Periodic Reporting for period 1 - MOLMIC (Molecular Biology of Sulfide-Oxidizing Nitrate-Reducing Microorganisms Involved in Microbiologically-Influenced Corrosion) Reporting period: 2016-01-06 to 2018-01-05 Summary of the context and overall objectives of the project Modern society is striving to orient our energy use towards sustainable ‘green’ energy sources, however, this transition is occurring slowly. Due to the increasing energy demand, fossil fuels will remain crucial for several years during the transition phase towards ‘greener’ solutions. The inevitable depletion of fossil fuel reserves and the devastating effects of oil spills to ecosystem services requires a responsible and sustainable development of the remaining resources during this transition period. Corrosion of steel infrastructure is a multi-billion Euro problem for the oil and gas industry, which can lead to significant costs due to equipment failure as well as risk to the environment. Microorganism can significantly influence corrosion reactions on steel surfaces and this phenomenon is referred to as microbiologically-influenced corrosion (MIC). MIC in the oil industry is often linked to activity of sulfate-reducing microorganisms (SRM), which produce toxic, explosive and corrosive hydrogen sulfide, thus contributing to oil reservoir souring. The formation of sulfide poses a significant threat for worker’s health and safety and decreases product values due to higher sulfur content. Nitrate injection into sour oil fields biologically removes hydrogen sulfide by promoting activity of sulfide-oxidizing nitrate-reducing microorganisms (soNRM). However, recent reports involved soNRM in MIC, threatening application of nitrate as a souring control strategy. The MOLMIC project addressed the Molecular Biology of Sulfide-Oxidizing Nitrate-Reducing Microorganisms Involved in Microbiologically-Influenced Corrosion. The project developed i) a sound understanding of the sulfur metabolism of oil field soNRM, ii) linked different soNRM metabolisms to corrosion and iii) evaluated nitrate-mediated MIC in complex microbial communities. MOLMIC developed an unprecedented understanding of the factors and mechanisms by which soNRMs contribute to corrosion during the injection of nitrate. This information increased our understanding of this bioengineering strategy and is invaluable for the development of targeted gene assays to monitor soNRM activity where nitrate-mediated corrosion might be an issue. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far MOLMIC investigated the role of sulfide-oxidizing nitrate-reducing microorganisms (soNRM) during microbiologically influenced corrosion. The project focused on determine the key-factors by which soNRM can cause corrosion and to identify the enzymatic mechanisms behind their sulfur and nitrogen metabolisms that cause accumulation of corrosive metabolites.The soNRM metabolism was studied by analyzing the formation of key metabolites and by sequencing the genomes of different oil field soNRMs. Furthermore, the genes likely involved in the conversion of sulfur and nitrogen compounds in soNRM was studied by genome-wide gene expression analysis to reveal which genes that are turned on during nitrate-mediated souring control. The effects on corrosion and the response of soNRM to corrosion was investigated by exposing steel coupons to growing soNRM cultures, determining the weight loss corrosion rates and comparing the expression of genes in corroding cultures with expression in non-corroding cultures. The results obtained from those pure cultures was supplemented by experiments with complex microbial communities. The effect on corrosion of souring and nitrate addition was studied with microbial communities retrieved from brackish and coastal sediments, simulating onshore and offshore water injection systems, respectively. The effects on the community structure and the expression of key genes was assessed by RNA sequencing of steel coupon biofilm samples and compared to microorganisms in the liquid phase of the cultures. The analysis showed that corrosion by soNRM is mainly mediated by the formation of biogenic sulfur particles during oxidation of sulfide and accumulation of nitrite further increased the corrosion by nearly 50%. Based on detailed analysis of key corrosion factors a conceptual model for soNRM corrosion have been proposed. Gene expression analysis showed that all tested soNRM use the same system to oxidize sulfide and form biogenic sulfur particles. Further oxidation of the biogenic sulfur seems to be more complicated and required at least the presence and expression of four genes of a sulfur oxidation multi-enzyme system. In addition, the presence of steel coupons resulted in expression of several genes in soNRM, which are involved in hydrogen metabolism or heavy metal resistance. The expression of those genes was likely triggered by accumulation of ferrous iron and hydrogen due to ongoing corrosion. The results and insights from MOLMIC have been presented to researchers from industry and academia at numerous conferences, as well as to students and the general public at different events and lecturers. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) Oil fields are complex ecosystems inhabited by phylogenetically and physiologically diverse microorganisms. The common detection of Epsilonproteobacteria (a phylum which contains various soNRM) in oil reservoirs and production facilities, implies a potentially significant corrosion risk during souring control with nitrate. While the biochemical pathways and genes involved in S oxidation have been extensively studied in other bacterial lineages, information from Epsilonproteobacteria was still scarce at the onset of MOLMIC. The project expanded our understanding of the metabolism of epsilonproteobacterial soNRM by providing novel genomes and metagenomes from oil systems. Furthermore, the project obtained unprecedented insights into the genetic mechanisms behind accumulation of different corrosive sulfur and nitrogen compounds by obtaining several (meta)transcriptomic datasets during nitrate-mediated souring control.The end products of soNRM metabolism vary depending on the prevailing level of sulfide (i.e. the extent of souring) and nitrate (i.e. nitrate-dosing strategy). MOLMIC showed that irrespective of the soNRM tested or nitrate to sulfide ratio applied, accumulated of biogenic sulfur was unavoidable. However, the amount of nitrate added had a profound impact on the corrosion when nitrite accumulated at high nitrate doses. The results obtained from numerous experiments performed during MOLMIC have been used to develop a conceptual model of how the activity of soNRM can potentially influence corrosion and identified several key-factors that can sometimes dramatically affect the overall corrosion. The genetic and (bio)chemical data obtained from oil field soNRM increased our understanding of this important component of nitrate-mediated souring control and will improve our understanding and prediction of metabolic processes in oil systems. This will likely improve the ‘green’ bioengineering strategy of nitrate injection for future oil productions, by potentially decreasing both the risk for corrosion and the economic costs arising from unexpected nitrate-mediated corrosion. Conceptual model of corrosion caused by sulfide oxidizing nitrate reducing microorganisms
