A Study on the Potential of Metal Corrosion by Sulfate-Reducing Bacteria in Animal Buildings

Abstract: The potential of sulfate-reducing bacteria (SRB) to cause metal corrosion in animal buildings was examined in this study. An analysis was done on the bacterial colonization and the corrosion products on the surfaces of metals exposed to three animal buildings and tone environmentally controlled building over a two-year period. Data from this study showed that the levels of SRB on metal surfaces were low after two-year's exposure (maximum count: 1.7 x 10⁴/cm²). SRB colonization levels after two years were not sufficient to corrode metal products exposed in animal environments. In addition, metal surface analysis data using X-ray photoelectron spectroscopy showed that the corrosion compounds formed on the surfaces of different metals were not due to the SRB-induced corrosion mechanisms. These compounds were mainly oxides and carbonates (FeO, Fe₂O₃, Fe₃O₄, and Fe(CO)₅ on iron samples; ZnO and ZnCo₃ on galvanized steel samples; Al₂O₃, ZnO, and ZnCO₃ on Galvalume samples, and were normally generated due to the classic types of corrosion mechanisms. Some sulfur was present to form ZnS on the galvanized steel samples, but might not be attributed to SRB. The origin of this sulfur was not clear.

Keywords. Animal housing, Sulfate reduction, Bacteria. Metal, Corrosion.

A considerable amount of research on microbial-induced corrosion has been conducted by industries that employ buried pipelines and underground structures (King and Miller, 1971; Tatnall, 1981; Iverson and Olson, 1983; Iverson, 1984; Ford and Mitchell, 1990). Since the sulfate-reducing bacteria (SRB) has been considered the major bacterial species in causing metal corrosion under anaerobic environments (Crombie et al., 1980; Pankhania, 1988; Ford and Mitchell, 1990; Hao et al., 1996), most of the past studies concentrated on the corrosion problems caused by SRB encountered in those environments. Little is known about the potential of this type of bacteria to corrode metals under aerobic situations. Recently, a few researchers have studied SRB corrosion problems in aerobic environments. Hardy and Bown (1984) suggested that the most aggressive conditions associated with SRB were those which were not entirely anaerobic, but where small quantities of oxygen might be present from time to time. Scott and Davies (1992) reported high counts of SRB on the corroded steel columns in a medical research building. They found that SRB could survive in an aerobic environment with the assistance of oxygen depletion bacteria. Zhu et al. (1994) reported findings of biofilms and the SRB counts on metal surfaces based on a preliminary survey conducted in animal buildings. The results from these studies showed that sulfate-reducing bacteria could survive and contribute to metal corrosion problems in environments with the presence of oxygen.

Feed material, dust, moisture, and animal feces are abundant in animal buildings and provide adequate nutrients for bacterial growth on metal surfaces in these buildings. No metal substratum can be totally immune from microbial colonization in animal building environments. A knowledge of the growth of SRB in animal buildings, as well as information regarding metal corrosion when exposed to these bacteria, will help estimate the probability of metal deterioration caused by these microbes and provide information on how to prevent it.

This article reports the experimental results of a study on the potential of metal corrosion by SRB during a two-year field exposure study in three animal buildings and one environmentally controlled building. The colonization, growth, and decay of SRB on metal surfaces were investigated. An analysis of the corrosion products on corroded metal surfaces using X-ray photoelectron spectroscopy was also done to determine if the corrosion products were produced by SRB.

Materials and Methods

Sample Materials and Placement

Six types of metal samples were used in the field exposure test (uncoated 1010 carbon steel, galvanized steel, prepainted G90 hot dipped galvanized steel, uncoated Galvalume, prepainted Galvalume , and pure zinc). The metal coupons were 25 mm x 63.5 mm. Ten sets of metal coupons were placed in each building. Each set consisted of six pieces (one of each metal type) so sets could be removed periodically for analysis over a period of two years.

The metal coupons were cut with a shear machine to avoid any influence on the micro-structure of metal as a result of overheating from the cutting operation. The cut areas of all the coupons were coated with MAB Rust Olastic primer (red) and then covered with MAB Rust Olastic finish coating (white) to avoid the initiation of corrosion from these disturbed areas. Coupons were suspended by nylon string through two small holes at each end to a bracket made from plexiglass. The six metal types were placed side by side in random order approximately 12.5 mm apart to ensure samples were uniformly exposed to the environment.

