Head of Laboratory

IBP1511_09 SHEWANELLA STRAIN ISOLATED FROM BLACK POWDER Márcia T.S Lutterbach1, Luciana S. Contador2 , Ana Lúcia C. Oliveira3, 4 5 Mariana M. Galvão , Gutemberg S. Pimenta Copyright 2009, Brazilian Petroleum, Gas and Biofuels Institute - IBP This Technical Paper was prepared for presentation at the Rio Pipeline Conference and Exposition 2009, held between September, 22-24, 2009, in Rio de Janeiro. This Technical Paper was selected for presentation by the Technical Committee of the event according to the information contained in the abstract submitted by the author(s). The contents of the Technical Paper, as presented, were not reviewed by IBP. The organizers are not supposed to translate or correct the submitted papers. The material as it is presented, does not necessarily represent Brazilian Petroleum, Gas and Biofuels Institute’ opinion, or that of its Members or Representatives. Authors consent to the publication of this Technical Paper in the Rio Pipeline Conference Proceedings. Abstract Black powder is a term frequently used to refer to residues formed by various types of iron sulfides mixed with contaminants eventually present in the natural gas flow. According to some researchers, the occurrence of black powder in gas pipelines, besides its chemical corrosion origin, can be directly related to the sulfate-reducing bacteria (SRB) metabolism in this environment. A black powder sample was inoculated in a Postgate E medium modified with the addition of thioglycolate. The resulting positive culture was kept in the laboratory for four years until its use. A dilution technique was then performed aiming to isolate an SRB strain. The bacterial strain isolated and identified through DNA sequencing was not an SRB but rather a Shewanella sp.. Compared to the sulfate-reducing bacteria group—traditionally considered the foremost responsible for microbially-influenced corrosion (MIC)—Shewanella is a facultative anaerobe and has a versatile metabolism. Shewanella is able to reduce ferric iron and sulfite, oxidize hydrogen gas, and produce hydrogen sulfide; therefore, these bacteria can be responsible for MIC and pite formation. The isolated Shewanella was used in a corrosion experiment, and the corrosion products were characterized by X-ray diffraction, identifying iron sulfides, iron oxides, and sulfur. Our results indicate that the strain isolated, S. putrefaciens, plays a key role in corrosion problems in gas pipelines. Keywords: Shewanella, blackpowder, gas pipeline, Microbiologically Influenced Corrosion Resumo Pó preto é um termo usualmente utilizado para denominar resíduos formados por vários tipos de sulfetos de ferro misturados a contaminantes eventualmente presentes em gasodutos. De acordo com algumas pesquisas, a presença do pó preto em gasodutos tem origem não apenas na corrosão eletroquímica, mas também pode ser diretamente relacionada ao metabolismo das bactérias redutoras de sulfato (BRS). Uma amostra de pó preto foi inoculada em meio de cultura Postgate E modificado com adição de tioglicolato. O cultivo positivo resultante foi mantido em laboratório por quatro anos. Uma técnica de isolamento por diluição foi realizada com o objetivo de isolar uma cepa de BRS. A cepa bacteriana isolada e identificada através de seqüenciamento de DNA não pertencia ao grupo das BRS, mas a uma bactéria produtora de H2S, do gênero Shewanella. Comparado ao grupo das BRS, considerado o principal responsável pela corrosão microbiologicamente induzida (CIM) – Shewanella é um anaeróbio facultativo de metabolismo versátil. Shewanella é capaz de reduzir íon férrico e sulfito, oxidar gás hidrogênio e produzir sulfeto de hidrogênio, sendo desta forma potencialmente causadora de CIM e formação de pites. A Shewanella isolada da amostra de pó preto foi utilizada em ensaios de corrosão. A caracterização dos produtos de corrosão por difração de raios X identificou a presença de sulfetos de ferro, óxidos de ferro e enxofre. Nossos resultados indicam que a cepa isolada, S. putrefaciens pode desempenhar um papel fundamental em problemas de corrosão em gasodutos onde há a formação do pó preto. ______________________________ 1 Ph.D., Biologist – head of Laboratory - National Institute of Technology 2 Ph.D., Biologist – researcher - National Institute of Technology 3 Biologist – researcher - National Institute of Technology 4 Master, Microbiologist – researcher - National Institute of Technology 5 Master, Engineer – senior consultant - Petrobrás Rio Pipeline Conference and Exposition 2009 1. Introduction The corrosion process was traditionally exclusively attributed to chemical and electrochemical origin. However, many studies revealed the importance of microbial activity on corrosion. Current consensus is that both chemical and microbiological mechanisms contribute