Microbiology of Spent Nuclear Fuel Storage Basins

Microbiological studies of spent nuclear fuel storage basins at Savannah River Site (SRS) were performed as a preliminary step to elucidate the potential for microbial-influenced corrosion (MIC) in these facilities. Total direct counts and culturable counts performed during a 2-year period indicated microbial densities of 10⁴ to 10⁷ cells/ml in water samples and on submerged metal coupons collected from these basins. Bacterial communities present in the basin transformed between 15% and 89% of the compounds present in Biolog™ plates. Additionally, the presence of several biocorrosion-relevant microbial groups (i.e., sulfate-reducing bacteria and acid-producing bacteria) was detected with commercially available test kits. Scanning electron microscopy and X-ray spectra analysis of osmium tetroxide-stained coupons demonstrated the development of microbial biofilm communities on some metal coupons submerged for 3 weeks in storage basins. After 12 months, coupons were fully covered by biofilms, with some deterioration of the coupon surface evident at the microscopical level. These results suggest that, despite the oligotrophic and radiological environment of the SRS storage basins and the active water deionization treatments commonly applied to prevent electrochemical corrosion in these facilities, these conditions do not prevent microbial colonization and survival. Such microbial densities and wide diversity of carbon source utilization reflect the ability of the microbial populations to adapt to these environments. The presumptive presence of sulfate-reducing bacteria and acid-producing bacteria and the development of biofilms on submerged coupons indicated that an environment for MIC of metal components in the storage basins may occur. However, to date, there has been no indication or evidence of MIC in the basins. Basin chemistry control and corrosion surveillance programs instituted several years ago have substantially abated all corrosion mechanisms. Received: 4 April 1998 / Accepted: 7 July 1998     

Microbiological induced corrosion of AA 6061 nuclear alloy in highly diluted media by Bacillus cereus RE 10

Microbiological studies of a spent nuclear fuel pool in Argentina were performed to evaluate the risk of microbiological induced corrosion and determine the cultivable bacterial population. Based on standard methods and sequencing of the 16sRNA gene, eighteen microorganisms were identified. Bacillus cereus RE 10 was the predominant organism isolated, and was selected to investigate the biofilm formation process and the corrosion effect on aluminum alloy AA 6061 and on pure aluminum (Al 99.999%). To simulate the environmental conditions, the experiments were performed using a highly diluted medium. After 20 days of exposure, major pits covered with deposits were found on AA 6061 samples exposed to B. cereus RE 10 but not on Al 99.999%. There was a close correlation between biofilm patches, corrosion deposits, pitting and Al-(Fe or Ti)-Si inclusions. We postulate that this correlation is a consequence of a light local alkalinization around the inclusions that produces changes in the expression pattern of B. cereus RE 10 and allows bacterial survival using other substrates. Under these conditions, the generated biofilm induces a crevice corrosion effect around the intermetallic inclusions of the alloy. Our results will be useful for further studies related to the microbial impact on nuclear safety in nuclear waste storage facilities in Argentina.     

Bacterial corrosion of iron in seawater in situ, and in aerobic and anaerobic model systems

Abstract A screening of twenty-two marine isolates was made to examine their effects on corrosion of carbon steel ASTM A619. In batch cultures, sixteen of the isolates gave a lower corrosion than the control. Aerobic and anaerobic biofilm populations were formed by immersing iron coupons in natural seawater under aerobic and anaerobic conditions. The effects of the biofilms depended on a balance between the presence of oxygen and the type of population. An anaerobic population attached to the surface increased the corrosion rate if immersed in a suspension of Vibrio sp. DW1. The vibrio population probably 'protected' the anaerobic population from oxygen and may have provided nutrients, thereby creating conditions that allowed production of corrosive metabolites close to the metal. In contrast, coupons without a biofilm showed a decrease in the corrosion when immersed in the same vibrio suspension. The protective effect of a dense suspension of bacteria found earlier [5,6] was tested in situ in seawater. Iron coupons were immersed in dialysis bags with a suspension of Vibrio sp. DW1. Coupons immersed in dialysis bags with DW1 showed a lower degree of corrosion than coupons immersed in bags with seawater.     

