Fernanda , A. Metal Corrosion and Biological H2S Cycling in Closed

Home , Euryarchaeota, Nitrospirae, Thermocladium, Thermodesulfobacteria

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

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. Controls which were maintained in dark were prepared. All cultures were maintained at 30oC at 850 nm. Controls were incubated at the same condition, but were covered with aluminum foil.

Evaluation of H2S concentration and SRB growth in the medium

The colorimetric method described in the laboratory manual was used to determine de

H2S concentration on selected microcosms and on liquid cultures. For estimation of SRB growth CARD-FISH was performed according to the laboratory manual using deltabacteria probes and its competitors (probes: Delta495-a; Delta495-B; Delta495-c).

Results and Discussion

Microcosms observations

Steel nail corrosion in the core

During core storage it was possible to see the growth of a green layer on the surface of the sediment (Figure 1a). Oxygen and hydrogen sulfide profile showed that the interface between water and sediment is an anaerobic environment with high

6 concentration of sulfide (Figure 1b). The steel nails which were at the top of the sediment were not corroded and still had a shine appearance after 8 days (Figure 1b and d). Portions of the nails in the first centimeters of the sediment were black, indication formation of iron sulfide, which is the product of corrosion by H2S (Figure 1c). After 8 days, bottom nails which were introduced in the deeper black layers of the sediment were retrieved and these were completely black (Figure 1e).

Figure 1 (A) Environmental sample from Trunk River showing where the steel nail were inserted; (B) Detail of the nail showing the Black area around it after 8 days (possibly FeS); Nail retrieved from top layer; (D) Nail retrieved from Bottom layer; (E) Oxygen , (F) Sulfide, (G) pH profile of the core sample. Black lines in the profiles represent the interface between water and sediment

One nail from each sediment layer was fixed in formaldehyde 1% and the cylindrical surface of the nail was completely scraped. The material obtained from scraping the nail was washed in PBS and filtered. A slice of the filter was stained with DAPI and cells quantification showed that the nail from the top and bottom layer of the sediment had approximately 4.2 x 104 and 1.1 x 105 cells/cm3, respectively (assuming the nail as a cylinder), suggesting that biofilm formation is more rapidly formed on the bottom layers of the sediment. It was also interesting to observe different cell morphologies

7 that could be observed exclusively on the top layers (Figure 2). In the nail sample from bottom layer of the sediment several cells are attached to metal pieces, supporting the idea that biofilm formation was more efficiently formed in this sediment layer (Figure 2d).

5 m

Figure 2 Fluorescence microscopy of nail samples from the (A and B) pink and (C and D) black layers of the core. In the first sample numerous filamentous bacteria could be observed (A); in the sample from deeper regions several bacteria were attached to metal particles, indicating biofilm formation (D).

Despite the fact that lost of weight was not observed in nails from neither layers, results indicate that corrosion is favored in the bottom layers of the sediment, where

H2S concentration is higher and were the SRB population is probably more abundant and forming biofilms.

Nails from both the top and bottom layers were washed in sterile PBS and inoculated on iron oxidizing bacteria gradient medium (Emerson and Floyd, 2005). After one week it was possible to see a white band formation in the nail from the top layer (arrow).

Rust was observed in both cases at the flat portion of the nail (arrowhead, Figure 3). Phase contrast microscopy of the top nail culture showed a variety in cell morphotypes; in the bottom nail cultured rod-shaped bacteria were the dominant (Figure3).

B C

5 m

Figure 3 (A) Gradient medium for iron oxidizing bacteria (Emerson and Floyd, 2005); (B) Tubes which where inoculated with the nails from the top and bottom layers of the core; (C) Microscopy image of the culture inoculated with the top nail; (D) Microscopy image of the culture inoculated with the top nail.

