Vanessa Marques Alvarez

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.

Comparative Bioremediation of Crude Oil-Amended Tropical Soil Microcosms by Natural Attenuation, Bioaugmentation, or Bioenrichment

Bioremediation is an efficient strategy for cleaning up sites contaminated with organic pollutants. In this study, we evaluated the effectiveness of monitored natural attenuation, bioenrichment, and bioaugmentation using a consortium of three actinomycetes strains in remediating two distinct typical Brazilian soils from the Atlantic Forest and Cerrado biomes that were contaminated with crude oil, with or without the addition of NaCl. Microcosms were used to simulate bioremediation treatments over a 120-day period. During this period, we monitored total petroleum hydrocarbons (TPHs) and n-alkanes degradation and changes in bacterial communities. Over time, we found the degradation rate of n-alkanes was higher than TPH in both soils, independent of the treatment used. In fact, our data show that the total bacterial community in the soils was mainly affected by the experimental period of time, while the type of bioremediation treatment used was the main factor influencing the actinomycetes populations in both soils. Based on these data, we conclude that monitored natural attenuation is the best strategy for remediation of the two tropical soils studied, with or without salt addition.

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.

Extracellular proteases of Halobacillus blutaparonensis strain M9, a new moderately halophilic bacterium

Halophilic microorganisms are source of potential hydrolytic enzymes to be used in industrial and/or biotechnological processes. In the present study, we have investigated the ability of the moderately halophilic bacterium Halobacillus blutaparonensis (strain M9), a novel species described by our group, to release proteolytic enzymes. This bacterial strain abundantly proliferated in Luria-Bertani broth supplemented with 2.5% NaCl as well as secreted proteases to the extracellular environment. The production of proteases occurred in bacterial cells grown under different concentration of salt, ranging from 0.5% to 10% NaCl, in a similar way. The proteases secreted by H. blutaparonensis presented the following properties: (i) molecular masses ranging from 30 to 80 kDa, (ii) better hydrolytic activities under neutral-alkaline pH range, (iii) expression modulated according to the culture age, (iv) susceptibility to phenylmethylsulphonyl fluoride, classifying them as serine-type proteases, (v) specific cleavage over the chymotrypsin substrate, and (vi) enzymatic stability in the presence of salt (up to 20% NaCl) and organic solvents (e.g., ether, isooctane and cyclohexane). The proteases described herein are promising for industrial practices due to its haloalkaline properties.

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.

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.

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)-β-d-glucopyranosyl and (1→3)-β-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)-β-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.