Athanasios Rizoulis

The potential impact of anaerobic microbial metabolism during the geological disposal of intermediate-level waste.

Abstract Microbial metabolism has the potential to control the biogeochemistry of redox-active radionuclides in a range of geodisposal scenarios. In this study, sediments from a high pH lime workings site were incubated under carefully controlled anaerobic conditions, at a range of alkali pH values with added electron donors and electron acceptors, to explore the limits and rates of bioreduction in a sediment system analogous to intermediate-level nuclear waste. There was a clear succession in the utilization of electron acceptors (in the order nitrate > Fe(III)-citrate > Fe(III) oxyhydroxide > sulfate), in accordance with calculated free energy yields and Eh values over the pH range 10-12. The rate and extent of bioreduction decreased at higher pH, with an upper limit for the processes studied at pH 12. The biochemical limits for such processes are discussed, alongside the potential impact of such forms of microbial metabolism on the solubility of a range of redox active radionuclides that will feature heavily in the safety case for the geological disposal of intermediate-level nuclear waste.

Microbial ecology of arsenic-mobilizing Cambodian sediments: lithological controls uncovered by stable-isotope probing.

Microbially mediated arsenic release from Holocene and Pleistocene Cambodian aquifer sediments was investigated using microcosm experiments and substrate amendments. In the Holocene sediment, the metabolically active bacteria, including arsenate-respiring bacteria, were determined by DNA stable-isotope probing. After incubation with (13) C-acetate and (13) C-lactate, active bacterial community in the Holocene sediment was dominated by different Geobacter spp.-related 16S rRNA sequences. Substrate addition also resulted in the enrichment of sequences related to the arsenate-respiring Sulfurospirillum spp. (13) C-acetate selected for ArrA related to Geobacter spp. whereas (13) C-lactate selected for ArrA which were not closely related to any cultivated organism. Incubation of the Pleistocene sediment with lactate favoured a 16S rRNA-phylotype related to the sulphate-reducing Desulfovibrio oxamicus DSM1925, whereas the ArrA sequences clustered with environmental sequences distinct from those identified in the Holocene sediment. Whereas limited As(III) release was observed in Pleistocene sediment after lactate addition, no arsenic mobilization occurred from Holocene sediments, probably because of the initial reduced state of As, as determined by X-ray Absorption Near Edge Structure. Our findings demonstrate that in the presence of reactive organic carbon, As(III) mobilization can occur in Pleistocene sediments, having implications for future strategies that aim to reduce arsenic contamination in drinking waters by using aquifers containing Pleistocene sediments.

Dynamic changes in microbial community structure and function in phenol-degrading microcosms inoculated with cells from a contaminated aquifer

Contamination of aquifers by organic pollutants threatens groundwater supplies and the environment. In situ biodegradation of organic pollutants by microbial communities is important for the remediation of contaminated sites, but our understanding of the relationship between microbial development and pollutant biodegradation is poor. A particular challenge is understanding the in situ status of microorganisms attached to solid surfaces, but not accessible via conventional sampling of groundwater. We have developed novel flow-through microcosms and examined dynamic changes in microbial community structure and function in a phenol-degrading system. Inoculation of these microcosms with a complex microbial community from a plume in a phenol-contaminated aquifer led to the initial establishment of a population dominated by a few species, most attached to the solid substratum. Initially, phenol biodegradation was incomplete, but as the microbial community structure became more complex, phenol biodegradation was more extensive and complete. These results were replicated between independent microcosms, indicating a deterministic succession of species. This work demonstrates the importance of examining community dynamics when assessing the potential for microbial biodegradation of organic pollutants. It provides a novel system in which such measurements can be made readily and reproducibly to study the temporal development and spatial succession of microbial communities during biodegradation of organic pollutants at interfaces within such environments.

Microbial reduction of U(VI) under alkaline conditions; implications for radioactive waste geodisposal

Although there is consensus that microorganisms significantly influence uranium speciation and mobility in the subsurface under circumneutral conditions, microbiologically mediated U(VI) redox cycling under alkaline conditions relevant to the geological disposal of cementitious intermediate level radioactive waste, remains unexplored. Here, we describe microcosm experiments that investigate the biogeochemical fate of U(VI) at pH 10-10.5, using sediments from a legacy lime working site, stimulated with an added electron donor, and incubated in the presence and absence of added Fe(III) as ferrihydrite. In systems without added Fe(III), partial U(VI) reduction occurred, forming a U(IV)-bearing non-uraninite phase which underwent reoxidation in the presence of air (O2) and to some extent nitrate. By contrast, in the presence of added Fe(III), U(VI) was first removed from solution by sorption to the Fe(III) mineral, followed by bioreduction and (bio)magnetite formation coupled to formation of a complex U(IV)-bearing phase with uraninite present, which also underwent air (O2) and partial nitrate reoxidation. 16S rRNA gene pyrosequencing showed that Gram-positive bacteria affiliated with the Firmicutes and Bacteroidetes dominated in the post-reduction sediments. These data provide the first insights into uranium biogeochemistry at high pH and have significant implications for the long-term fate of uranium in geological disposal in both engineered barrier systems and the alkaline, chemically disturbed geosphere.

