Pauliina Rajala

Rapid Reactivation of Deep Subsurface Microbes in the Presence of C-1 Compounds

Microorganisms in the deep biosphere are believed to conduct little metabolic activity due to low nutrient availability in these environments. However, destructive penetration to long-isolated bedrock environments during construction of underground waste repositories can lead to increased nutrient availability and potentially affect the long-term stability of the repository systems, Here, we studied how microorganisms present in fracture fluid from a depth of 500 m in Outokumpu, Finland, respond to simple carbon compounds (C-1 compounds) in the presence or absence of sulphate as an electron acceptor. C-1 compounds such as methane and methanol are important intermediates in the deep subsurface carbon cycle, and electron acceptors such as sulphate are critical components of oxidation processes. Fracture fluid samples were incubated in vitro with either methane or methanol in the presence or absence of sulphate as an electron acceptor. Metabolic response was measured by staining the microbial cells with fluorescent dyes that indicate metabolic activity and transcriptional response with RT-qPCR. Our results show that deep subsurface microbes exist in dormant states but rapidly reactivate their transcription and respiration systems in the presence of C-1 substrates, particularly methane. Microbial activity was further enhanced by the addition of sulphate as an electron acceptor. Sulphate- and nitrate-reducing microbes were particularly responsive to the addition of C-1 compounds and sulphate. These taxa are common in deep biosphere environments and may be affected by conditions disturbed by bedrock intrusion, as from drilling and excavation for long-term storage of hazardous waste.

Microbially induced corrosion of carbon steel in deep groundwater environment

The metallic low and intermediate level radioactive waste generally consists of carbon steel and stainless steels. The corrosion rate of carbon steel in deep groundwater is typically low, unless the water is very acidic or microbial activity in the environment is high. Therefore, the assessment of microbially induced corrosion of carbon steel in deep bedrock environment has become important for evaluating the safety of disposal of radioactive waste. Here we studied the corrosion inducing ability of indigenous microbial community from a deep bedrock aquifer. Carbon steel coupons were exposed to anoxic groundwater from repository site 100 m depth (Olkiluoto, Finland) for periods of 3 and 8 months. The experiments were conducted at both in situ temperature and room temperature to investigate the response of microbial population to elevated temperature. Our results demonstrate that microorganisms from the deep bedrock aquifer benefit from carbon steel introduced to the nutrient poor anoxic deep groundwater environment. In the groundwater incubated with carbon steel the planktonic microbial community was more diverse and 100-fold more abundant compared to the environment without carbon steel. The betaproteobacteria were the most dominant bacterial class in all samples where carbon steel was present, whereas in groundwater incubated without carbon steel the microbial community had clearly less diversity. Microorganisms induced pitting corrosion and were found to cluster inside the corrosion pits. Temperature had an effect on the species composition of microbial community and also affected the corrosion deposits layer formed on the surface of carbon steel.

Influence of Chlorination and Choice of Materials on Fouling in Cooling Water System under Brackish Seawater Conditions

Cooling systems remove heat from components and industrial equipment. Water cooling, employing natural waters, is typically used for cooling large industrial facilities, such as power plants, factories or refineries. Due to moderate temperatures, cooling water cycles are susceptible to biofouling, inorganic fouling and scaling, which may reduce heat transfer and enhance corrosion. Hypochlorite treatment or antifouling coatings are used to prevent biological fouling in these systems. In this research, we examine biofouling and materials’ degradation in a brackish seawater environment using a range of test materials, both uncoated and coated. The fouling and corrosion resistance of titanium alloy (Ti-6Al-4V), super austenitic stainless steel (254SMO) and epoxy-coated carbon steel (Intershield Inerta160) were studied in the absence and presence of hypochlorite. Our results demonstrate that biological fouling is intensive in cooling systems using brackish seawater in sub-arctic areas. The microfouling comprised a vast diversity of bacteria, archaea, fungi, algae and protozoa. Chlorination was effective against biological fouling: up to a 10–1000-fold decrease in bacterial and archaeal numbers was detected. Chlorination also changed the diversity of the biofilm-forming community. Nevertheless, our results also suggest that chlorination enhances cracking of the epoxy coating.

Nitrate and ammonia as nitrogen sources for deep subsurface microorganisms

We investigated the N-utilizing bacterial community in anoxic brackish groundwater of the low and intermediate level nuclear waste repository cave in Olkiluoto, Finland, at 100 m depth using 15N-based stable isotope probing (SIP) and enrichment with 14∕15N-ammonium or 14∕15N-nitrate complemented with methane. Twenty-eight days of incubation at 12°C increased the concentration of bacterial 16S rRNA and nitrate reductase (narG) gene copies in the substrate amended microcosms simultaneously with a radical drop in the overall bacterial diversity and OTU richness. Hydrogenophaga/Malikia were enriched in all substrate amended microcosms and Methylobacter in the ammonium and ammonium+methane supplemented microcosms. Sulfuricurvum was especially abundant in the nitrate+methane treatment and the unamended incubation control. Membrane-bound nitrate reductase genes (narG) from Polarimonas sp. were detected in the original groundwater, while Burkholderia, Methylibium, and Pseudomonas narG genes were enriched due to substrate supplements. Identified amoA genes belonged to Nitrosomonas sp. 15N-SIP revealed that Burkholderiales and Rhizobiales clades belonging to the minority groups in the original groundwater used 15N from ammonium and nitrate as N source indicating an important ecological function of these bacteria, despite their low number, in the groundwater N cycle in Olkiluoto bedrock system.

