Metal corrosion and biological H2S cycling in closed systems Fernanda Abreu* Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil; [email protected]* Abstract Sulfate reducing bacteria produce H2S during growth. This gas is toxic and is associated with corrosion in industrial systems. In the environment purple sulfur bacteria, green sulfur bacteria and sulfur oxidizing bacteria use the H2S produced by sulfate reducing bacteria as electron donors. The major aim of this project is to evaluate the possibility of using H2S consuming bacteria to lower H2S concentration and prevent corrosion. Introduction In metal corrosion process the surface of the metal is destroyed due to certain external factors that lead to its chemical or electrochemical change to form more stable compounds. The simplified explanation of the corrosion process is the oxidation of at an anode (corroded end releasing electrons) and the reduction of a substance at a cathode. Corrosion mechanisms are very diverse and can be based on inorganic physicochemical reactions and/or biologically influenced. Microbiologically influenced corrosion (MIC) is a natural process that occurs in the environment as a result of metabolic activity of microorganisms. Microbial colonization and biofilm formation on metal surfaces modify the electrochemical conditions at the metal–solution interface, which usually have positive influence on corrosion process. MIC of steel generates approximately US$ 100 million financial losses per annum in the United States (Muyzer and Stams, 2008). In industrial settings, especially in petroleum, gas and shipping industries, sulfate reducing bacteria (SRB) are a major concern. SRB are ubiquitous in anoxic habitats and have an important role in both the sulfur and carbon cycles (Muyzer and Stams, 2008). According to rrs gene phylogenetic analysis, 1 the majority of sulfate reducing bacteria are grouped in Bacteria domain within Deltaproteobacteria subgroup; there are some SRB species localized within the Clostridia (Desulfotomaculum, Desulfosporosinus and Desulfosporomusa genera) and within three of thermophilic SRB lineages (Nitrospirae, Thermodesulfobacteria and Thermodesulfobiaceae). Sulfate reduction is also described for Archaea domain and belong to the genus Archaeoglobus (Euryarchaeota) and to the genera Thermocladium and Caldirvirga (Crenarchaeota) (Muyzer and Stams, 2008). During growth SRB produce corrosive metabolic products that will increase corrosion rates (Videla and Herrera, 2005). Hydrogen sulfide gas (H2S) is the product of sulfate reduction by SRB in anaerobic respiration to obtain energy. This gas is extremely toxic and corrosive. Inhalation of low concentrations of hydrogen sulfide cause irritation to mucous membranes, headaches, dizziness, and nausea; inhalation of higher concentrations (200-300 ppm) result in respiratory arrest leading to coma, unconsciousness, pulmonary paralysis, collapse and death. Hydrogen sulfide removal in industrial systems is usually done by chemical methods, which are expensive and energy consuming (Sayed et al., 2006). In this way, biological methods are considered a desirable as an alternative to chemical treatment. Clues for the development of hydrogen sulfide removal methods using biological methods are based on sulfur cycle. SRB play a huge hole in sulfur cycling and are responsible for the consumption of the most significant oxidation state of sulfur specimen in nature, the +6 oxidation state (sulfate). In anaerobic regions in aquatic environment, for example, SRB promotes the conversion of sulfate to sulfide, using the first sulfur specimen as an electron acceptor in metabolic pathways (Tang et al., 2009). In anaerobic regions where light is available, the H2S generated by SRB is used by anaerobic phototrophic bacteria (ANP) as electron donors in order to obtain energy. Chemolitotrophic sulfur-oxidizing bacteria (SOB) also utilize H2S during growth and are located in the interface between oxygen and sulfide. They can use sulfide as an electron donors and oxygen as an electron acceptor. In these sob the oxidation of sulfide usually leads to the production of phase-bright globules of elemental sulfur in the periplasm. In the environment aerobic regions, chemotrophic bacteria can obtain 0 2- their energy from oxidation of H2S and S to form SO4 (Sayed et al., 2006). Therefore, 2 interesting candidates to be used in biological H2S removal are the ANP and SOB, which can consume H2S during growth. Here H2S concentration and metal corrosion were evaluated in environmental samples and also SRB, ANP and SOB relation was studied in vitro to determine the possible effect of sulfide consumption in (1) H2S concentration in a closed system, (2) SRB growth (i.e. inhibition or stimulation) and (3) metal corrosion process. Community analysis on a sulfide rich environmental sample was performed in order to determine the diversity of ANP, SOB and SRB. Materials and Methods Sampling Samples of sediment and water were collected in Trunk River using cores. The structure of the sediment layers were observed