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 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.

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.

Bioremediation potential of a tropical soil contaminated with a mixture of crude oil and production water.

A typical tropical soil from the northeast of Brazil, where an important terrestrial oil field is located, was accidentally contaminated with a mixture of oil and saline production water. To study the bioremediation potential in this area, molecular methods based on PCR-DGGE were used to determine the diversity of the bacterial communities in bulk and in contaminated soils. Bacterial fingerprints revealed that the bacterial communities were affected by the presence of the mixture of oil and production water, and different profiles were observed when the contaminated soils were compared with the control. Halotolerant strains capable of degrading crude oil were also isolated from enrichment cultures obtained from the contaminated soil samples. Twenty-two strains showing these features were characterized genetically by amplified ribosomal DNA restriction analysis (ARDRA) and phenotypically by their colonial morphology and tolerance to high NaCl concentrations. Fifteen ARDRA groups were formed. Selected strains were analyzed by 16S rDNA sequencing, and Actinobacteria was identified as the main group found. Strains were also tested for their growth capability in the presence of different oil derivatives (hexane, dodecane, hexadecane, diesel, gasoline, toluene, naphthalene, o-xylene, and p-xylene) and different degradation profiles were observed. PCR products were obtained from 12 of the 15 ARDRA representatives when they were screened for the presence of the alkane hydroxylase gene (alkB). Members of the genera Rhodococcus and Gordonia were identified as predominant in the soil studied. These genera are usually implicated in oil degradation processes and, as such, the potential for bioremediation in this area can be considered as feasible.