Characterization of Metagenomic Sequences from a Brazilian Petroleum Reservoir with Potential for Hydrocarbon Biodegradation
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1 Characterization of Metagenomic Sequences from a Brazilian Petroleum Reservoir with Potential for Hydrocarbon Biodegradation Isabel Natalia Sierra García* Microbial Resource Division -DRM Research Center for Chemistry, Biology and Agriculture CPQBA / UNICAMP State University of Campinas UNICAMP Campinas, SP, Brazil
2 Characterization of Metagenomic Sequences from a Brazilian Petroleum Reservoir with Introduction Methodology and results Conclussions
3 Characterization of Metagenomic Sequences from a Brazilian Petroleum Reservoir with Introduction Alkanes Crude oil Saturated hydrocarbons Linear alkanes Branched Cyclo alkanes Alkanes are the major components of crude oil and the most abundant and available carbon and energy sources in reservoirs.
4 Characterization of Metagenomic Sequences from a Brazilian Petroleum Reservoir with Introduction Microbial degradation of crude oil in reservoirs BIODEGRADATION Crude oil Heavy oil Increase the oil viscosity Amount of sulfur Acidity Worldwide problem for petroleum industry recovering and refining difficult and costly
5 Characterization of Metagenomic Sequences from a Brazilian Petroleum Reservoir with Introduction Microbial degradation of crude oil in reservoirs Effects on the composition and physical properties of crude oil are well-known Processes of oil biodegradation in petroleum reservoirs, mechanisms, microorganisms or metabolic pathways involved are poorly understood Limited knowledge is due to the use of Culture based methods Access only a small part of microbial communities Continuous isolation of the same species
6 Characterization of Metagenomic Sequences from a Brazilian Petroleum Reservoir with Introduction Pathways for aerobic and anaerobic bacterial alkane degradation Several bacterial strains can degrade alkanes using enzyme systems that have still not been characterized and that may include new proteins not related to those currently known (Rojo, 2009). Wentzel et al. (2007). Appl Microbiol Biotechnol 76:
7 Characterization of Metagenomic Sequences from a Brazilian Petroleum Reservoir with Introduction Metagenomic approach Culture independent method which allows the access to the metabolic potential of previosly uncultured microorganisms In petroleum environments would allow the access to new genes or complete metabolic pathways
8 Characterization of Metagenomic Sequences from a Brazilian Petroleum Reservoir with Introduction Metagenomic fosmid library constructed clones Fosmid 45 Kb Cloning-Ready Copy Control pcc2fos (EPICENTRE) Petroleum sample collected at the Potiguar Basin in Brazil clones screened for hexadecane degradation FOS1A 91% High hexadecane degradation rate
9 Characterization of Metagenomic Sequences from a Brazilian Petroleum Reservoir with Introdution METAGENOMIC LIBRARY 1 clone:fos1a Hexadecane degradation = 91% Identification of metagenomic sequences responsible for the observed hydrocarbon degradation Information about mechanisms of biodegradation in a Brazilian petroleum reservoir New biocatalysts Biorremediation MEOR
10 Characterization of Metagenomic Sequences from a Brazilian Petroleum Reservoir with Introduction Methodology and results Conclusions
11 Methodology and Results Characterization of Metagenomic Sequences from a Brazilian Petroleum Reservoir with Fosmidial clone FOS1A Random shotgun library construction Sequencing Data analysis Assembly and gene chracterization
12 Characterization of Metagenomic Sequences from a Brazilian Petroleum Reservoir with Methodology and Results Random shotgun library construction Fosmid DNA purification DNA Fragments pb Sonication Well 1: Sonicated DNA Well 2: Leader 1. Fosmid DNA isolation 2. DNA shearing by sonication 3. Size selection and gel purification 4. End repair and phosphorylation 5. Ligation pbluescript II SK+ (Stratagene) / Eco RV CiAP (Invitrogen) T4 DNA polymerase 6. Transformation into E. coli by electroporation Escherichia coli DH10B
13 Methodology and Results Characterization of Metagenomic Sequences from a Brazilian Petroleum Reservoir with Shotgun library construction Clone FOS1A Fosmid 40kb DNA shearing and Size selection Ligation with pbluescript KS+ and Electroporation into E. coli DH10B Fosmid DNA isolation clones ranging from bp from a 40kb fosmid
14 Materials and methods Characterization of Metagenomic Sequences from a Brazilian Petroleum Reservoir with Origin of the metagenomic library Fosmidial clone FOS1A Random shotgun library construction Sequencing Data analysis Assembly and gene chracterization
