Identification of algal rich microbial blooms in the Sellafield Pile Fuel Storage Pond and the application of ultrasonic treatment to control the formation of blooms.

The presence of microorganisms in a range of nuclear facilities has been known for many years. In this study the microbial community inhabiting the Pile Fuel Storage Pond (PFSP), which is a legacy open-aired facility on the Sellafield nuclear site, Cumbria, UK, was determined to help target microbial bloom management strategies in this facility. The PFSP is currently undergoing decommissioning and the development of prolonged dense microbial blooms reduces the visibility within the water. Such impairment in the pond water visibility can lead to delays in pond operations, which also has financial implications. Efforts to control the microbial population within the PFSP are ongoing, with the installation of ultrasonic treatment units. Here next generation sequencing techniques focussing on broad targets for both eukaryotic and prokaryotic organisms were used to identify the microbial community. On-site monitoring of photosynthetic pigments indicated when microbial blooms formed and that eukaryotic algae were most likely to be responsible for these events. The sequencing data suggested that the blooms were dominated by members of the class Chrysophyceae, a group of golden algae, while evidence of cyanobacteria and other photosynthetic bacteria was limited, further supporting eukaryotic organisms causing the blooms. The results of sequencing data from 2018 was used to inform a change in the operational settings of the ultrasonic units, while monitoring of the microbial community and photosynthetic pigments trends was extended. Since the changes were made to the ultrasonic treatment, the visibility in the pond was significantly improved, with an absence of a spring bloom in 2020 and an overall reduction in the number of days lost due to microbial blooms annually. This work extends our knowledge of the diversity of microbes able to colonise nuclear fuel storage ponds, and also suggests that sequencing data can help to optimise the performance of ultrasonic treatments, to control algal proliferation in the PFSP facility and other inhospitable engineered systems.

The interplay between Cs and K in Pseudanabaena catenata; from microbial bloom control strategies to bioremediation options for radioactive waters.

Pseudanabaena dominates cyanobacterial blooms in the First-Generation Magnox Storage Pond (FGMSP) at a UK nuclear site. The fission product Cs is a radiologically significant radionuclide in the pond, and understanding the interactions between Cs and Pseudanabaena spp. is therefore important for determining facility management strategies, as well as improving understanding of microbiological responses to this non-essential chemical analogue of K. This study evaluated the fate of Cs following interactions with Pseudanabaena catenata, a laboratory strain most closely related to that dominating FGMSP blooms. Experiments showed that Cs (1 mM) exposure did not affect the growth of P. catenata, while a high concentration of K (5 mM) caused a significant reduction in cell yield. Scanning transmission X-ray microscopy elemental mapping identified Cs accumulation to discrete cytoplasmic locations within P. catenata cells, indicating a potential bioremediation option for Cs. Proteins related to stress responses and nutrient limitation (K, P) were stimulated by Cs treatment. Furthermore, selected K+ transport proteins were mis-regulated by Cs dosing, which indicates the importance of the K+ transport system for Cs accumulation. These findings enhance understanding of Cs fate and biological responses within Pseudanabaena blooms, and indicate that K exposure might provide a microbial bloom control strategy.

Identification of a Stable Hydrogen-Driven Microbiome in a Highly Radioactive Storage Facility on the Sellafield Site.

