Abstract
It is believed that iron reducing bacteria can be active in a deep underground where the overpack will be placed, being in a reducing condition with less oxygen or anaerobic environment. A buffer material (such as bentonite) surrounding the overpack will react with groundwater to produce alkaline pore water. We need some information to judge whether the iron reducing bacteria would be a factor to promote the corrosion of the overpack if iron reducing bacteria inhabits in alkaline condition. Also, it is very important to evaluate effect of anions, which promote corrosion of the carbon steel on growth of iron reducing bacteria and on the corrosion behaviour of carbon steel. In this study, we conducted microbial corrosion experiments of carbon steel both in an alkaline liquid medium and in a liquid medium with anions. The results indicate that iron reducing bacteria can be active, even where it is in alkaline conditions with or without anions.
Introduction
Japan Atomic Energy Agency has been conducting research on the long term stability of an overpack, a metallic outer container for high level waste. The overpack is one of the components of the engineered barrier system for geological disposal and is expected to prevent the high level waste from contact with groundwater for 1000 years. The primary candidate material for the overpack is carbon steel and its stability has been studied.1–4 The overpack containing vitrified waste will be placed in a deep underground which is regarded as an anaerobic environment where iron reducing bacteria can inhabit. Iron reducing bacteria works with the energy generated when it reduces Fe(III) ion in the pore water in soil to Fe(II) ion. 5 Growth of bacteria has been confirmed in the soil where iron archaeological artifacts were buried for over a millennium. 6 Therefore, we need to determine the impact of iron reducing bacteria on the corrosion behaviour of carbon steel. When overpack is placed in deep underground, the environment gets covered with bentonite. The pH of porewater in the buffer material is around 8-10. 7 Iron is relatively stable in an alkaline condition due to formation of passivation film, 8 therefore it is suggested that bentonite pore water would not greatly influence the corrosion in the iron. However, when iron reducing bacteria exists in bentonite pore water in an alkaline condition, the way this factor would alter the corrosion behaviour of carbon steel has not been investigated or studied sufficiently. Consequently, we need information about the possibility of the growth of iron reducing bacteria in alkaline conditions and the corrosion behaviour of carbon steel. The corrosion process of iron begins when iron gets ionised in the presence of water and oxygen. In this regard, if chloride ion and sulphate ion gets involved, the corrosion progresses and eventually corrosion products will be formed. 9 Chloride ion is a cause of pitting corrosion 10 and destroys the passivation film of carbon steel,11,12 which is known as a factor promoting corrosion. We need to know how the corrosion behaviour of carbon steel would change when chloride ion and iron reducing bacteria coexist.
In this study, we conducted microbial corrosion experiments using an alkaline liquid medium and a liquid medium with anions to confirm the activity of iron reducing bacteria and to determine the corrosion products and their relation to the corrosion behaviour of carbon steel.
Experimental
Experiment on microbial corrosion in carbon steel in alkaline liquid medium
We ground the carbon steel (SS 400) and defatted it with acetone in an ultrasonic cleaner. It was dry-heat sterilised at 170°C for 30 min in a constant temperature reservoir. The liquid medium used an iron reducing medium. The medium composition is as shown in Table 1. The medium got an autoclave sterilisation at 121°C for 20 min.
Chemical compositions of iron reducing medium (400 mL)
The iron reducing bacteria used for the experiments was obtained from the soil stratum in the Hinata ruins in Hitachi Ota city, Ibaraki prefecture where some iron artifacts were excavated. A dwelling site was excavated and determined to be the one from the early Tumulus period to the Heian period by examinations. The iron reducing medium (pH 6) was adjusted to be pH 8 and pH 9 by 10% (w/v) NaOH solution. We dispensed 20 mL of pH 8 iron reducing medium in vial containers, soaked carbon steel in them and added iron reducing bacterial liquid. As a sample for comparison, we dispensed 20 mL of iron reducing medium alone and soaked carbon steel in it. In the same way as above, another sample was prepared with pH 9 iron reducing medium.