A total of 240 metal samples was were placed in three animal buildings (dairy loafing, poultry laying, and swine finishing) and one environmentally controlled laboratory (control). The dairy building was naturally ventilated while the poultry and swine buildings were mechanically ventilated. In each building, samples were placed at two different heights (upper level and lower level) to study the effect of variations of the environment at the different heights on bacterial colonization. The upper level was defined as immediately underneath the ceiling or roof. In the swine, poultry, and the controlled buildings, the upper level samples were placed about 30 cm below the insulated ceiling. In the dairy building, since there was no ceiling, all the upper level samples were placed horizontally at the same height level (about 2 m from the ground) so the distance between a specific sample and the inclined roof varied from location to location. The lower level was defined as about 80 to 100 cm above the floor. In the dairy building, the sample sets were placed close to the end wall of the building and a metal wire mesh was used to cover all the lower level samples to prevent them from being licked or damaged by cows. In the swine building, the lower level samples were placed immediately above the partition fence and in the poultry building, the sample brackets were suspended vertically in a wood frame immediately under the chicken cages and about 80 cm above the manure collection pit. A metal sheet was used to cover the wood frame to protect samples from being contaminated by manure falling directly on them. The lower level samples from the environmentally controlled laboratory were located in a well ventilated area, about 1 m above the floor.

Sample Analysis

For the first six months, one set of coupons was selected randomly and removed every month for analysis. For the second six months, one set of coupons was removed every three months; then one set of coupons was taken out every six months. The full test period was two years.

Bacterial Analysis

The metal samples for the bacterial analysis were brought to an anaerobic microbiology laboratory immediately after removal. The recipes for anaerobic diluent and culture media for SRB and the detailed procedures in preparing the media were presented by Zhu et al. (1994) Surface scrapings from a 1 cm2 area of each sample were homogenized and diluted to different concentrations from 10^-1 to 10^-10. Then, the solution was moved into an anaerobic cabinet. Droplets of 20uL of this solution were spotted on the nutrient agar plates. When the droplets were absorbed into the agar plates, the plates were inverted and incubated at 38°C to 39°C in a N2 cabinet for SRB counts.

After incubation for five to seven days, the number of bacterial colonies on the nutrient agar plates was counted and the bacterial concentration on the given area was calculated by multiplying the counts by the dilution level.

Analysis of Corrosion Product.

X-ray photoelectron spectroscopy (XPS) was employed to analyze the corrosion products because this technique was able to reveal the types of compounds formed on the corroded metal surfaces based on the molecular binding energies. According to Ford and Mitchell (1990). The corrosion products caused by SRB for iron due to the metabolic oxidation of hydrogen are mainly iron sulfides. Iverson (1984) proposed that the corrosion products were phosphides because of a highly corrosive metabolic product containing phosphorus which initiated a general type of corrosion. Thus, it can be inferred that the corrosion products caused by SRB will be either sulfides or phosphides, or the combination of both. Therefore, whether these corrosion compounds were present on the corroded metal surfaces would give an indication of the level of SRB corrosion.

Environment Measurements

Environmental indices that are possibly related to bacterial growth were monitored at the sample locations so their influence on the SRB-induced corrosion could be studied. The indices monitored included air temperature, relative humidity, and dust mass levels.

Temperature and relative humidity were measured continuously in the center of each building during the entire exposure period by hygrothermographs (Model 37250-00, Cole Parmer Co.). Dust mass concentrations in the air of the three animal buildings were sampled once a week and those in the controlled building were sampled biweekly using an air pump and filters (Rotary vane oilless vacuum pump from Grainger Company, Part No. 4z335, filter membrane from Nuclepore Polycarbonate, pore size: 0.1 um, diameter: 47 mm, Catalog No. 111105). Measurements were made by pumping a measured volume of air through filters and measuring the weight difference of the filter membrane before and after sampling. Air volume passing through the filters was measured using a Wet-Test Gas Meter (Precision Scientific Petroleum Instruments Co., Fisher Scientific Catalog No. 11-166-2).

