Eugenio V. Santos Neto

Analysis of the composition of bacterial communities in oil reservoirs from a southern offshore Brazilian basin

The aim of this study was to characterize and compare the bacterial community structure of two distinct oil samples from a petroleum field in Brazil by using both molecular, based on the construction of 16S rRNA gene libraries, and cultivation methods. Statistical comparisons of libraries based on Amplified Ribosomal DNA Restriction Analysis (ARDRA) data revealed no significant differences between the communities recovered in the non-biodegraded (NBD) and highly biodegraded oils (HBD). BlastN analysis of the 16S rRNA gene sequences representative of distinct ribotypes from both oils showed the presence of nine different bacterial genera in these samples, encompassing members of the genera Arcobacter, Halanaerobium, Marinobacter, Propionibacterium, Streptomyces, Leuconostoc, Acinetobacter, Bacillus and Streptococcus. Enrichments obtained using oil as inoculum and sole carbon source yielded bacterial isolates showing high 16S rRNA gene sequence similarity with Achromobacter xylosoxidans, Bacillus subtilis, Brevibacillus sp., Dietzia sp. and Methylobacterium sp. Comparison between the data obtained using cultivation-independent and enrichment cultures suggests that different selection of community members may occur when using distinct approaches. All the organisms found, except for Leuconostoc sp. and Streptococus sp., have been previously reported in the literature as hydrocarbon degraders and/or associated to oil field environments.

New Hydrocarbon Degradation Pathways in the Microbial Metagenome from Brazilian Petroleum Reservoirs

Current knowledge of the microbial diversity and metabolic pathways involved in hydrocarbon degradation in petroleum reservoirs is still limited, mostly due to the difficulty in recovering the complex community from such an extreme environment. Metagenomics is a valuable tool to investigate the genetic and functional diversity of previously uncultured microorganisms in natural environments. Using a function-driven metagenomic approach, we investigated the metabolic abilities of microbial communities in oil reservoirs. Here, we describe novel functional metabolic pathways involved in the biodegradation of aromatic compounds in a metagenomic library obtained from an oil reservoir. Although many of the deduced proteins shared homology with known enzymes of different well-described aerobic and anaerobic catabolic pathways, the metagenomic fragments did not contain the complete clusters known to be involved in hydrocarbon degradation. Instead, the metagenomic fragments comprised genes belonging to different pathways, showing novel gene arrangements. These results reinforce the potential of the metagenomic approach for the identification and elucidation of new genes and pathways in poorly studied environments and contribute to a broader perspective on the hydrocarbon degradation processes in petroleum reservoirs.

Could petroleum biodegradation be a joint achievement of aerobic and anaerobic microrganisms in deep sea reservoirs

Several studies suggest that petroleum biodegradation can be achieved by either aerobic or anaerobic microorganisms, depending on oxygen input or other electron acceptors and appropriate nutrients. Evidence from in vitro experiments with samples of petroleum formation water and oils from Pampo Field indicate that petroleum biodegradation is more likely to be a joint achievement of both aerobic and anaerobic bacterial consortium, refining our previous observations of aerobic degradation. The aerobic consortium depleted, in decreasing order, hydrocarbons > hopanes > steranes > tricyclic terpanes while the anaerobic consortium depleted hydrocarbons > steranes > hopanes > tricyclic terpanes. The oxygen content of the mixed consortia was measured from time to time revealing alternating periods of microaerobicity (O2 ~0.8 mg.L-1) and of aerobicity (O2~6.0 mg.L-1). In this experiment, the petroleum biodegradation changed from time to time, alternating periods of biodegradation similar to the aerobic process and periods of biodegradation similar to the anaerobic process. The consortia showed preferences for metabolizing hydrocarbons > hopanes > steranes > tricyclic terpanes during a 90-day period, after which this trend changed and steranes were more biodegraded than hopanes. The analysis of aerobic oil degrading microbiota by the 16S rRNA gene clone library detected the presence of Bacillus, Brevibacterium, Mesorhizobium and Achromobacter, and the analysis of the anaerobic oil degrading microbiota using the same technique detected the presence of Bacillus and Acinetobacter (facultative strains). In the mixed consortia Stenotrophomonas, Brevibacterium, Bacillus, Rhizobium, Achromobacter and 5% uncultured bacteria were detected. This is certainly a new contribution to the study of reservoir biodegradation processes, combining two of the more important accepted hypotheses.