during sampling. Only samples which had visible differentiation of layers were kept and stored in the laboratory. Micro sensor measurement Oxygen, H2S and pH profiles in the Trunk River core sample were done using Uniscience electrodes. Calibrations were performed according to each sensor manual. Corrosion of steel in the different layers of sediment sample Three stainless steel nails (GripRite Fas’Ners) were introduced in the pink and black layers of the sediment in a core of approximately 6 cm in diameter. The core was maintained in the hood under artificial light illumination. After 8 days the two nails of each sediment layer were removed from the core and fixed in formaldehyde 1% for CARD-FISH analysis. There other nail of each layer was inoculated in the gradient medium for iron oxidizing bacteria (Emerson and Floyd, 2005). Corrosion assay in microcosms A core of approximately 4 cm in diameter was also sampled in Trunk River and used in microcosms corrosion assays. The pink and black layers of the sediment were carefully separated and 5g of each layer was transferred to 50 mL glass vials. The pink layer of 3 the sediment was used for purple sulfur bacteria (PSB) enrichment; the sediment just below pink layer was used for green sulfur bacteria (GSB) and sulfur-oxidizing bacteria (SOB) enrichment. The black layer of the sediment was used for SRB enrichment. Marine phototrophic base (MPB) was used for bacterial enrichment and especial conditions were defined for each type of enrichment. The following substances were added to MPB according to the enrichment: (1) PSB enrichment sodium thiosulfate (5 mM) and sodium sulfide (1 mM) was added to MPB; (2) GSB sodium sulfide (3 mM); (3) SOB sodium nitrate (2 mM) and sodium sulfide (1 mM) was added; (4) SRB sodium sulfate (20 mM) and sodium acetate (5 mM) was added. Controls were done by the addition of filtered sterilized water from the sampling site. All enrichments and respective control (except for SOB) were incubated at 30oC. SOB enrichment and control were incubated at room temperature. PSB enrichment and control were illuminated at 850 nm and GSB enrichment and control at 770 nm. Corrosion control was performed adding MPB or filtered sterilized water from the sampling site to 50 mL glass vials. The remaining pink and black layers of the sediment were mixed and 10g of the mixed sediment was added to a 50 mL glass vial. The enrichment conditions and controls described above were maintained for PSB, GSB, SOB and SRB enrichments. DNA extraction, PCR amplification and 454 Pyrosequencing DNA extraction was done from pink and black layers of the sediment collected at Trunk River. DNA extraction was performed with the PowerSoilTM DNA isolation kit (Mo BIO laboratories, Inc.) according to the kit instructions. 454 barcode 16S PCR was performed for both pink and black layers for the sediment according to Microbial diversity laboratory manual instructions. PSB, GSB, SOB and SRB isolation and growth The bacteria used in these work have been isolated during the first weeks of the 2012 Microbial diversity summer course. ANP, SOB and BRS were gently provided by Brian Brigham, Stefan Thiele and Florence Schubotz, respectively. A colony of plates containing each bacterium was obtained and transferred to specific liquid medium 4 described in the laboratory manual. For SOB liquid medium was developed based on the overlay medium for SOB described in the laboratory manual; sodium sulfide (1 mM) and sodium nitrate (2 mM) were added to the overlay medium. Corrosion experiment The medium used in corrosion experiment in liquid and in gradient tubes was developed based on the media described for each bacterium in the laboratory manual. For the semisolid medium the sulfide agarose plug used was prepared as described in the laboratory manual (page 7.4). The overlay medium is described below: Overlay medium for SRB, PSB, GSB and SOB: 1x Sea water base 400 mL 100x NH4Cl 0.4 mL 100x Kphosphate 0.04 mL 1% resazurin 2 mL Na thiosulfate 0.48 g Na bicarbonate 2,32 g Na acetate 0.4 g Na2SO4 1,5 g NaNO3 0.2 g Cystein 0.24g 1000x Trace elements 500 l 1000x Vitamin solution 500 l Agarose 0.08% Autoclave 5 Add Na2S 1M 400 l Check pH. It should be around 7. If needed add sterile HCl or NaOH. For 400 mL of the liquid medium: the overlay medium described above was used, but agarose was omitted. Glass vials were used to add 5 mL of the liquid medium; the medium was flushed with nitrogen gas for 5 minutes, caped with butyl caps and the head space was flushed for 2 minutes with the same gas. Growth was monitored by checking turbidity and by phase-contrast microscopy. Before inoculation in liquid and semisolid medium cell quantification per mL of each culture was performed in order to add know cell concentration of H2S producing and consuming bacteria. One of each liquid and gradient medium was inoculated as follow: (1) only 2.8 x 107 SRB cells; (2) 1.9 x 107 cell of PSB and 0.9 x 107 cell of SOB (PSB + SOB will be called PSSOB); (3) PSSOB and SRB in the relation 1:1; (4) PSSOB and SRB in the relation 1:3.