Results and Discussion

Environmental Data

Unless otherwise indicated, a statistical student t test at a significance level of 0.05 was employed in the comparisons of data throughout the following text.

Temperature and Relative Humidity

The means and standard deviations of temperature and relative humidity in the three animal buildings and one environmentally controlled laboratory over the two-year period are shown in table 1.

Table 1. The means and standard deviations of dust levels and the maximum and minimum temperatures and relative humidities in the test buildings (data collected from October 1992 to October 1994)*

The temperatures in all buildings were below the optimum range for the growth of SRB, which is between 28 and 32°C (Hao et al., 1996). It would be expected that the growth rate of SRB in the southern part of the U.S. could be higher due to warm temperatures. There were significant differences in the maximum means of relative humidities between the dairy, swing, poultry, and control buildings. The rank of means from high to low was: dairy, poultry and swine, and control. There were significant differences in the minimum means of relative humidity among the buildings with a rank from high to low being: dairy, poultry, swine, and control. The dairy building had the lowest temperatures (peak mean was 17.2°C) among all the buildings over the entire test period. In addition, the dairy building showed a larger fluctuation in temperature than the other buildings (mean variation was 10.2°C).

Dust

The average dust mass concentrations during the two years in all the test buildings are presented in table 1. According to table 1 the dust mass levels were significantly different among the four exposure areas. The poultry building had the highest dust level (4.32 mg/m3), while the control had the lowest (0.05 mg/m3).

Bacterial Data

The sulfate-reducing bacterial counts on different metal samples at both upper and lower levels in all the test buildings are presented in figures 1 to 4. It can be seen from these figures that SRB, without preference for metal types, started colonizing metal surfaces immediately after the samples were placed, and maintained high growth rates on most of the test samples in the three animal buildings for the first nine months of exposure. The colonization rates decreased slightly for all the samples thereafter. In the environmentally controlled laboratory, the bacterial colonization rates showed a much lower level than those in animal buildings and leveled off after about one year's exposure. The reason why the bacterial growth on the metal surfaces reached a maximum level after a certain period of exposure time was probably due to the limited nutrients on the metal surfaces. Therefore, it could be inferred that this limitation could hinder the bacterial growth and reduce the potential of metal corrosion caused by the bacteria.

According to figures 1 to 4, the SRB counts on all the samples were low after two year's exposure (maximum: about 1.7x 10⁴/cm² for an uncoated 1010 carbon steel sample at upper level in the dairy building). In a previous study (Zhu et al., 1994), a level of 1.6 x 10⁴/cm² was observed by analyzing the surface scrapings of metal products which were assumed to be exposed in animal buildings for at least five years. Comparing the SRB counts in these two studies showed that it required at least one year for SRB to reach the presumed long-term level (five years). In addition, the trend found was that the SRB counts did not increase after one year's exposure. Thus, the next question is whether SRB counts at a level of 1.7 x 10⁴/cm² are sufficient to cause corrosion of the metals tested. Information on levels of SRB at which metals start to corrode is limited. Gaylard (1992) conducted a laboratory study on the corrosive activity of SRB. He tested mild steel coupons with dimensions of 4 cm x 1.5 cm x 0.1 com in the media where SRB were inoculated. He reported that no significant corrosion was observed until the SRB concentrations had reached about 10⁷/mL for more than 20 days. Although the two numbers (1.7 x 10⁴/cm² and 10⁷ mL) are not directly comparable due to the different units, it still can be inferred that the SRB levels in this study after a two-year period of colonization was not high. At this low level, it is doubtful that the long-term effect of SRB on metal corrosion in animal buildings would be significant.

Figure 1. Dairy building: Semi-log plot of the sulfate-reducing bacterial counts (1 cm² scrapings of metal coupons); (a) upper level, (b) lower level. Symbols: -diamond- 1010 uncoated carbon steel, -square- galvanized, -triangle- painted galvanized, -x- uncoated Galvalume, -*- paintd Galvalume, -circle- pure zinc.