Bacterial diversity characterization in petroleum samples from Brazilian reservoirs

This study aimed at evaluating potential differences among the bacterial communities from formation water and oil samples originated from biodegraded and non-biodegraded Brazilian petroleum reservoirs by using a PCR-DGGE based approach. Environmental DNA was isolated and used in PCR reactions with bacterial primers, followed by separation of 16S rDNA fragments in the DGGE. PCR products were also cloned and sequenced, aiming at the taxonomic affiliation of the community members. The fingerprints obtained allowed the direct comparison among the bacterial communities from oil samples presenting distinct degrees of biodegradation, as well as between the communities of formation water and oil sample from the non-biodegraded reservoir. Very similar DGGE band profiles were observed for all samples, and the diversity of the predominant bacterial phylotypes was shown to be low. Cloning and sequencing results revealed major differences between formation water and oil samples from the non-biodegraded reservoir. Bacillus sp. and Halanaerobium sp. were shown to be the predominant components of the bacterial community from the formation water sample, whereas the oil sample also included Alicyclobacillus acidoterrestris, Rhodococcus sp., Streptomyces sp. and Acidithiobacillus ferrooxidans. The PCR-DGGE technique, combined with cloning and sequencing of PCR products, revealed the presence of taxonomic groups not found previously in these samples when using cultivation-based methods and 16S rRNA gene library assembly, confirming the need of a polyphasic study in order to improve the knowledge of the extent of microbial diversity in such extreme environments.

Cultivation-independent methods applied to the microbial prospection of oil and gas in soil from a sedimentary basin in Brazil

The upper parts of oil field structures may leak gas which is supposed to be indirectly detected by the soil bacterial populations. Such microorganisms are capable of consuming this gas, supporting the Microbial Prospection of Oil and Gas (MPOG) methodology. The goal of the present work was to characterize microbial communities involved in short-chain alkane metabolism, namely methane, ethane and propane, in samples from a petroliferous (P) soil through clone libraries of the 16S rRNA gene of the Domains Bacteria and Archaea and the catabolic gene coding for the soluble di-iron monooxygenase (SDIMO) enzyme alpha subunit. The microbial community presented high abundance of the bacterial phylum Actinobacteria, which represented 53% of total clones, and the Crenarchaeota group I.1b from the Archaea Domain. The analysis of the catabolic genes revealed the occurrence of seven Operational Protein Families (OPF) and higher richness (Chao = 7; Ace = 7.5) and diversity (Shannon = 1.09) in P soil when compared with a non-petroliferous (Np) soil (Chao = 2; Ace = 0, Shannon = 0.44). Clones related to the ethene monooxygenase (EtnC) and methane monooxygenase (MmoX) coding genes occurred only in P soil, which also presented higher levels of methane and lower levels of ethane and propane, revealed by short-chain hydrocarbon measures. Real-time PCR results suggested that the SDIMO genes occur in very low abundance in the soil samples under study. Further investigations on SDIMOs genes in natural environments are necessary to unravel their still uncharted diversity and to provide reliable tools for the prospection of degrading populations.

3-Alkyl and 3-carboxyalkyl steranes in marine evaporitic oils of the Potiguar Basin, Brazil

Abstract The geochemical characterization of marine evaporitic oils from the Fazenda Belem oil field, Potiguar Basin, Brazil showed good correlation between both acidic and neutral fractions, and led to the identification of several biomarkers including the homologous series of 3-alkyl- and 3-carboxyalkyl-5α(H) steranes and also the new series of 3-alkyl- and 3-carboxyalkyl-5β(H) steranes. These compounds were identified using GC/MS and their structures were confirmed by coinjection with synthetic standards. A minor series of 4-carboxyalkyl steranes was also detected. The analysis of the acidic biomarkers allowed a more accurate evaluation of maturation, biodegradation and migration of the studied oils.