Bacterial Diversity in the Hyperalkaline Allas Springs (Cyprus), a Natural Analogue for Cementitious Radioactive Waste Repository

The biogeochemical gradients that will develop across the interface between a highly alkaline cementitious geological disposal facility for intermediate level radioactive waste and the geosphere are poorly understood. In addition, there is a paucity of information about the microorganisms that may populate these environments and their role in biomineralization, gas consumption and generation, metal cycling, and on radionuclide speciation and solubility. In this study, we investigated the phylogenetic diversity of indigenous microbial communities and their potential for alkaline metal reduction in samples collected from a natural analogue for cementitious radioactive waste repositories, the hyperalkaline Allas Springs (pH up to 11.9), Troodos Mountains, Cyprus. The site is situated within an ophiolitic complex of ultrabasic rocks that are undergoing active low-temperature serpentinization, which results in hyperalkaline conditions. 16S rRNA cloning and sequencing showed that phylogenetically diverse microbial communities exist in this natural high pH environment, including Hydrogenophaga species. This indicates that alkali-tolerant hydrogen-oxidizing microorganisms could potentially colonize an alkaline geological repository, which is predicted to be rich in molecular H2, as a result of processes including steel corrosion and cellulose biodegradation within the wastes. Moreover, microbial metal reduction was confirmed at alkaline pH in this study by enrichment microcosms and by pure cultures of bacterial isolates affiliated to the Paenibacillus and Alkaliphilus genera. Overall, these data show that a diverse range of microbiological processes can occur in high pH environments, consistent with those expected during the geodisposal of intermediate level waste. Many of these, including gas metabolism and metal reduction, have clear implications for the long-term geological disposal of radioactive waste.

Microbially mediated reduction of FeIII and AsV in Cambodian sediments amended with 13C-labelled hexadecane and kerogen

Environmental context The use of groundwater with elevated concentrations of arsenic for drinking, cooking or irrigation has resulted in the worst mass poisoning in human history. This study shows that organic compounds that can be found in arsenic rich subsurface sediments may be used by indigenous microorganisms, contributing to the release of arsenic from the sediments into the groundwater. This study increases our understanding of the range of organic substrates (and their sources) that can potentially stimulate arsenic mobilisation into groundwaters. Abstract Microbial activity is generally accepted to play a critical role, with the aid of suitable organic carbon substrates, in the mobilisation of arsenic from sediments into shallow reducing groundwaters. The nature of the organic matter in natural aquifers driving the reduction of AsV to AsIII is of particular importance but is poorly understood. In this study, sediments from an arsenic rich aquifer in Cambodia were amended with two 13C-labelled organic substrates. 13C-hexadecane was used as a model for potentially bioavailable long chain n-alkanes and a 13C-kerogen analogue as a proxy for non-extractable organic matter. During anaerobic incubation for 8 weeks, significant FeIII reduction and AsIII mobilisation were observed in the biotic microcosms only, suggesting that these processes were microbially driven. Microcosms amended with 13C-hexadecane exhibited a similar extent of FeIII reduction to the non-amended microcosms, but marginally higher AsIII release. Moreover, gas chromatography–mass spectrometry analysis showed that 65% of the added 13C-hexadecane was degraded during the 8-week incubation. The degradation of 13C-hexadecane was microbially driven, as confirmed by DNA stable isotope probing (DNA-SIP). Amendment with 13C-kerogen did not enhance FeIII reduction or AsIII mobilisation, and microbial degradation of kerogen could not be confirmed conclusively by DNA-SIP fractionation or 13C incorporation in the phospholipid fatty acids. These data are, therefore, consistent with the utilisation of long chain n-alkanes (but not kerogen) as electron donors for anaerobic processes, potentially including FeIII and AsV reduction in the subsurface.

Impacts of Repeated Redox Cycling on Technetium Mobility in the Environment

Technetium is a problematic contaminant at nuclear sites and little is known about how repeated microbiologically mediated redox cycling impacts its fate in the environment. We explore this question in sediments representative of the Sellafield Ltd. site, UK, over multiple reduction and oxidation cycles spanning ∼1.5 years. We found the amount of Tc remobilised from the sediment into solution significantly decreased after repeated redox cycles. X-ray Absorption Spectroscopy (XAS) confirmed that sediment bound Tc was present as hydrous TcO2-like chains throughout experimentation and that Tcs increased resistance to remobilization (via reoxidation to soluble TcO4-) resulted from both shortening of TcO2 chains during redox cycling and association of Tc(IV) with Fe phases in the sediment. We also observed that Tc(IV) remaining in solution during bioreduction was likely associated with colloidal magnetite nanoparticles. These findings highlight crucial links between Tc and Fe biogeochemical cycles that have significant implications for Tcs long-term environmental mobility, especially under ephemeral redox conditions.