Microbial diversity in the anaerobic tank of a full-scale produced water treatment plant

Microbial characteristics in the anaerobic tank of a full-scale produced water treatment plant capable of anaerobic hydrocarbon removal were analyzed and compared to those in the influent produced water using cultivation-independent molecular methods. Clones related to methanogens including the methylotrophic Methanomethylovorans thermophila and hydrogen- and the formate-utilizing Methanolinea tarda were in abundance in both samples, but greater numbers of M. tarda-like clones were detected in the biofilm library. Both DGGE and cloning analysis results indicated that the archaea in the biofilm were derived from the influent produced water. Bacterial communities in the influent and biofilm samples were significantly different. Epsilonproteobacteria was the dominant bacterial group in the influent while Nitrospira and Deltaproteobacteria were the predominant groups in the biofilm. Many clones related to syntrophic bacteria were found among the Deltaproteobacteria. One Deltaproteobacteria clone was related to Syntrophus, which is commonly found in methanogenic hydrocarbon-degrading consortia. A number of Deltaproteobacteria clones were assigned to the clone cluster group TA, members of which predominate in various methanogenic consortia that degrade aromatic compounds. These results suggest that a microbial community associated with methanogenic hydrocarbon degradation may have been established in the biofilm.     

Atomic force microscopy examination of the topography of a hydrated bacterial biofilm on a copper surface

A bacterium, designated CCI#8, that was isolated from a corroded copper coupon colonized both polished and unpolished copper surfaces under batch culture conditions. Atomic Force Microscopy (AFM) images revealed that the biofilm was heterogeneous in nature, both in depth and in cell distribution. Bacterial cells were shown to be associated with pits on the surface of the unpolished copper coupons. These observations support previous studies that CCI#8 is associated with the pitting corrosion of copper.     

Importance of biofilm formation for corrosion inhibition of SAE 1018 steel by axenic aerobic biofilms

To investigate if corrosion inhibition by aerobic biofilms is a general phenomenon, carbon steel (SAE 1018) coupons were exposed to a complex liquid medium (Luria–Bertani) and seawater-mimicking medium (VNSS) containing fifteen different pure-culture bacterial suspensions representing seven genera. Compared to sterile controls, the mass loss in the presence of these bacteria (which are capable of developing a biofilm to various degrees) decreased by 2- to 15-fold. The extent of corrosion inhibition in LB medium depended on the nature of the biofilm: an increased proportion of live cells, observed with confocal scanning laser microscopy (CSLM) and image analysis, decreased corrosion. Corrosion inhibition in LB medium was greatest with Pseudomonas putida (good biofilm formation), while metal coupons exposed to Streptomyces lividans in LB medium (poor biofilm formation) corroded in a manner similar to the sterile controls. Pseudomonas mendocina KR1 reduced corrosion the most in VNSS. It appears that only a small layer of active, respiring cells is required to inhibit corrosion, and the corrosion inhibition observed is due to the attached biofilm. Received 09 December 1996/ Accepted in revised form 19 March 1997     

The corrosion behaviour of galvanized steel in cooling tower water containing a biocide and a corrosion inhibitor

The corrosion behaviour of galvanized steel in cooling tower water containing a biocide and a corrosion inhibitor was investigated over a 10-month period in a hotel. Planktonic and sessile numbers of sulphate reducing bacteria (SRB) and heterotrophic bacteria were monitored. The corrosion rate was determined by the weight loss method. The corrosion products were analyzed by energy dispersive X-ray spectroscopy and X-ray diffraction. A mineralized, heterogeneous biofilm was observed on the coupons. Although a biocide and a corrosion inhibitor were regularly added to the cooling water, the results showed that microorganisms, such as SRB in the mixed species biofilm, caused corrosion of galvanized steel. It was observed that Zn layers on the test coupons were completely depleted after 3?months. The Fe concentrations in the biofilm showed significant correlations with the weight loss and carbohydrate concentration (respectively, p?<?0.01 and p?<?0.01).     

Extracellular Electron Transfer Is a Bottleneck in the Microbiologically Influenced Corrosion of C1018 Carbon Steel by the Biofilm of Sulfate-Reducing Bacterium Desulfovibrio vulgaris

Carbon steels are widely used in the oil and gas industry from downhole tubing to transport trunk lines. Microbes form biofilms, some of which cause the so-called microbiologically influenced corrosion (MIC) of carbon steels. MIC by sulfate reducing bacteria (SRB) is often a leading cause in MIC failures. Electrogenic SRB sessile cells harvest extracellular electrons from elemental iron oxidation for energy production in their metabolism. A previous study suggested that electron mediators riboflavin and flavin adenine dinucleotide (FAD) both accelerated the MIC of 304 stainless steel by the Desulfovibrio vulgaris biofilm that is a corrosive SRB biofilm. Compared with stainless steels, carbon steels are usually far more prone to SRB attacks because SRB biofilms form much denser biofilms on carbon steel surfaces with a sessile cell density that is two orders of magnitude higher. In this work, C1018 carbon steel coupons were used in tests of MIC by D. vulgaris with and without an electron mediator. Experimental weight loss and pit depth data conclusively confirmed that both riboflavin and FAD were able to accelerate D. vulgaris attack against the carbon steel considerably. It has important implications in MIC failure analysis and MIC mitigation in the oil and gas industry.     

