The influence of nitrate on the short-term corrosion of steel catalyzed by marine microorganisms

Microbiologically influenced corrosion (MIC) is of global concern and a threat due to its economic, environmental as well as public health implications. Identifying the factors that govern or influence MIC is beneficial in predicting such effects and for taking early preventive measures. Although the basic process of MIC is electrochemical, when biotic components and environmental factors are included, MIC becomes a collection of complex interactions that requires individual components to be identified and studied in order to understand their effects. This dissertation focuses on the effect of elevated levels of nitrate on the MIC of steel exposed to seawater. Metal exposed to seawater is known to be susceptible to MIC. Based on the analysis of field data of steel pilings exposed to coastal marine waters, Professor Robert Melchers put forward an empirical multi-phase phenomenological model of steel corrosion. The model proposes two periods where MIC occurs whereby the rate of corrosion depends on bacterial metabolic activity. The activity of the corrosive microbes is in turn thought to be largely a function of the nutrient supply. Oftentimes, the major nutrient limiting bacterial activity in seawater is dissolved inorganic nitrogen (DIN), most notably nitrate, ammonium and nitrite concentrations. The overall objective of my study was to investigate the effect of elevated levels of nitrate on marine biofilms that are associated with the corrosion of carbon steel. Based on the phenomenological model, the hypothesis was that nitrate enhances corrosion by general stimulation of microbial activity. The first section of the dissertation interrogated the microbial communities of biofilms on carbon steel coupons exposed to shallow warm marine waters at four locations around the globe. Metagenomic analysis was performed with DNA extracted from the biofilms growing on coupons immersed at each location. Two of the locations (#5 and #7) had normal and relatively low levels of nitrate while the marine waters off the locations #4 and #6 had an unusually high level of dissolved inorganic nitrogen (DIN) (2.4 – 2.6 mg/L). However, the largest weight losses and rates of corrosion were independent of the DIN status of the ambient seawaters, thus the results did not support the hypothesis. Location #6 had the highest rate of general corrosion, as measured by weight loss by mild steel coupons, but both location #5 and #6 had more severe pitting corrosion, indicative of MIC. It was subsequently hypothesized that the high DIN levels may have enriched for nitrate-reducing sulfate-reducing bacteria that led to relatively high sulfide-driven corrosion rates in location #6. Bacterial communities of both locations #4 and #6 had relatively high biomass based on DNA concentration as well as 16S qPCR. Moreover, they had higher frequencies of nitrate/nitrite reducing gene reads compared to the other two sites. The taxonomic analysis of nitrogen cycling genes revealed that high levels of nitrate was associated with the increase of nitrifying, facultative anaerobic as well as aerobic Alphaproteobacteria. The biofilms from location # 6 had a higher relative abundance of Deltaproteobacteria (majority were Desulfovibrio) and sulfur-oxidizing bacteria that is at least consistent with the higher corrosion rate compared to the other locations, but few of the sulfate-reducing bacteria also had nitrate reduction genes. Therefore, the second hypothesis was also disproven. Additionally, the relative frequency of ccoN genes (an indicator of aerobic respiration potential) was relatively high in the location #4 compared to the samples of other locations. The effect of elevated levels of nitrate on carbon steel corrosion was also tested with the common marine organism Marinobacter hydrocarbonoclasticus SP17 as the biotic component. In light of unpublished observations we hypothesized that Marinobacter biofilms protect 1018 carbon steel under aerobic conditions. Furthermore, it was hypothesized that the biofilm cells inhibit or reduce corrosion specifically by the removal of oxygen. Carbon steel coupons were exposed to Marinobacter cells that were amended with nitrate or ammonium as the nitrogen source and incubated under aerated (unsealed) or oxygen limited (sealed) conditions. The weight loss of coupons with biofilm was significantly lower than that without a biofilm regardless of the nitrogen source or oxygen supply status. The coupon weight loss difference was significant between the sealed (oxygen-limited) and unsealed (oxygen-unlimited) incubations, with less weight lost from coupons incubated under oxygen-limited conditions. Dissolved ferrous iron measurements as an indicator of corrosion showed the same trends as measurements of weight loss with less corrosion associated with coupons with biofilms compared to biofilm-free coupons, regardless of the oxygen and nitrogen status, i.e., dissolved iron measurements were lower with coupons in sealed (limited-oxygen) treatments compared to unsealed treatments with or without a biofilm. The coupon weight loss and the dissolved ferrous iron measurements did not exhibit significant differences between abiotic treatments with nitrate, ammonium or no nitrogen source added. The observations supported the hypothesis that Marinobacter biofilm protects 1018 carbon steel under aerobic conditions. The most likely mechanism for corrosion inhibition by the organism appeared to be oxygen respiration thereby limiting the ability of this gas to reach and interact with the coupon surface. In any event, the study did not support the hypothesis of enhanced corrosion in the presence of elevated levels of DIN, either nitrate or ammonium. This dissertation relied on both field and laboratory experiments to interrogate the impact of elevated levels of nitrate in seawater on carbon steel corrosion. The observations did not support the contention that elevated levels of nitrate were associated with higher corrosion. Further, high levels of nitrate compared to ammonium as a DIN source were also not associated with significantly more corrosion in the presence of a common marine heterotrophic bacterium. The metagenomic analyses of biofilms from the field support the hypothesis of a generic stimulation of microbial proliferation. However, since both locations with high DIN did not experience same amount of corrosion and microbial activity was not tested, it does not explain increases in corrosion as proposed by Melchers. Admittedly, the examination of only four marine coastal areas can hardly be considered exhaustive. Similarly, the impact of a pure culture heterotrophic bacterium that is capable of utilizing nitrogen under both aerobic and anaerobic conditions can only partially reflect the many intricacies of entire marine microbial communities. However, the observations described herein suggest that the presence of high DIN or nitrate in local marine environment cannot be used as the reliable indicator of high corrosion rates as selection for nitrate-reducing microbes can include those that enhance MIC and those that do not. Additionally, the use of nitrate/nitrite reducing gene frequency and/or nitrogen cycling genes as a marker for corrosion should be reconsidered