Microbial ecology of arsenic-mobilizing Cambodian sediments: lithological controls uncovered by stable-isotope probing: As mobilization in contrasting Cambodian sediments

Microbially mediated arsenic release from Holocene and Pleistocene Cambodian aquifer sediments was investigated using microcosm experiments and substrate amendments. In the Holocene sediment, the metabolically active bacteria, including arsenate-respiring bacteria, were determined by DNA stable-isotope probing. After incubation with (13) C-acetate and (13) C-lactate, active bacterial community in the Holocene sediment was dominated by different Geobacter spp.-related 16S rRNA sequences. Substrate addition also resulted in the enrichment of sequences related to the arsenate-respiring Sulfurospirillum spp. (13) C-acetate selected for ArrA related to Geobacter spp. whereas (13) C-lactate selected for ArrA which were not closely related to any cultivated organism. Incubation of the Pleistocene sediment with lactate favoured a 16S rRNA-phylotype related to the sulphate-reducing Desulfovibrio oxamicus DSM1925, whereas the ArrA sequences clustered with environmental sequences distinct from those identified in the Holocene sediment. Whereas limited As(III) release was observed in Pleistocene sediment after lactate addition, no arsenic mobilization occurred from Holocene sediments, probably because of the initial reduced state of As, as determined by X-ray Absorption Near Edge Structure. Our findings demonstrate that in the presence of reactive organic carbon, As(III) mobilization can occur in Pleistocene sediments, having implications for future strategies that aim to reduce arsenic contamination in drinking waters by using aquifers containing Pleistocene sediments.

The microbial ecology of a hyper-alkaline spring, and impacts of an alkali-tolerant community during sandstone batch and column experiments representative of a geological disposal facility for intermediate level radioactive waste

ABSTRACT Naturally occurring hyper-alkaline springs and associated hyper-alkaline environments may have components that are analogous to a cement-based deep geological disposal facility (GDF) for intermediate level radioactive waste (ILW). Such high pH environments could give insights into the biogeochemical processes that could occur in the region of a GDF environment after the ingress of GDF-derived groundwater leads to the formation of a hyper-alkaline plume in the surrounding rock mass. This study focuses on the microbial community composition found at a highly alkaline spring near Buxton, Derbyshire, England, and the variation in community structure across spatially separated sample points of contrasting pH values (ranging from pH 7.5–13). Communities containing alkaliphilic and alkalitolerant bacteria were observed across the site by PCR amplification and 16S rRNA gene pyrosequencing and included members of the families Comamonadaceae and Xanthomonadaceae. At pH 13, the sequence library was dominated by Gammaproteobacteria of the families Pseudomonadaceae and Enterobacteriaceae. Bacterial communities from the site demonstrated the ability to reduce Fe(III) in microcosm experiments up to pH 11.5, suggesting the potential to reduce other metals and radionuclides of relevance to cement-encapsulated intermediate level radioactive waste (ILW) disposal. In laboratory column flow-through experiments, microbial communities present at the field site were also able to colonize crushed sandstone. Bacterial community composition varied between columns that had been supplied with alkali surface waters from the site amended with carbon (lactate and acetate, as proxies for products of cellulose degradation from ILW), and control columns that were not supplied with added carbon. Members of the family Clostridiaceae dominated the sequence library obtained from the carbon amended column inlet (45.8% of library), but became less dominant at the outlet (20.8%). Members of the family Sphingomonadaceae comprised 11.8% of the sequence library obtained from the control column inlet, but were not present in sediments collected from the column outlet, whereas the relative abundance of members of the family Comamonadaceae increased from the column inlet (35.2%) to the column outlet (57.2%). The spatial variation in community composition within the columns is indicative of discrete biogeochemical zonation in these flow-through systems.

Microbial ecology of arsenic-mobilizing Cambodian sediments

Microbially mediated arsenic release from Holocene and Pleistocene Cambodian aquifer sediments was investigated using microcosm experiments and substrate amendments. In the Holocene sediment, the metabolically active bacteria, including arsenate-respiring bacteria, were determined by DNA stable-isotope probing. After incubation with (13) C-acetate and (13) C-lactate, active bacterial community in the Holocene sediment was dominated by different Geobacter spp.-related 16S rRNA sequences. Substrate addition also resulted in the enrichment of sequences related to the arsenate-respiring Sulfurospirillum spp. (13) C-acetate selected for ArrA related to Geobacter spp. whereas (13) C-lactate selected for ArrA which were not closely related to any cultivated organism. Incubation of the Pleistocene sediment with lactate favoured a 16S rRNA-phylotype related to the sulphate-reducing Desulfovibrio oxamicus DSM1925, whereas the ArrA sequences clustered with environmental sequences distinct from those identified in the Holocene sediment. Whereas limited As(III) release was observed in Pleistocene sediment after lactate addition, no arsenic mobilization occurred from Holocene sediments, probably because of the initial reduced state of As, as determined by X-ray Absorption Near Edge Structure. Our findings demonstrate that in the presence of reactive organic carbon, As(III) mobilization can occur in Pleistocene sediments, having implications for future strategies that aim to reduce arsenic contamination in drinking waters by using aquifers containing Pleistocene sediments.