Inhibiting mild steel corrosion from sulfate-reducing and iron-oxidizing bacteria using gramicidin-S-producing biofilms

A gramicidin-S-producing Bacillus brevis 18-3 biofilm was shown to reduce corrosion rates of mild steel by inhibiting both the sulfate-reducing bacterium Desulfosporosinus orientis and the iron-oxidizing bacterium Leptothrix discophora SP-6. When L. discophora SP-6 was introduced along with D. orientis to a non-antimicrobial-producing biofilm control, Paenibacillus polymyxa ATCC 10401, a corrosive synergy was created and mild steel coupons underwent more severe corrosion than when only D. orientis was present, showing a 2.3-fold increase via electrochemical impedance spectroscopy (EIS) and a 1.8-fold difference via mass-loss measurements. However, when a gramicidin-S-producing, protective B. brevis 18-3 biofilm was established on mild steel, the metal coupons were protected against the simultaneous attack of D. orientis and L. discophora SP-6. EIS data showed that the protective B. brevis 18-3 biofilm decreased the corrosion rate about 20-fold compared with the non-gramicidin-producing P. polymyxa ATCC 10401 biofilm control. The mass loss for the protected mild steel coupons was also significantly lower than that for the unprotected ones (4-fold decrease). Scanning electron microscope images corroborated the corrosion inhibition by the gramicidin-S-producing B. brevis biofilm on mild steel by showing that the metal surface remained untarnished, i.e., the polishing grooves were still visible after exposure to the simultaneous attack of the sulfate-reducing bacterium and the iron-oxidizing bacterium.     

Biofilms and corrosion interactions on stainless steel in seawater

Biofouling and biocorrosion lead to an important modification of the metal/ solution interface inducing changes in the type and concentration of ions, pH values, oxygen levels, flow velocity, etc. Metal dissolution in seawater is mainly conditioned by two different processes: (a) biofouling settlement and (b) corrosion products formation.Corrosion-resistant alloys such as stainless steel present an ideal substratum for microbial colonization, rather similar to inert non-metallic surfaces, due to the lack of corrosion products. Stainless steels are sensitive to pitting and other types of localized corrosion in chloride-containing media such as seawater. Biofilms and bacterial metabolism may accelerate the initiation of crevice attack by depletion of oxygen in the crevice solution due to microbial respiration. Bacterial colonization occurs within a period of 24–72 h on stainless steel samples exposed to natural seawater and, depending on environmental conditions, a copious and patchy biofilm is generally formed.Different interpretations of biofilms' effects on corrosion are critically discussed. A practical case, involving polluted harbour seawater, is reported to illustrate biofilm and corrosion interactions on stainless steel samples.     

Diverse bacterial groups are associated with corrosive lesions at a Granite Mountain Record Vault (GMRV)

Aims: This study applied culture‐dependent and molecular approaches to examine the bacterial communities at corrosion sites at Granite Mountain Record Vault (GMRV) in Utah, USA, with the goal of understanding the role of microbes in these unexpected corrosion events. Methods and Results: Samples from corroded steel chunks, rock particles and waters around the corrosion pits were collected for bacterial isolation and molecular analyses. Bacteria cultivated from these sites were identified as members of Alphaproteobacteria, Gammaproteobacteria, Firmicutes and Actinobacteria. In addition, molecular genetic characterization of the communities via nested‐polymerase chain reaction‐denaturing gradient gel electrophoresis (DGGE) indicated the presence of a broad spectrum of bacterial groups, including Alphaproteobacteria, Betaproteobacteria, Deltaproteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. However, neither cultivation nor molecular approaches identified sulfate‐reducing bacteria (SRB), the bacteria commonly implicated as causative organisms were found associated with corrosive lesions in a process referred to as microbially influenced corrosion (MIC). The high diversity of bacterial groups at the corrosion sites in comparison with that seen in the source waters suggested to us a role for the microbes in corrosion, perhaps being an expression of a redox‐active group of microbes transferring electrons, harvesting energy and producing biomass. Conclusions: The corrosion sites contained highly diverse microbial communities, consistent with the involvement of microbial activities along the redox gradient at corrosion interface. We hypothesize an electron transport model for MIC, involving diverse bacterial groups such as acid‐producing bacteria (APB), SRB, sulfur‐oxidizing bacteria (SOB), metal‐reducing bacteria (MRB) and metal‐oxidizing bacteria (MOB). Significance and Impact of the Study: The characterization of micro‐organisms that influence metal‐concrete corrosion at GMRV has significant implications for corrosion control in high‐altitude freshwater environments. MIC provides a potential opportunity to further our understandings of extracellular electron transfer and interspecies communications.     