The use of nuclear power has been a significant part of the United Kingdom's energy portfolio with the Sellafield site being used for power production and more recently reprocessing and decommissioning of spent nuclear fuel activities. Before being reprocessed, spent nuclear fuel is stored in water ponds with significant levels of background radioactivity and in high alkalinity (to minimize fuel corrosion). Despite these challenging conditions, the presence of microbial communities has been detected. To gain further insight into the microbial communities present in extreme environments, an indoor, hyper-alkaline, oligotrophic, and radioactive spent fuel storage pond (INP) located on the Sellafield site was analyzed. Water samples were collected from sample points within the INP complex, and also the purge water feeding tank (FT) that supplies water to the pond, and were screened for the presence of the 16S and 18S rRNA genes to inform sequencing requirements over a period of 30 months. Only 16S rRNA genes were successfully amplified for sequencing, suggesting that the microbial communities in the INP were dominated by prokaryotes. Quantitative Polymerase Chain Reaction (qPCR) analysis targeting 16S rRNA genes suggested that bacterial cells in the order of 104-106 mL-1 were present in the samples, with loadings rising with time. Next generation Illumina MiSeq sequencing was performed to identify the dominant microorganisms at eight sampling times. The 16S rRNA gene sequence analysis suggested that 70% and 91% from of the OTUs samples, from the FT and INP respectively, belonged to the phylum Proteobacteria, mainly from the alpha and beta subclasses. The remaining OTUs were assigned primarily to the phyla Acidobacteria, Bacteroidetes, and, Cyanobacteria. Overall the most abundant genera identified were Hydrogenophaga, Curvibacter, Porphyrobacter, Rhodoferax, Polaromonas, Sediminibacterium, Roseococcus, and Sphingomonas. The presence of organisms most closely related to Hydrogenophaga species in the INP areas, suggests the metabolism of hydrogen as an energy source, most likely linked to hydrolysis of water caused by the stored fuel. Isolation of axenic cultures using a range of minimal and rich media was also attempted, but only relatively minor components (from the phylum Bacteroidetes) of the pond water communities were obtained, emphasizing the importance of DNA-based, not culture-dependent techniques, for assessing the microbiome of nuclear facilities.

Radiation Tolerance of Pseudanabaena catenata, a Cyanobacterium Relevant to the First Generation Magnox Storage Pond.

Recently a species of Pseudanabaena was identified as the dominant photosynthetic organism during a bloom event in a high pH (pH ∼11.4), radioactive spent nuclear fuel pond (SNFP) at the Sellafield Ltd., United Kingdom facility. The metabolic response of a laboratory culture containing the cyanobacterium Pseudanabaena catenata, a relative of the major photosynthetic microorganism found in the SNFP, to X-ray irradiation was studied to identify potential survival strategies used to support colonization of radioactive environments. Growth was monitored and the metabolic fingerprints of the cultures, during irradiation and throughout the post-irradiation recovery period, were determined using Fourier transform infrared (FT-IR) spectroscopy. A dose of 95 Gy delivered over 5 days did not significantly affect growth of P. catenata, as determined by turbidity measurements and cell counts. Multivariate statistical analysis of the FT-IR spectral data revealed metabolic variation during the post-irradiation recovery period, with increased polysaccharide and decreased amide spectral intensities. Increases in polysaccharides were confirmed by complementary analytical methods including total carbohydrate assays and calcofluor white staining. This observed increased production of polysaccharides is of significance, since this could have an impact on the fate of the radionuclide inventory in the pond via biosorption of cationic radionuclides, and may also impact on downstream processes through biofilm formation and biofouling.

Microbial bloom formation in a high pH spent nuclear fuel pond.

Microorganisms are able to colonise a wide range of extreme environments, including nuclear facilities. In this study, the First Generation Magnox Storage Pond (FGMSP) a high pH, legacy spent nuclear fuel pond (SNFP) situated at Sellafield, Cumbria, UK, was studied. Despite the inhospitable conditions in the FGMSP, microorganisms can cause "blooms" within the facility which to date have not been studied. These microbial blooms significantly reduce visibility in the engineered facility, disrupting fuel retrieval operations and slowing decommissioning. The microbial community colonising the pond during two microbial bloom periods was determined by using physiological measurements and high throughput next generation sequencing techniques. In situ probes within the ponds targeting photosynthetic pigments indicated a cyanobacterial bloom event. Analysis of the 16S rRNA gene data suggested that a single cyanobacterial genus was dominant during the bloom events, which was most closely related to Pseudanabaena sp. Comparisons between the microbial community of FGMSP and an adjacent SNFP that is periodically purged into the FGMSP, showed different community profiles. Data confirm the onset of the microbial blooms occurred when the pond purge rate was reduced, and blooms could be controlled by re-establishing the purging regime. The presence of Pseudanabaena sp. that can colonise the pond and dominate during bloom periods is notable since they have received little attention for their role in cyanobacterial bloom formation. This work also informs bioremediation efforts to treat waters contaminated with radionuclides, which could benefit from the use of cyanobacteria able to tolerate extreme environments and accumulate priority radionuclides.