Reactivation of Deep Subsurface Microbial Community in Response to Methane or Methanol Amendment

Microbial communities in deep subsurface environments comprise a large portion of Earth’s biomass, but the microbial activity in these habitats is largely unknown. Here, we studied how microorganisms from two isolated groundwater fractures at 180 and 500 m depths of the Outokumpu Deep Drillhole (Finland) responded to methane or methanol amendment, in the presence or absence of sulfate as an additional electron acceptor. Methane is a plausible intermediate in the deep subsurface carbon cycle, and electron acceptors such as sulfate are critical components for oxidation processes. In fact, the majority of the available carbon in the Outokumpu deep biosphere is present as methane. Methanol is an intermediate of methane oxidation, but may also be produced through degradation of organic matter. The fracture fluid samples were incubated in vitro with methane or methanol in the presence or absence of sulfate as electron acceptor. The metabolic response of microbial communities was measured by staining the microbial cells with fluorescent redox sensitive dye combined with flow cytometry, and DNA or cDNA-derived amplicon sequencing. The microbial community of the fracture zone at the 180 m depth was originally considerably more respiratory active and 10-fold more numerous (105 cells ml-1 at 180 m depth and 104 cells ml-1 at 500 m depth) than the community of the fracture zone at the 500 m. However, the dormant microbial community at the 500 m depth rapidly reactivated their transcription and respiration systems in the presence of methane or methanol, whereas in the shallower fracture zone only a small sub-population was able to utilize the newly available carbon source. In addition, the composition of substrate activated microbial communities differed at both depths from original microbial communities. The results demonstrate that OTUs representing minor groups of the total microbial communities play an important role when microbial communities face changes in environmental conditions.

Microbial fouling and corrosion of carbon steel in deep anoxic alkaline groundwater

Abstract Understanding the corrosion of carbon steel materials of low and intermediate level radioactive waste under repository conditions is crucial to ensure the safe storage of radioactive contaminated materials. The waste will be in contact with the concrete of repository silos and storage containers, and eventually with groundwater. In this study, the corrosion of carbon steel under repository conditions as well as the microbial community forming biofilm on the carbon steel samples, consisting of bacteria, archaea, and fungi, was studied over a period of three years in a groundwater environment with and without inserted concrete. The number of biofilm forming bacteria and archaea was 1,000-fold lower, with corrosion rates 620-times lower in the presence of concrete compared to the natural groundwater environment. However, localized corrosion was detected in the concrete–groundwater environment indicating the presence of local microenvironments where the conditions for pitting corrosion were favorable.

Ennoblement, corrosion, and biofouling in brackish seawater: Comparison between six stainless steel grades

In this work, six common stainless steel grades were compared with respect to ennoblement characteristics, corrosion performance and tendency to biofouling in brackish sea water in a pilot-scale cooling water circuit. Two tests were performed, each employing three test materials, until differences between the materials were detected. Open circuit potential (OCP) was measured continuously in situ. Potentiodynamic polarization and electrochemical impedance spectroscopy (EIS) measurements were conducted before and after the tests. Exposed specimens were further subjected to examinations by scanning electron microscopy (SEM) and energy-dispersive spectroscopy (EDS), and the biofouling was studied using epifluorescence microscopy, quantitative polymerase chain reaction (qPCR) and high-throughput sequencing (HTP sequencing). The results revealed dissimilarities between the stainless steel grades in corrosion behaviour and biofouling tendency. The test material that differed from the most of the other studied alloys was grade EN 1.4162. It experienced fastest and most efficient ennoblement of OCP, its passive area shrank to the greatest extent and the cathodic reaction was accelerated to a significant degree by the development of biofilm. Furthermore, microbiological analyses revealed that bacterial community on EN 1.4162 was dominated by Actinobacteria, whereas on the other five test materials Proteobacteria was the main bacterial phylum.

Influence of Carbon Sources and Concrete on Microbiologically Influenced Corrosion of Carbon Steel in Subterranean Groundwater Environment

Microbiologically influenced corrosion of carbon steel was assessed in a laboratory environment simulating the deep geological repository of radioactive waste. A dense and diverse biofilm was forme...

CO 2 and carbonate as substrate for the activation of the microbial community in 180 m deep bedrock fracture fluid of Outokumpu Deep Drill Hole, Finland

Microbial communities in deep subsurface environments comprise a large portion of Earths biomass, but the metabolic activities in these habitats are largely unknown. Here the effect of CO2 and carbonate on the microbial community of an isolated groundwater fracture zone at 180 m depth of the Outokumpu Deep Scientific Drill Hole (Finland) was tested. Outokumpu groundwater at 180 m depth contains approximately 0.45 L L−1 dissolved gas of which methane contributes 76%. CO2, on the other hand, is scarce. The number of microbial cells with intracellular activity in the groundwater was low when examined with redox staining. Fluorescence Assisted Cell Sorting (FACS) analyses indicated that only 1% of the microbial community stained active with the redox sensing dye in the untreated groundwater after 4 weeks of starvation. However, carbon substrate and sulfate addition increased the abundance of fluorescent cells up to 7%. CO2 and CO2 + sulfate activated the greatest number of microbes, especially increasing the abundance of Pseudomonas sp., which otherwise was present at only low abundance in Outokumpu. Over longer exposure time (2 months) up to 50% of the bacterial cells in the groundwater were shown to incorporate inorganic carbon from carbonate into biomass. Carbon recapture is an important feature in this ecosystem since it may decrease the rate of carbon loss in form of CO2 released from cellular processes.