Influence of sulfate reducing bacterial biofilm on corrosion behavior of low-alloy,high-strength steel (API-5L X80)

The utilization of high strength carbon steels in oil and gas transportation systems has recently increased. This work investigates microbiologically influenced corrosion (MIC) of API 5L X80 linepipe steel by sulfate reducing bacteria (SRB). The biofilm and pit morphology that developed with time were characterized with field emission scanning electron microscopy (FESEM). In addition, electrochemical impedance spectroscopy (EIS), polarization resistance (R_p) and open circuit potential (OCP) were used to analyze the corrosion behavior. Through circuit modeling, EIS results were used to interpret the physicoelectric interactions between the electrode, biofilm and solution interfaces. The results confirmed that the extensive localized corrosion activity of SRB is due to a formed biofilm and a porous iron sulfide layer on the metal surface. Energy Dispersive Spectroscopy (EDS) revealed the presence of different sulfide and oxide constituents in the corrosion products for the system exposed to SRB.     

Effect of separator and inoculum type on electricity generation and microbial community in single-chamber microbial fuel cells

Single-chamber microbial fuel cell (SMFC)-I consisted of 4 separator-electrode assemblies (SEAs) with two types of cation exchange membrane (CEM: Nafion and CMI 7000) and an anion exchange membrane (AEM: AMI 7001). SMFC-II consisted of 4 SEAs with Nafion and three types of nonwoven fabric. SMFC-I and -II were inoculated with anaerobic digested and activated sludge, respectively, and operated under fed-batch mode. In SMFC I, AEM-SEA showed a maximum power density (PD_max). Nafion-SEA showed a PD_max in SMFC II, which was similar to that of Nafion–SEA of SMFC I. Although different bacteria were developed in SMFC-I (Deltaproteobacteria and Firmicutes) and SMFC-II (Gammaproteobacteria, Betaproteobacteria and Bacteroidetes), the inoculum type little affects electricity generation. Variations of pH and oxygen in biofilm have influenced microbial community structure and electricity generation according to the electrode and separator material. Although the electricity generation of non-woven fabric-SEA was less than that of Nafion-SEA, the use of non-woven fabrics is expected to reduce the construction and operating costs of MFCs.     

Experimental investigation of microbiologically influenced corrosion of selected steels in sugarcane juice environment

In the current study, ferritic stainless grades AISI 439 and AISI 444 were investigated as possible construction materials for machinery and equipment in the cane-sugar industry. Their performance in corrosive cane-sugar juice environment was compared with the presently used low carbon steel AISI 1010 and austenitic stainless steel AISI 304. The Tafel plot electrochemical technique was used to evaluate general corrosion performance. Microbiologically influenced corrosion (MIC) behaviour in sugarcane juice environment was studied. Four microbial colonies were isolated from the biofilms on the metal coupon surfaces on the basis of their different morphology. These were characterized as Brevibacillus parabrevis, Bacillus azotoformans, Paenibacillus lautus and Micrococcus sp. The results of SEM micrographs showed that AISI 439 and AISI 304 grades had suffered maximum localized corrosion. MIC investigations revealed that AISI 444 steel had the best corrosion resistance among the tested materials. However from the Tafel plots it was evident that AISI 1010 had the least corrosion resistance and AISI 439 the best corrosion resistance.     