15 Methodology and Results Characterization of Metagenomic Sequences from a Brazilian Petroleum Reservoir with 604 clones Sequencing Sanger sequencing PCR tested Primers M13F and M13R Sequencing MegaBace 500 System 304 clones
16 Methodology and Results Characterization of Metagenomic Sequences from a Brazilian Petroleum Reservoir with Origin of the metagenomic library Fosmidial clone FOS1A Random shotgun library construction Sequencing Data analysis Assembly and gene chracterization
17 Methodology and Results Caracterização Estrutural e Funcional de Genes de Degradação de Petróleo Originados de Metagenoma Microbiano de Reservatórios de Petróleo Data analysis Assembly and gene chracterization Local BLAST (blastn, blastx) Genebank Database E-value: < 10-5 Identity > 80% Alignment length > 100bp Phred/Phrad/Consed Vector and contamination screening and removal Quality trimming Assembly Open reading frame ORF Genemark NCBI, SwissProt, and EMBL
18 Methodology and Results Assembly Characterization of Metagenomic Sequences from a Brazilian Petroleum Reservoir with Assembly statistics Total length bp #Clusters 100 #Contigs 24 #singlets 76 Mean contig size 1058 bp Largest contig 1774 bp N50: contigs
19 Methodology and Results Assembly Contig Contig size (bp) Characterization of Metagenomic Sequences from a Brazilian Petroleum Reservoir with Number of sequences in Contig Contig Alcohol dehydrogenase zinc-binding domain protein Contig succinate-semialdehyde dehydrogenase [NADP+] Top BLASTX hit Identity (%) Organism type Target Accesion 76.8% Thauera sp. MZ1T YP_ % Bradyrhizobium japonicum USDA 110 NP_ Contig flavoprotein 54.4% Azoarcus sp. BH72 YP_ Contig uroporphyrinogen decarboxylase Contig putative Orn/Arg/Lys decarboxylase Contig Orn/Arg/Lys decarboxylase Contig glycosyltransferase, group 2 family protein 73.3% Aromatoleum aromaticum EbN1 YP_ % Azoarcus sp. BH72 YP_ % Azoarcus sp. BH72 YP_ % Thiobacillus denitrificans ATCC YP_
20 Methodology and Results Characterization of Metagenomic Sequences from a Brazilian Petroleum Reservoir with Alcohol dehydrogenases (ADHs) Catalyzes the oxidation of alcohols to aldehydes, is widely present in all organisms In the context of alkane biodegradation, ADH catalyzes the second reaction step of the oxidation pathway
21 Methodology and Results Assembly Contig Contig size (bp) Characterization of Metagenomic Sequences from a Brazilian Petroleum Reservoir with Number of sequences in Contig Contig Alcohol dehydrogenase zinc-binding domain protein Contig succinate-semialdehyde dehydrogenase [NADP+] Top BLASTX hit Identity (%) Organism type Target Accesion 76.8% Thauera sp. MZ1T YP_ % Bradyrhizobium japonicum USDA 110 NP_ Contig flavoprotein 54.4% Azoarcus sp. BH72 YP_ Contig uroporphyrinogen decarboxylase Contig putative Orn/Arg/Lys decarboxylase Contig Orn/Arg/Lys decarboxylase Contig glycosyltransferase, group 2 family protein 73.3% Aromatoleum aromaticum EbN1 YP_ % Azoarcus sp. BH72 YP_ % Azoarcus sp. BH72 YP_ % Thiobacillus denitrificans ATCC YP_
22 Methodology and Results Characterization of Metagenomic Sequences from a Brazilian Petroleum Reservoir with Aldehyde dehydrogenases (ADLHs) Catalyzes the oxidation of aldehydes to carboxilic acids ADLHs are involved in the third step of the oxidation of alkanes Li X, et al. Microbiol Res (2010) Alkane Alkyl alcohol Alkyl aldehyde Fatty acid
23 Characterization of Metagenomic Sequences from a Brazilian Petroleum Reservoir with Introduction Methodology and results Conclusions
24 Characterization of Metagenomic Sequences from a Brazilian Petroleum Reservoir with Conclusions Metagenomic approach have allowed the access to potential new sequences involved with hydrocarbon biodegradation Complete sequencing of the shotgun library may improve the assembly and may allow a complete coverage for the metagenomic fosmid responsible for hydrocarbon degradation
25 Characterization of Metagenomic Sequences from a Brazilian Petroleum Reservoir with Conclusions Accessing new catabolic properties from such a hostile environment may represent an interesting source for biotechnological use in bioremediation For the first time in Brazil, gene sequences responsible for the degradation processes in oil reservoirs are being identified from microbial metagenomic data
26 Acknowledgements Dr. Valéria Maia de Oliveira Dr. Anete Pereira de Souza Dr. Suzan Pantaroto de Vasconcellos
27 Questions?
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