Steel nail corrosion in enrichment microcosms

After 14 days of incubation the appearance of the steel nail and the color and turbidity of the microcosms were different among each other depending on the type of enrichment. For PSB enrichment in microcosms with the mixed sediment and with the pink layer the nail characteristics were preserved and the liquid and sediment layers were pink, indicating the growth of the target bacteria. PSB microcosms controls (that means no enrichment) with mixed sediment layers and pink sediment presented

9 different results in relation to nail preservation and microcosms characteristics. The nail inside the microcosm containing the mixed sediment layers was very damaged by corrosion and rust formation was observed (Figure 4). The sulfide quantification in the microcosms with mixed sediment layers (control and enrichment for PSB) indicates similar levels of the gas, suggesting that the corrosion observed in the control might be caused by the action of other bacteria. An aliquot of the control microcosm where rust was observed was inoculated on iron oxidizing gradient medium described by Emerson and Floyd (2005). After 7 days a white band was formed in the tube and microscopy observation confirmed the presence of bacterial growth (cocci and spirilla) (Figure 5). In the microcosm containing the pink layer sediment corrosion was not observed for both enrichment and control. The presence of pink pigmentation was only observed in the enrichment microcosm.

Figure 4 (A and B) Different microcosms used to evaluate metal corrosion and possible prevention by PSB; (C) Comparison between medium and water controls and PSB enrichment and control.

5 m

Figure 5 (A) Gradient tube for iron oxidizing bacteria isolation; (B) Phase-contrast microscopy of the white band showed in (A).

In the microscosms for GSB enrichment turbidity and green color in the liquid phase was observed for both mixed sediment layers and sediment fraction below pink layer, suggesting GSB growth. Corrosion in the nail and rust was only detected on the GSB enrichment microcosm containing mixed sediment layers. However this corrosion might not be related to SRB H2S production because H2S concentration was low (Figure 9). Controls for GSB enrichments showed similar results; both with liquid phase clear and no apparent corrosion (Figure 6).

Figure 6 (A and B) Different microcosms used to evaluate metal corrosion and possible prevention by GSB; (C) Comparison GSB enrichment and control from mixed sediment layers samples.

In the microcosms for SOB enrichment nail changes in appearance were similar, they had some black portions along the nail, but no rust formation was observed. Nails from both enrichments were completely black, suggesting the production of H2S, which indicates SRB growth. In the mixed sediment microcosms a green color was observed in both control and enrichment. Possibly GSB grew in these microcosms because the inoculated sample had some H2S and this sample was maintained at room temperature in the bench, so there was light availability (Figure 7).

Figure 7 (A and B) Different microcosms used to evaluate metal corrosion and possible prevention by SOB; (C) Comparison SOB enrichment and control from mixed sediment layers samples.

For SRB enrichments, the nails in the microcosms containing the black layer of the core sediment (enrichment and control) were completely black, suggesting that this is the maximum of steel corrosion that can happen in the time frame used in this study. Interestingly, nails containing the mixed sediment layers from the core were not completely corroded, indicating that H2S concentrations were lower than the microcosms containing the black layer only. Possible explanations for this corrosion result are related to lower growth rate of SRB because of the presence of populations from the pink layer or simply by H2S consumption by bacteria which are present in the pink layers of the sediment (Figure 8). Sulfide concentration in each of the microcosms containing mixed sediment layers are in agreement with the observed nail corrosion aspects (Figure 9).

Figure 8 (A and B) Different microcosms used to evaluate metal corrosion and possible prevention by SRB; (C) Comparison SRB enrichment and control from mixed sediment layers samples.

Figure 9 Sulfide concentrations in microcosms with mixed sediment layers.

CARD-FISH was performed on selected microcosms containing mixed sediment layers based on different nail corrosion characteristics. Deltaproteobacteria quantification based on CARD-FISH results suggests that SRB are more abundant in the PSB enrichment (Figure 10). SRB are in low number in the microcosm where rust was formed, indication that the corrosion of the nail in this condition was generated by other bacteria. Oxygen presence in this microcosm is not discarded because the rust that was observed in usually indicative of iron oxidizing bacteria activity. In the microscosm of PSB enrichment the number of bacteria labeled with the Deltaproteobacteria probe was higher, suggesting higher number of SRB. This suggests that PSB are able to consume H2S and prevent corrosion.

