Using novel high pressure bioreactors to probe the microbiology of shale gas extraction
Hydraulic fracturing is widely used in the US for the production of shale gas. This approach uses horizontal drilling with pumping water-based fluids at high pressure to artificially fracture the host rock and allow the gas to flow freely. The UK government is looking to exploit its own shale gas reserves using this same method. It is important to understand how microbial activity may affect this process, in particular, the formation of hydrogen sulfide. This is a corrosive and harmful gas formed from the microbial reduction of sulfate, and has recently been recognised as a problem in US shale gas wells. It has been suggested that the organic chemicals that are added to the fracturing fluid may be responsible for stimulating microbial sulfate reduction and linked microbially-induced corrosion processes, therefore we conducted experiments to test this hypothesis.
The most widely used fracturing fluid additive is guar gum, a natural polymer used to thicken the fluid and keep sand in suspension. Together with colleagues at Rawwater Engineering, we tested whether guar gum could stimulate the microbial production of hydrogen sulfide, using a laboratory-based bioreactor approach. We set up two high pressure bioreactor experiments, both with guar gum as the only organic substrate supplied for microbial metabolism. The first experiment contained a synthetic fluid with a high concentration of added sulfate. The second experiment used surface water collected from a seasonal pond with a naturally low sulfate concentration. We also added microorganisms originating from a drinking water reservoir to each bioreactor.
In both experiments we detected the production of hydrogen sulfide over the course of several weeks, indicating that even when the level of sulfate in the system is naturally low, microbes can use guar gum for their energy. We looked at how the microbial communities changed over time, and in both experiments we saw not only an increase in sulfate-reducing microorganisms (more so in the added sulfate experiment), but also in bacteria that are able to use a diverse range of organic compounds to fuel their metabolisms and produce fatty acids as by-products.
This study demonstrates that this type of laboratory approach can be successfully used to investigate how the organic additives used in shale gas extraction can stimulate microbial activity. Bioreactors could prove to be a valuable tool for studying how other fracturing fluid additives may stimulate undesirable microbial activity, such as the generation of harmful gases and biofilm formation that could hinder gas extraction.
Guar gum stimulates biogenic sulfide production at elevated pressures: Implications for shale gas extraction. Nixon SL, Walker L, Streets MDT, Eden B, Boothman C, Taylor KG, and Lloyd JR. 2017 Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2017.00679
How to remove radioactive technetium from groundwater
Groundwater at nuclear sites can be contaminated with technetium, a radioactive metal that can be mobile in the environment and toxic. Microbial processes can cause technetium to be removed from groundwater by forming minerals in the subsurface, which may prevent technetium from causing harm. This study assessed different ways of stimulating this beneficial microbial activity using sediments collected from the Sellafield nuclear site. The results showed that the three materials used in the experiments were successful in causing technetium to be removed from groundwater, and this effect was long lived even after the minerals were exposed to very different environmental conditions. This suggests that these materials might be effective in remediating technetium-contaminated groundwater at nuclear sites.
Summary diagram showing the results of this study
Under oxygenated conditions technetium is mobile in groundwater as the pertechnetate [Tc(VII)] ion, but it precipitates out of solution as Tc(IV)O2 in anoxic conditions. Supplying sediment bacteria with organic substrates (“foodstuffs”) can lead to the development of anoxic conditions in the subsurface, as can adding certain materials that consume oxygen such as iron (ZVI, or zero valent iron).
In this study we took three products that are commercially available for treating groundwater and assessed whether they were suitable for remediating technetium contamination at nuclear sites using sediment samples collected from Sellafield in microcosm experiments. Two materials were supplied by Regensis® and contained slow-release organic substrates designed to dissolve slowly in groundwater [HRC®] and also containing a sulphur compound [MRC®]. The third material was supplied by Peroxychem® and contained zero valent iron and organics in the form of plant matter [EHC®].