to the corrosion process, but the proportion of losses caused by microbiologically induced corrosion (MIC) is still difficult to estimate. Studies indicate that microbial activity is responsible for approximately 40% of internal corrosion in gas pipeline industry (Graves & Sullivan, 1996; Pound, 1998), however new studies are needed to confirm or revise this estimate. In general, the presence of microorganisms accelerates and aggravates the abiotic corrosion processes. Microbiologically influenced corrosion (MIC) in pipelines and other equipment is a serious problem for the oil and gas industry worldwide (Graves & Sullivan, 1996; Pound, 1998). Failures caused by MIC can result in safety problems, health risks and environmental hazards. The economic costs of MIC include loss of product by leaks, stoppages of production, costs of environmental cleaning, environmental fines and preventive actions against MIC, like biocidal treatment (Koch et al., 2001). One of the main aspects of corrosion process in gas pipelines is the generation of black powder. Black powder is a generic definition used to classify all particulate material found in pipelines and is usually a mixture of iron sulfides and oxides. The presence of CO2, H2S, acid pH, and water in gas pipelines may result in the abiotic generation of black powder. Microbiological activities may lead to a biotic origin of black powder. The black powder can cause several problems such as product contamination, instruments and filter clogging and flow reduction (Sherik, 2008). Despite the acknowledgement and acceptance of the consequences of MIC, the mechanisms of MIC and black powder production remain unclear. The sulfate reducing bacteria (SRB) are traditionally identified as the major contributors to MIC (Iverson, 1987; Ford & Michell, 1990; Beech & Gaylard, 1999; Hamilton, 2003; Videla et al., 2005). However, beside SRB, there are many different types of microorganisms responsible for MIC. Some of the main bacterial groups implicated in MIC are sulfur-oxidizing bacteria, methanogens, iron-oxidizing/reducing bacteria, manganese-oxidizing bacteria, and bacteria secreting organic acids and slime (Beech & Coutinho, 2003). MIC is rarely associated to one single mechanism or to one single species of microorganism (Videla et al., 2005). The general concern about SRB is mostly due to their capacity of producing hydrogen sulfide (H2S), which directly attack metal surfaces leading to anaerobic corrosion and pit formation (Hamilton, 1985). Nevertheless there are other bacteria capable of producing H2S. The present study aim the isolation of bacteria from a black powder sample, which will be stored in cryogenic collection and used in corrosion essays to a better understanding of the role of MIC in black powder generation. 2. Methodology A black powder sample was inoculated in a Postgate E medium modified with the addition of thioglycolate. The Postgate E medium has the following composition (g/L): KH2PO4 0.5; NH4Cl 1.0; Na2SO4 1.0; CaCl2.2H2O 0.67; MgCl2.6H2O 1.68; Yeast extract 1.0; Ascorbic acid 0.1; Agar 1.9; NaCl 5.0; FeSO4.7H2O 0.5. Sodium lactate (7ml/L) is employed as the carbon source and 4 ml of resarzurin (0.025%) was used as an oxidation indicator. The resulting positive culture was kept in the laboratory for four years until its use. To obtain a pure culture, the dilution to extinction technique was employed. Successive decimal dilutions were prepared from the positive culture; aliquots of each dilution were then inoculated in Postgate E medium. The dilution technique was performed twice from the last positive dilution tube. This methodology was followed by streaking onto plates and the isolate was purified by repeating the streaking three times. Plates were incubated in anaerobic jar. The purity of the isolate was confirmed by microscopic observations. The isolated bacterium was also inoculated in an ammonium-iron citrate culture medium designed for iron-precipitating bacteria (CETESB – L5 207) and in a non- selective agar (Plate count Agar OXOID CM0325). The DNA from the pure strain was extracted using a Microbial DNA isolation kit. After extraction, the 16S rDNA was selectively amplified by polymerase chain reaction (PCR) from purified genomic DNA using the bacterial forward primer SAdir (5’-AGAGTTTGATCATGGCTCAGA-3’) corresponding to positions 8–28 of Escherichia coli 16S rRNA, and the bacterial reverse primer, S17rev (5’-GTTACCTTGTTACGACTT-3’). The PCR reaction was performed as previously described (Agogué et al., 2005). The PCR products were further purified using a PCR cleaning kit and sent to the University of São Paulo (USP) for sequencing. The sequence of the 16S rDNA was compared to those available in the GenBank database by using the

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