Lactobacillus rhamnosus GG protects the intestinal epithelium from radiation injury through release of lipoteichoic acid, macrophage activation and the migration of mesenchymal stem cells.

OBJECTIVE: Lactobacillus rhamnosus GG (LGG), a probiotic, given by gavage is radioprotective of the mouse intestine. LGG-induced radioprotection is toll-like receptor 2 (TLR2) and cyclooxygenase-2 (COX-2)-dependent and is associated with the migration of COX-2+mesenchymal stem cells (MSCs) from the lamina propria of the villus to the lamina propria near the crypt epithelial stem cells. Our goals were to define the mechanism of LGG radioprotection including identification of the TLR2 agonist, and the mechanism of the MSC migration and to determine the safety and efficacy of this approach in models relevant to clinical radiation therapy. DESIGN: Intestinal radioprotection was modelled in vitro with cell lines and enteroids as well as in vivo by assaying clinical outcomes and crypt survival. Fractionated abdominal and single dose radiation were used along with syngeneic CT26 colon tumour grafts to assess tumour radioprotection. RESULTS: LGG with a mutation in the processing of lipoteichoic acid (LTA), a TLR2 agonist, was not radioprotective, while LTA agonist and native LGG were. An agonist of CXCR4 blocked LGG-induced MSC migration and LGG-induced radioprotection. LGG given by gavage induced expression of CXCL12, a CXCR4 agonist, in pericryptal macrophages and depletion of macrophages by clodronate liposomes blocked LGG-induced MSC migration and radioprotection. LTA effectively protected the normal intestinal crypt, but not tumours in fractionated radiation regimens. CONCLUSIONS: LGG acts as a 'time-release capsule' releasing radioprotective LTA. LTA then primes the epithelial stem cell niche to protect epithelial stem cells by triggering a multicellular, adaptive immune signalling cascade involving macrophages and PGE2 secreting MSCs. TRIAL REGISTRATION NUMBER: NCT01790035; Pre-results.

Effect of humic acid & bacterial exudates on sorption-desorption interactions of 90Sr with brucite.

One of the nuclear fuel storage ponds at Sellafield (United Kingdom) is open to the air, and has contained a significant inventory of corroded magnox fuel and sludge for several decades. As a result, some fission products have also been released into solution. 90Sr is known to constitute a small mass of the radionuclides present in the pond, but due to its solubility and activity, it is at risk of challenging effluent discharge limits. The sludge is predominantly composed of brucite (Mg(OH)2), and organic molecules are known to be present in the pond liquor with occasional algal blooms restricting visibility. Understanding the chemical interactions of these components is important to inform ongoing sludge retrievals and effluent management. Additionally, interactions of radionuclides with organics at high pH will be an important consideration for the evolution of cementitious backfilled disposal sites in the UK. Batch sorption-desorption experiments were performed with brucite, 90Sr and natural organic matter (NOM) (humic acid (HA) and Pseudanabaena catenata cyanobacterial growth supernatant) in both binary and ternary systems at high pH. Ionic strength, pH and order of addition of components were varied. 90Sr was shown not to interact strongly with the bulk brucite surface in binary systems under pH conditions relevant to the pond. HA in both binary and ternary systems demonstrated a strong affinity for the brucite surface. Ternary systems containing HA demonstrated enhanced sorption of 90Sr at pH 11.5 and vice versa, likely via formation of strontium-humate complexes regardless of the order of addition of components. The distribution coefficients show HA sorption to be reversible at all pH values studied, and it appeared to control 90Sr behaviour at pH 11.5. Ternary systems containing cyanobacterial supernatant demonstrated a difference in 90Sr behaviour when the culture had been subjected to irradiation in the first stages of its growth.

High interfacial activity of polymers "grafted through" functionalized iron oxide nanoparticle clusters.