Searching for monooxygenases and hydrolases in bacteria from an extreme environment.

Microbial oxidation potentials of extremophiles recovered from Pampo Sul oil field, Campos Basin, Brazil, in pure culture or in consortia, were investigated using high-throughput screening (HTS) and multibioreactions. Camphor (1), cis-jasmone (2), 2-methyl-cyclohexanone (3), 1,2-epoxyoctane (4), phenylethyl acetate (5), phenylethyl propionate (6), and phenylethyl octanoate (7) were used to perform multibioreaction assays. Eighty-two bacterial isolates were recovered from oil and formation water samples and those presenting outstanding activities in HTS assays were identified by sequencing their 16S rRNA genes. These results revealed that most microorganisms belonged to the genus Bacillus and presented alcohol dehydrogenase, monooxygenase, epoxide hydrolase, esterase, and lipase activities.

Trace element distributions in biodegraded crude oils and fractions from the Potiguar Basin, Brazil

Trace element distribution patterns were determined in crude oils from onshore biodegraded reservoirs along a secondary migration trend (Potiguar Basin, Brazil). Samples were fractionated into asphaltenes, paraffins, aromatics and polars and resins for the determination of 22 trace elements by ICP-MS. Most of them are strongly enriched in the resin and asphaltene fractions, but V and Ni are also associated with the aromatic and polar fractions. V, Co, Ni and Mo were influenced by biodegrading processes and accumulate in the most affected and migrated samples. Variations of the Ni/V ratio in aromatics and polars, matching those of homohopane and gammacerane indexes in paraffin, evidenced also the influence of biodegradation. Very similar elemental distribution patterns in oil and corresponding asphaltenes were observed, suggesting that the latter fraction, more refractory than the original oil, can be used as a robust inorganic geochemical fingerprint for oil characterization.

Diversity of Hydrocarbon-Related Catabolic Genes in Oil Samples from Potiguar Basin (Rn, Brazil)

Biodegradation may result in physicochemical changes in crude oil and natural gas properties, being responsible for the decrease of saturated hydrocarbons and yielding heavy oil with low economic value. Studies on the diversity of microbial catabolic genes in oil reservoirs are scarce and could help to predict the potential of a petroleum sample to be biodegraded. The aim of this study was to evaluate the diversity of genes involved in hydrocarbon degradation in Brazilian petroleum samples (biodegraded and non-biodegraded) through the construction and analysis of gene libraries (alkane monooxygenase – alk, dioxygenase – ARHDs and 6-oxocyclohex-1-ene-1-carbonyl-CoA hydrolase - bamA). The results showed a differential distribution of catabolic genes between the sites, being the biodegraded oil more diverse for the alk and bamA genes. Sequences were similar to the alkB genes from Geobacillus thermoleovorans and several species of Acinetobacter, to ARHD genes from Pseudomonas spp. and two species of Burkholderia, and to bamA genes from deltaproteobacteria. Interestingly, most of the catabolic sequences recovered from both petroleum reservoirs grouped together forming distinct clusters in the phylogenetic tree reconstruction and may correspond to potentially new genes, possibly harbored by yet uncultivated microorganisms. This is the first report on the detection of alk, ARHD and bamA genes in petroleum reservoir environments, demonstrating the genetic potential of such microbial communities to biodegrade the oil.

Geosteranes: Identification and synthesis of a novel series of 3-substituted steranes

Abstract A novel series of 3-alkyl and 3-carboxyalkyl-5β(H)-steranes 7–10 along with a full homologous series of carboxyalkyl-sterane (C 1 to C 6 ) 4–6 with 3α(H)5α(H) configuration have been identified in marine-evaporitic oils from Fazenda Belem, Potiguar Basin (Brazil) on the basis of mass spectral interpretation. The synthesis of enantiomerically pure 3α-alkyl-5β(H)-cholestane and 3β-alkyl-5α(H)-cholestane standards and their coinjection with petroleum fractions confirmed the structural assignments.