Table 2 presents the means and standard deviations of the SRB counts at upper and lower levels in the different test buildings. Due to large variation in data, there were no significant differences in the SRB counts between upper and lower levels in all the test buildings. Also, there were no significant differences among the three animal buildings for any of the sampling times, although the levels of dust, temperature, and relative humidity in the four test buildings differed significantly (table 1). Therefore, it could be assumed that the growth of SRB was not directly related to the environmental conditions that were monitored in the animal buildings. The SRB counts in the control were significantly lower than those in the animal buildings after six months of exposure. Possible nutrients in the air for bacterial growth were not monitored and may have played a role in the differences in SRB growth rates between the animal buildings and the control.

Table 2. The means and standard deviations of the SRB counts at the upper and lower levels in different buildings*

Figure 2. Swine building: Semi-log plot of the sulfate-reducing bacterial counts (1 cm2 scrapings of metal coupons); (a) upper level, (b) lower level. Symbols: -diamond- 1010 uncoated carbon steel, -square- galvanized, -triangle- painted galvanized, -x- uncoated Galvalume, -*- paintd Galvalume, -circle- pure zinc.	Figure 3. Poultry Building: Semi-log plot of the sulfate-reducing bacterial counts (1 cm2 scrapings of metal coupons); (a) upper level, (b) lower level. Symbols: -diamond- 1010 uncoated carbon steel, -square- galvanized, -triangle- painted galvanized, -x- uncoated Galvalume, -*- paintd Galvalume, -circle- pure zinc.

Figure 4. Control Building: Semi-log plot of the sulfate-reducing bacterial counts (1 cm² scrapings of metal coupons); (a) upper level, (b) lower level. Symbols: -diamond- 1010 uncoated carbon steel, -square- galvanized, -triangle- painted galvanized, -x- uncoated Galvalume, -*- paintd Galvalume, -circle- pure zinc.

Sample Surface Analysis (XPS) Data

A visual check was performed on all test samples. When cleaned of dirt, both galvanized steel and Galvalume samples showed little corrosion, although they were less shiny due to some kind of oxide film formed on the surfaces (most likely ZnCO3 and Al2O3). There were no visible pits found on the surfaces. Uncoated 1010 carbon steel samples had a uniform rust layer covering the entire area. The cleaned surfaces of these samples showed that they were evenly attacked. However, pure zinc samples were corroded only in the localized form, i.e., pits. The corrosion was not uniform. The deepest pit measured about 0.1mm on upper level samples from the dairy building. There was no visible damage to the prepainted samples.

A total of nine samples from the three animal buildings were studied using the XPS technique (uncoated 1010 carbon steel from upper levels of dairy, swine, poultry, and control; galvanized steel from upper levels of dairy, swine, and poultry; uncoated 1010 carbon steel and Galvalume from lower level of poultry). The poultry lower level samples were examined because of the highly adverse environment at that exposure area (right above the fresh manure). The samples were exposed for 1.5 years. Detailed XPS data for different metal samples are presented in the following sub-sections.

Uncoated 1010 Carbon Steel

A surface survey revealed identical chemical elements on the uncoated 1010 carbon steel samples among the different buildings. These elements included carbon (C), oxygen (O), iron (Fe), and nitrogen (N). There was no nitrogen found on the samples from the controlled laboratory and poultry lower level. The binding energies for these samples are listed in table 3.

According to Iverson (1984) and Ford and Mitchell (1990), the corrosion products caused by SRB included either FeS, FeP, or both. Since no sulfur and phosphorus were detected on these samples by XPS, it could be concluded that the corrosion products of uncoated 1010 carbon steel after 1.5 year's exposure in animal buildings were not produced by SRB.

Since the binding energy for iron compounds was about 710 eV (table 3), all potential compounds with binding energies close to 710 eV in the Handbook of X-ray Photoelectron Spectroscopy (Perkin, 1993) were examined. These compounds include Fe2O3 (710.9 eV), FeO (709.4 eV), Fe3O4 (710.4 eV), and Fe(CO)5 (709.6 eV). To further determine the types of compounds, the binding energies of oxygen and carbon were checked. For iron oxides, the binding energies for oxygen ranged from 529.6 eV to 530.8 eV. For iron carbonates, the binding energies ranged from 288.0 eV to 288.2 eV. These numbers were very close to those listed in table 3. Thus, it appeared that the most likely compounds existing on the surfaces of uncoated 1010 carbon steel samples from all test buildings were Fe2O3, Fe4O4, FeO, and Fe(CO)5. Based on the above analysis, it can be concluded that the corrosion of uncoated 1010 carbon steel samples could be classified as the classic corrosion type (rust). There were no compounds matching the binding energies of nitrogen so the existence of nitrogen based corrosion compounds seemed questionable according to this analysis. The source of nitrogen on metal surfaces was not determined in this study.