In order to maintain the reducing condition necessary for the growth of iron reducing bacteria, a gas barrier bag (brand name: Escal by Mitsubishi Gas Chemical Company, Inc.) containing deoxygenating agent and dehydrating agent (brand name: RP-A by Mitsubishi Gas Chemical Company, Inc.) was placed. We cultured them at 30°C for 14 days. After the culture, we washed the surface of the carbon steel with distilled water and dried it. The products on the surface of the carbon steel were observed by an optical microscope (KEYENCE, VHX-900) and a scanning electron microscope (SEM; KEYENCE, VE9800). The product was reduced to powder and analysed by X-ray diffraction (XRD) analysis equipment (Bruker AXS Corporation, D8 ADVANCE, Target: Cu). The analysis condition was as follows. Ni filter, tube voltage: 40 kV, tube current: 20 mA, measuring range: 5-90° (2θ), measuring speed: 1 s/step.
Experiment on microbial corrosion in carbon steel in liquid medium with anions
The preparation protocol for carbon steel was the same as the section on ‘Experiment on microbial corrosion in carbon steel in alkaline liquid medium’. The iron reducing medium was prepared based on the medium composition in Table 1. NaCl solution [3% (w/v)] and Na2SO4 solution [3% (w/v)] of anions were added to the medium. Corrosion on iron is hastened the most with around 3% salt concentration. 13 Therefore, 3% (w/v) NaCl solution and 3% (w/v) Na2SO4 solution of anions were added to the medium. The iron reducing medium (20 mL) and 10 mL of 3% (w/v) NaCl solution were dispensed into vial containers. Carbon steel was soaked in the medium and iron reducing bacterial liquid was added. As a sample for comparison, 20 mL of iron reducing medium and 10 mL of 3% (w/v) NaCl solution alone were dispensed in vial containers and carbon steel was soaked there. In the same method as above, a sample was prepared with iron reducing medium and 3% (w/v) Na2SO4 solution. To maintain the reducing condition necessary for growth of the iron reducing bacteria, the same gas barrier bag, deoxygenating agent and dehydrating agent as the section on ‘Experiment on microbial corrosion in carbon steel in alkaline liquid medium’ were chosen. The culture period was 120 days at a temperature of 30°C.
Observational method and analytical method were the same as the section on ‘Experiment on microbial corrosion in carbon steel in alkaline liquid medium’. Only XRD analytical condition was different: Ni filter, tube voltage: 40 kV, tube current: 40 mA, measuring range: 5-60° (2θ), measuring speed: 1 s/step.
Results and discussion
Microbial corrosion experiment for carbon steel in alkaline liquid medium
While the iron reducing medium was being composed, it looked yellow since iron citrate hydrate was added as a nutrient for iron reducing bacteria. After the culture, only the pH 8 and pH 9 mediums appeared colourless with iron reducing bacteria added. It was considered that the colour change of the medium was brought by iron reducing bacteria growing in alkaline medium. We confirmed that iron reducing bacteria can grow in both pH 8 and pH 9 mediums. See Fig. 1 for the iron reducing mediums before and after the culture.
Change in iron reducing medium with iron reducing bacteria before and after culture
As a result of observation on the carbon steel surface by a SEM, oval-shaped iron reducing bacteria (1-2 μm in length) were identified (Fig. 2). Bacteria are classified according to
Iron reducing bacteria are 1·6 μm in size and long and thin oval at both ends
oxygen tolerance
nutrient requirement
metabolism of bacteria
shape
gram staining and so on.
The result of the experiment did not show any differences in the form of the iron reducing bacteria14,15 observed in iron reducing medium in neutral condition. However, in this result, we could not find any differences from the iron reducing bacterial group cultured in neutral condition.
Microbe begins to multiply when environmental conditions such as temperature, pH and oxygen partial pressure and nutritional conditions such as carbon source and nitrogen source get prepared. It is said that once multiplication starts, it makes rapid progress. 16 One of the reasons iron reducing bacteria can be active in the alkaline condition of this experiment is the nutrient supply for the bacteria from the medium. This result shows that iron reducing bacteria can be active if there is an iron reducing bacterial nutrient in an alkaline condition. It is considered that even though alkaline pore water was produced from the buffer material of the overpack installed deep underground, microbial corrosion of carbon steel would be difficult to take place unless nutritional conditions for iron reducing bacteria get prepared. However, we cannot deny the possibility that iron reducing bacteria would grow and influence the corrosive activities in carbon steel, when burying environment for the overpack meets growth conditions (nutrients, pH, oxygen level, etc.).