The mechanism by which polymers, when grafted to inorganic nanoparticles, lower the interfacial tension at the oil-water interface is not well understood, despite the great interest in particle stabilized emulsions and foams. A simple and highly versatile free radical "grafting through" technique was used to bond high organic fractions (by weight) of poly(oligo(ethylene oxide) monomethyl ether methacrylate) onto iron oxide clusters, without the need for catalysts. In the resulting ∼1 µm hybrid particles, the inorganic cores and grafting architecture contribute to the high local concentration of grafted polymer chains to the dodecane/water interface to produce low interfacial tensions of only 0.003 w/v % (polymer and particle core). This "critical particle concentration" (CPC) for these hybrid inorganic/polymer amphiphilic particles to lower the interfacial tension by 36 mN/m was over 30-fold lower than the critical micelle concentration of the free polymer (without inorganic cores) to produce nearly the same interfacial tension. The low CPC is favored by the high adsorption energy (∼10(6) kBT) for the large ∼1 µm hybrid particles, the high local polymer concentration on the particles surfaces, and the ability of the deformable hybrid nanocluster cores as well as the polymer chains to conform to the interface. The nanocluster cores also increased the entanglement of the polymer chains in bulk DI water or synthetic seawater, producing a viscosity up to 35,000 cP at 0.01 s(-1), in contrast with only 600 cP for the free polymer. As a consequence of these interfacial and rheological properties, the hybrid particles stabilized oil-in-water emulsions at concentrations as low as 0.01 w/v %, with average drop sizes down to 30 µm. In contrast, the bulk viscosity was low for the free polymer, and it did not stabilize the emulsions. The ability to influence the interfacial activity and rheology of polymers upon grafting them to inorganic particles, including clusters, may be expected to be broadly applicable to stabilization of emulsions and foams.

Modified montmorillonite clay microparticles for stable oil-in-seawater emulsions.

Environmentally benign clay particles are of great interest for the stabilization of Pickering emulsions. Dodecane-in-synthetic seawater (SSW) emulsions formed with montmorillonite (MMT) clay microparticles modified with bis(2-hydroxyethyl)oleylamine were stable against coalescence, even at clay concentrations down to 0.1% w/v. Remarkably, as little as 0.001% w/v surfactant lowered the hydrophilicity of the clay to a sufficient level for stabilization of oil-in-SSW emulsions. The favorable effect of SSW on droplet size reduction and emulsion stability enhancement is hypothesized to be due to reduced electrostatic repulsion between adsorbed clay particles and a consequent increase in the continuous phase (an aqueous clay suspension) viscosity. Water/oil (W/O) emulsions were inverted to O/W either by decreasing the mass ratio of surfactant-to-clay (transitional inversion) or by increasing the water volume fraction (catastrophic inversion). For both types of emulsions, coalescence was minimal and the sedimentation or creaming was highly correlated with the droplet size. For catastrophic inversions, the droplet size of the emulsions was smaller in the case of the preferred curvature. Suspensions of concentrated clay in oil dispersions in the presence of surfactant were stable against settling. The mass transfer pathways during emulsification of oil containing the clay particles were analyzed on the droplet size/stability phase diagrams to provide insight for the design of dispersant systems for remediating surface and subsurface oceanic oil spills.

Synergistic formation and stabilization of oil-in-water emulsions by a weakly interacting mixture of zwitterionic surfactant and silica nanoparticles.

Oil-in-water emulsions were formed and stabilized at low amphiphile concentrations by combining hydrophilic nanoparticles (NPs) (i.e., bare colloidal silica) with a weakly interacting zwitterionic surfactant, caprylamidopropyl betaine, to generate a high hydrophilic-lipophilic balance. The weak interaction of the NPs with surfactant was quantified with contact angle measurements. Emulsions were characterized by static light scattering to determine the droplet size distributions, optical photography to quantify phase separation due to creaming, and both optical and electron microscopy to determine emulsion microstructure. The NPs and surfactant acted synergistically to produce finer emulsions with a greater stability to coalescence relative to the behavior with either NPs or surfactant alone. As a consequence of the weak adsorption of the highly hydrophilic surfactant on the anionic NPs along with the high critical micelle concentration, an unusually large surfactant concentration was available to adsorb at the oil-water interface and lower the interfacial tension. The synergy for emulsion formation and stabilization for the two amphiphiles was even greater in the case of a high-salinity synthetic seawater aqueous phase. Here, higher NP adsorption at the oil-water interface was caused by electrostatic screening of interactions between (1) NPs and the anionic oil-water interface and (2) between the NPs. This greater adsorption as well as partial flocculation of the NPs provided a more efficient barrier to droplet coalescence.