Galvanized Steel and Galvalume.

Data from the surface survey for different elements are listed in table 4. The presence of ZnS on the surface of galvanized steel was fairly certain because the binding energies listed in the columns of zinc and sulfur in table 4 were consistent with those values in the Handbook (Perkin, 1993), in which the binding energies for Zn and S were 1022.0 eV and 164.0 eV, respectively. Although phosphorus was detected on a Galvalume sample in the poultry building, it did not match any binding energies of the rest of the elements in table 4 related to the potential compounds. Thus, the existence of phosphides can be eliminated.

One point that needs to be mentioned here is why there were no iron sulfides and aluminum sulfides found on uncoated 1010 carbon steel and Galvalume samples, while there was ZnS found on galvanized samples. It is not believed that this was caused by SRB because, according to past research (Iverson, 1987), aluminum was more susceptible to SRB corrosion due to its higher oxidation potential than zinc. Thus, if SRB did play a role in corroding these samples, aluminum should be the first victim. However, no aluminum-sulfur compounds were detected in this study. The sulfur which formed ZnS on galvanized steel could be from sources other that SRB. Since sulfur in the test environment was not measured, the origin of this sulfur was unknown. Furthermore, why sulfur would only attack galvanized steel is not clear.

According to Perkin (1993), the binding energies of carbon for carbonates ranged from 290.0 eV to 291.5 eV, and the binding energies of oxygen for carbonates ranged from roughly 530.5 eV to 531.5 eV. Thus, the values in table 4 showed that the carbon and oxygen could exist in some forms of carbonates. Since a thin film of zinc carbonate would normally form and protect fresh zinc underneath from reacting further with the outside corrosives, it could be assumed that the unknown carbonate on the surfaces of galvanized and Galvalume samples was most likely zinc carbonate. Also, there was a possibility of the presence of ZnO because the binding energy of oxygen for ZnO listed in the Handbook (Perkin, 1993) was 530.4 eV.

For the Galvalume sample from the poultry lower level, the binding energy of aluminum in table 4 closely matched that of Al2O3 (74.4 eV) in the Handbook (Perkin, 1993). So the presence of Al2O3 on Galvalume was certain, which was produced by the same corrosion mechanism as that under atmospheric conditions. Since Galvalume is composed of 55% Al and 45% Z, the other corrosion products on Galvalume samples should include ZnO and ZnCo3 according to table 4 and the Handbook (Perkin, 1993). No compounds with nitrogen were found.

Conclusions

1. Data of environmental indices and bacterial colonization in this study showed that the levels of dust, temperature, and relative humidity did not have a direct effect of the sulfate-reducing bacterial colonization rates. Based on the cultures of 1 cm2 scrapings from sample surfaces, the sulfate-reducing bacterial levels in all the test buildings were low after two year's exposure (maximum: 1.7 x 10⁴/cm²) and did not show signs of further increase thereafter. Since the growth for sulfate-reducing bacteria on metal surfaces was limited (due possibly to the lack of sufficient nutrients), it is not believed that this type of bacteria can cause significant metal corrosion in animal buildings.
2. According to the X-ray Photoelectron Spectroscopy analysis, the corrosion products on uncoated 1010 carbon steel samples were mainly Fe2O3, Fe3O4, FeO, and Fe(CO)5. The corrosion products on galvanized steel and Galvalume consisted on ZnO, ZnS, ZnCO3 and A2O3 (on Galvalume only). Since no SRB-related mechanisms of corrosion were found in this study in the formation of these corrosion compounds, it could be concluded that the contribution of SRB to the metal corrosions in all test buildings is insignificant.
3. The Zinc sulfides found on the galvanized steel samples could be due to other corrosion mechanisms. More research is needed to determine the sulfur source and the potential effects of ZnS on the service life of the galvanized steel.

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