Biofilm Colonization Dynamics and Its Influence on the Corrosion Resistance of Austenitic UNS S31603 Stainless Steel Exposed to Gulf of Mexico Seawater

Viable bacterial counts, chemical markers, phospholipid fatty acid analysis (PLFA), and Fourier-transformed infrared spectroscopy (FTIR), together with electrochemical methods, were used to study biofilm dynamics and its impact on the corrosion resistance of UNS S31603 stainless steels exposed to the Gulf of Mexico seawater. Biofilms progressively accumulated, peaking on day 20, but finally detached. The extracellular polysaccharide (EPS)/cellular biomass ratio remained low most of the time, but reached its highest level (4.2 ± 1.9) also on day 20. Viable bacterial cells reached their highest abundance earlier (∼800 CFU/cm²), on day 15. Biofilms were seen covering the stainless steel surfaces heterogeneously and were composed mainly of gram-negative rods, presumably EPS-producing bacteria. Despite the different levels of biofilm biomass and attachment state, field-exposed steel coupons ennobled significantly and showed more active pitting potentials (∼+500 mV_SCE) than on the abiotic control (+650 mV_SCE), where no significant ennoblement occurred. These results suggest that the heterogeneous distribution of biofilms, as opposed to the quantity of surface-associated biomass, promotes formation of differential aeration cells, and that this in turn contributes to the ennoblement of these steels.     

Microbially influenced corrosion of galvanized steel pipes in aerobic water systems

Aims: To investigate the role of heterotrophic bacteria in the corrosion of galvanized steel in the presence of water. Methods and Results: Samples were taken from corroding galvanized steel pipes conveying water for specialist applications, and heterotrophic bacteria were isolated and cultured. The majority of bacteria were Gram‐negative aerobes and included Pseudomonas sp., Bacillus pumilus, Afipia spp. and Blastobacter denitrificans/Bradyrhizobium japonicum. Zinc tolerance was assessed through growth and zinc disc diffusion experiments. In general, zinc negatively influenced growth rates. An unidentified yeast also isolated from the system demonstrated a high tolerance to zinc at concentrations up to 4 g l^?1. Coupon experiments were performed to assess corrosion by the bacteria on galvanized steel and steel coupons. The majority of isolates as pure culture biofilms (69%) accelerated corrosion of galvanized coupons, assessed as zinc release, relative to sterile control coupons (P < 0·05). Pure culture biofilms did not increase the corrosion of steel, with four isolates demonstrating protective effects. Conclusions: Pure culture biofilms of heterotrophic bacteria isolated from a corroding galvanized pipe system were found to accelerate the corrosion of galvanized steel coupons. Significance and Impact of the Study: Microbially influenced corrosion is a potential contributor to sporadically occurring failures in galvanized steel systems containing water. Management strategies should consider microbial control as a means for corrosion prevention in these systems.     

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

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.     

Microbially initiated pitting on 316L stainless steel

Pitting corrosion of 316L stainless steel ennobled in the presence of manganese-oxidizing bacteria, Leptothrix discophora, was studied in a low-concentration sodium chloride solution. Corrosion coupons were first exposed to the microorganisms in a batch reactor until ennoblement occurred, then sodium chloride was added, which initiated pitting. The pits had aspect ratios (length divided by width) and shapes closely resembling the aspect ratio and the shape of the bacteria, which suggested that the microorganisms were involved in pit initiation.     

Metal corrosion by aerobic bacteria isolated from stimulated corrosion systems: Effects of additional nitrate sources

Many microorganisms are reported to influence the corrosive behaviour of mild steel and stainless steel in different habitats. In this study, 40 bacterial strains were isolated from corroded mild steel and stainless steel coupons in the nitrate supplemented environments. The corrosion abilities of the isolates against the mild steel and stainless steel coupons were tested with or without additional nitrate sources. The presence of bacterial isolates alone stimulated the corrosion of mild steel coupons. Most of the bio-corrosion processes of mild steel coupons were mitigated by adding nitrate supplement with bacterial isolates. The effects of bacterial isolates and additional nitrogen sources on corrosion of stainless steels were varied. Not all bacterial isolates stimulated the corrosion on stainless steel during the study period. Unlike the effects on mild steel coupons, additional NaNO₃ might stimulate, retard the corrosion rate by the bacterial isolates or have limited effects. Similar results were obtained when NH₄NO₃ was used. Phylogenetic analysis demonstrated that all isolates were closely related. The majority of the bacterial isolates from corroded metal coupons were identified as Bacillus species. Others were identified as Pseudomonas sp., Marinobacter sp., and Halomonas species. The results prove that the isolated aerobic microorganisms do play a role in the corrosion process of stainless and mild steel. Adding additional nitrate sources might be a tool to mitigate corrosion of mild steel which was stimulated by the presence of bacteria. However, to prevent the corrosion of stainless steels, it might need a trial and errors approach in each case.