d-glucopyranosyl and (1→3)-ß-D-galactopyranosyl alternating with (1→4)-α-d-mannopyranosyl units that branch from the structure at O-2. Side chains are composed of units of (1→6)-ß-d-glucopyranosyl and 3-O-linked galactopyranosyl bearing a pyruvic acid acetal substitution at O-4 and O-6. Our results indicate that the EPS produced by Ensifer adhaerens JHT2 is a promising option for improving and maintaining stable emulsions in food prepared with edible oils.

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.

Whole-Genome Sequence of Rummeliibacillus stabekisii Strain PP9 Isolated from Antarctic Soil.

The whole genome of Rummeliibacillus stabekisii PP9, isolated from a soil sample from Antarctica, consists of a circular chromosome of 3,412,092 bp and a circular plasmid of 8,647 bp, with 3,244 protein-coding genes, 12 copies of the 16S-23S-5S rRNA operon, 101 tRNA genes, and 6 noncoding RNAs (ncRNAs).

Response of the bacterial community in oil-contaminated marine water to the addition of chemical and biological dispersants.

The use of dispersants in different stages of the oil production chain and for the remediation of water and soil is a well established practice. However, the choice for a chemical or biological dispersant is still a controversial subject. Chemical surfactants that persist long in the environment may pose problems of toxicity themselves; therefore, biosurfactants are considered to constitute an environmentally friendly and effective alternative. Nevertheless, the putative effects of such agents on the microbiomes of oil-contaminated and uncontaminated marine environments have not been sufficiently evaluated. Here, we studied the effects of the surfactant Ultrasperse II® and the surfactin (biosurfactant) produced by Bacillus sp. H2O-1 on the bacterial communities of marine water. Specifically, we used quantitative PCR and genetic fingerprint analyses to study the abundance and structure of the bacterial communities in marine water collected from two regions with contrasting climatic conditions. The addition of either chemical surfactant or biosurfactant influenced the structure and abundance of total and oil-degrading bacterial communities of oil-contaminated and uncontaminated marine waters. Remarkably, the bacterial communities responded similarly to the addition of oil and/or either the surfactant or the biosurfactant in both set of microcosms. After 30 days of incubation, the addition of surfactin enhanced the oil-degrading bacteria more than the chemical surfactant. However, no increase of hydrocarbon biodegradation values was observed, irrespective of the dispersant used. These data contribute to an increased understanding of the impact of novel dispersants on marine bacteriomes before commercial release into the environment.

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

BACKGROUND: Biosurfactants 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. METHODS: In 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. RESULTS: A 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 into 18 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. CONCLUSION: The 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.

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.

BACKGROUND: Despite 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). RESULTS: The 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. CONCLUSION: The 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.

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 (14)C-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 (14)C-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.

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.

Bacterial community response to petroleum hydrocarbon amendments in freshwater, marine, and hypersaline water-containing microcosms.

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.

Amino acid treatment enhances protein recovery from sediment and soils for metaproteomic studies.

Characterization of microbial protein expression provides information necessary to better understand the unique biological pathways that occur within soil microbial communities that contribute to atmospheric CO2 levels and the earth's changing climate. A significant challenge in studying the soil microbial community proteome is the initial dissociation of bacterial proteins from the complex mixture of particles found in natural soil. The differential extraction of intact bacterial cells limits the characterization of the complete representation of a microbial community. However, in situ lysis of bacterial cells in soil can lead to potentially high levels of protein adsorption to soil particles. Here, we investigated various amino acids for their ability to block soil protein adsorption sites prior to in situ lysis of bacterial cells, as well as their compatibility with both tryptic digestion and mass spectrometric analysis. The treatments were tested by adding proteins from lysed Escherichia coli cells to representative treated and untreated soil samples. The results show that it is possible to significantly increase protein identifications through blockage of binding sites on a variety of soil and sediment textures; use of an optimized desorption buffer further increases the number of identifications.