According to the XRD analysis, vivianite-group phosphate [vivianite (Fe3(PO4)2.8H2O) or baricite ((Mg, Fe, Mn)3(PO4)2.8H2O] and magnetite (Fe3O4) were detected in the carbon steel soaked in both pH 8 and pH 9 mediums with iron reducing bacteria added (Figs. 3a and 4a). The broad peak at the region of 2θ = 15-35° indicates the presence of amorphous phase. On the other hand, regardless of the medium pH, only Fe was detected in the carbon steel soaked in the medium without iron reducing bacteria and the corrosion in the carbon steel made little progress (see Figs. 3b and 4b). In the medium with iron reducing bacteria, as products magnetite was detected regardless of the medium pH.
a medium with iron reducing bacteria; b medium without iron reducing bacteria
a medium with iron reducing bacteria; b medium without iron reducing bacteria
It is considered that the formation of magnetite in the medium with iron reducing bacteria was influenced by carbon steel and iron citrate. The carbon steel soaked in the medium generates Fe(II) ion in the corrosion process (Formula 1). In the medium with iron reducing bacteria, the activities of iron reducing bacteria help Fe(II) ion reduce Fe(III) ion which is iron citrate hydrate in the medium component. In the culture medium, FeOOH is generated in the corrosion process but FeOOH is reduced to magnetite by Fe(II) ion supply. Magnetite, being stable in reducing condition, would contribute to corrosion control for the overpack (such as carbon steel) installed deep underground. The formulas below show the production process of magnetite
Experiment on microbial corrosion in carbon steel in liquid medium with anions
Culturing iron reducing medium with iron reducing bacteria resulted in the medium being colourless regardless of the kind of anions in the medium (chloride ion, sulphate ion). It was found that under the circumstance with nutrients for iron reducing bacteria, both 3% chloride ion and 3% sulphate ion would not influence the activities of iron reducing bacteria. According to the XRD analysis, vivianite-group phosphate and magnetite were detected when iron reducing bacteria existed, regardless of the kind of anions. Vivianite-group phosphate was detected when iron reducing bacteria did not exist. Magnetite was detected only when iron reducing bacteria existed in the medium. Figure 5 shows the XRD pattern in the medium with chloride ion. Figure 6 shows the XRD pattern in the medium with sulphate ion.
a medium with iron reducing bacteria; b medium without iron reducing bacteria
a medium with iron reducing bacteria; b medium without iron reducing bacteria
It has been reported that β-FeOOH would be formed in the presence of chloride ion 17 and α-FeOOH and γ-FeOOH in the presence of sulphate ion.18,19 Although there are chloride ion and sulphate ion added to the medium in this study, no corrosion products in FeOOH system were detected. There could possibly be a different corrosion process in forming corrosion products in FeOOH system being in a common solution, under the experimental condition with and without iron reducing bacteria.
Conclusions
We made it clear that iron reducing bacteria can inhabit in a liquid medium both in an alkaline liquid medium and in a liquid medium with anions, and also found that neither an alkaline condition nor anions would influence the activities of iron reducing bacteria. In the range of this experiment, an alkaline condition has nothing to do with the size or form of iron reducing bacteria. It has been found that iron reducing bacteria can inhabit if there are nutrients for iron reducing bacteria in bentonite pore water in an alkaline condition, and that magnetite can form in the presence of iron reducing bacteria.
We found that corrosion products in FeOOH system cannot be detected when anions and iron reducing bacteria coexist and that the corrosion process is different from the one of carbon steel in a solution. When we used a liquid media with anions for a microbial experiment with iron reducing bacteria, magnetite was detected only in the experiment, and the corrosion process was different depending on whether the existence of iron reducing bacteria or not.
Footnotes
Acknowledgements
This paper presents a part of the results of a joint study ‘Microbial Corrosion Evaluation Research on Archaeological Iron Objects’ by Japan Atomic Energy Agency (JAEA) and the University of Tsukuba of Comprehensive Human Sciences. The study was supported by the JAEA Cooperative Research Scheme on the Nuclear Fuel Cycle. The authors wish to thank the staff at University of Tsukuba for their support of this study.