Figure 10: Quantification of Deltaproteobacteria (CARD-FISH) 6.0×104

4.0×104

cells/mL 2.0×104

PSB control

PSB enrichment GSB enrichment Culture related results

Cell quantification in cultures for determination of the initial inoculums in the gradient and liquid media

After 7 days of growth in the appropriate culture conditions described in materials and methods section, cell quantification was performed based on DAPI staining. GSB didn’t grow so well, so it was not used in the gradient and liquid cultures. According to these results the inoculums were determine for the experiments of steel wire corrosion and

H2S production in gradient and anaerobic liquid cultures as mentioned in the materials and methods section.

CARD-FISH using deltaproteobacteria probe was performed in liquid cultures after 7 days of inoculum to check if the relative abundance of SRB was maintained according to inoculation or if it has any fluctuations. According to these results SRB growth is not affected by PSB and SOB populations. As expected SRB abundance is higher in the cultures maintained at dark. Interestingly, the inoculums of 1:3 (SRB: H2S consumers), had an increase in SRB population when compared with 1:1 inoculum. One explanation to this observation could be related to a stimulus in SRB growth because of H2S consumption (Figure 12).

Figure 11 Relative abundance of SRB in different inoculums conditions.

Sulfide concentration measurements showed that H2S concentration was higher in the cultures maintained under dark, suggesting that SOB population was not enough to lower sulfide concentration (Figure 12).

Figure 13 Sulfide concentrations in the liquid cultures.

In the gradient tubes inoculated only with SRB the oxidized fraction on the top of the medium was completely reduce in a few days, indicating SRB growth and H2S production. Culture tubes with 1:1 and 3:1(SRB: PSSOB) were similar in growth and turbidity. No band formation was observed in any of these tubes, but an irregular growth was seen around the steel wire (Figure 13). CARD-FISH using deltaproteobacteria probe indicate that PSSOB and SRB are growing on the gradient tubes (Figure 13). Unfortunately total and SRB quantification was not possible because of the high background of DAPI on the images. Cultures which were maintained in dark conditions had to be transferred to light conditions because those that were illuminated were accidentally autoclaved, so H2S and oxygen measurements along the gradient media were not compared between light and dark conditions.

A B

Figure 13 (A) Gradient tubes for corrosion experiment; (B) CARD-FISH image of semisolid medium inoculated with SRB.

Corrosion and rust were not observed in any of the culture conditions described above. These processes might take more time in these conditions (cultures), because SRB might have to reach high densities to have a corrosive effect on steel.

454 Pyrosequencing

A preliminary analysis of 454 16S pyrosequencing showed that PSB are very abundant in top layer of the sediment. Despite the fact that the color of the sediment already gives the idea that PSB are very abundant in this microenvironment, I wasn’t expecting the observed dominance (Figure 14a). The Deltaproteobacteria diversity on the black layer of the sediment seems to be distributed. The dominant group in this case is Desulfobacteraceae (Figure 14b).

Conclusions

According to the results observed in this preliminary study, PSB and GSB are capable to decrease H2S concentration in the environment. In experiments related to PSB enrichment metal corrosion was also prevented. Many research groups are aiming in the use of these bacteria to decrease sulfide concentration in industrial systems. A good review on the subject is Sayed et al., 2006.

Acknowledgments

I would like to thank the course co-directors Dan Buckley and Steve Zinder, the course coordinator Asheley Campbell and all the instructors, especially Sara Kleindienst, Verena Salman. I also would like to thank Suzanna Brauer for helping me with media development and all my course friends. MBL, Daniel S. and Edith T. Grosch Scholarship Fund, The Gordon & Betty Moore Foundation and CNPq are acknowledged.

References

Emerson, D. and Floyd, M.M. Enrichment and isolation of iron-oxidizing bacteria at neutral pH. Methods Enzymol. 397:112-23.

Muyzer, G. and Stams, A. J. M. 2008. The ecology and biotechnology of sulphate- reducing bacteria. Nature Reviews Microbiology, vol. 6, no. 6, pp. 441–454.

Sayed, M., Soreanu, G., Falleta, P. and Beland, M. 2006. Removal of hydrogen sulfide from gas streams using biological processes-A review, Canadian Biosystems Engineering, 48, 2.2-2.14.

Tang, K., Baskaran, V. and Nemati. M. 2009. Bacteria of the sulphur cycle: An overview of microbiology, biokinetics and their role in petroleum and mining industries. Biochemical Engineering Journal, vol. 44, no1, pp. 73–94.