The results showed that anoxic conditions developed in microcosms containing each of the three materials, and this stimulated the removal of technetium from groundwater, in the form of an oxide [TcO2] or as a sulfide [TcS2]. We also looked at changes in the sediment microbial communities and found that before amendment the sediment contained a diverse range of soil bacteria, but after the addition of each material the microbial communities became dominated by bacteria and archaea typically found in anoxic environments.
After this we subjected the sediments to strongly oxidising conditions to assess whether the technetium would become oxidised and released back to groundwater. The results showed that under oxic conditions the technetium remained associated with sediment as TcO2, and that it was likely that the presence of slow-release organics /iron from the substrates that were designed to be long-lived in the environment that protected the technetium from becoming oxidised and remobilised.
Long-term immobilization of technetium via bioremediation with slow-release substrates. Newsome L, Cleary A, Morris K and Lloyd JR. 2017 Environmental Science & Technology, 51 (3), 1595–1604. http://dx.doi.org/10.1021/acs.est.6b04876
Shewanella can reduce radionuclides at a faster rate when riboflavin is added
Bacteria have developed different mechanisms to obtain energy under the versatile conditions found in the environment. Shewanella oneidensis MR-1 is a facultative bacterium that in the absence of oxygen can couple the oxidation of organic matter to the reduction of a wide range of electron acceptors such as NO3–, Fe(III) minerals, Mn (IV), U (VI), among others. For this reason, it has been used as one of the model organisms for the study of electron transfer processes. There are at least two mechanisms by which S. oneidensis MR-1 reduces solid Fe(III): by direct contact with the mineral surface (via cytochromes) and by releasing redox mediators, often called “electron shuttles”. It was recently observed that S. oneidensis MR-1 releases riboflavin and flavin mononucleotide (FMN) as electron shuttles, and these molecules play a key role in this process. Until today, most of the studies on electron transfer by S. oneidensis MR-1 have been focused on the reduction of Fe(III) and Mn(IV), but it has been observed that this strain of Shewanella can also reduce a range of radionuclides, although this mechanism has been poorly studied.
In a recent paper, researchers of the University of Manchester tested the effect of the addition of riboflavin on radionuclides reduction by S. oneidensis MR-1. Their results show that the addition of this flavin improved the reduction rates of Tc(VII), Np(V) and Pu(IV), although it had no significant effect on the reduction of U(VI). This is an interesting finding because it gives an insight at to what kind of mechanism is utilised in the reduction of radionuclides by this bacterium. Ultimately, it means that riboflavin (a cheap food additive) could be used in the remediation of radionuclide contaminated groundwater and wastes. But of course, more studies are needed before a system like this can be used in a wider scale. If you want to learn more about how flavins can enhance the rates of reduction of radionuclides by bacteria click here.
Influence of riboflavin on the reduction of radionuclides by Shewanella oneidenis MR-1.
Cherkouk A, Law GT, Rizoulis A, Law K, Renshaw JC, Morris K, Livens FR, Lloyd JR (2016). Dalton Transactions. 45 (12) : 5030-7. doi: 10.1039/c4dt02929a.
Could bacteria retard Cs and Sr contamination?
Cs-137 and Sr-90 are important products from the nuclear industry that accumulate and largely contribute to radioactivity in spent fuel, waste from spent fuel storage ponds and fuel reprocessing plants, such as at Sellafield, UK. The discharge of Cs-137 and Sr-90 into the environment can be planned or accidental, but once there, they turn into an environmental hazard that can persist for hundreds of years after their release. Additionally, Cs-137 has biochemical behaviour similar to K (an essential nutrient) and Sr-90 readily exchanges for Ca in living organisms. For all these reasons, the behaviour of these radionuclides in natural environments needs to be understood.
A recently published study from researchers of the University of Manchester aimed at investigating the effect on mineral properties of microbial reduction of Fe(III) in biotite and chlorite, and how this affects the sorption of Cs and Sr. Bacteria could affect the sorption by physical (blockage of sorption sites) or chemical (change in pH) means, but surprisingly, the results suggest that Cs and Sr sorption to both minerals was not affected or was even reduced by the presence of bacteria and/or the subsequent changes in the chemistry of the solution. The reduction of metals carried out by bacteria is a technique that has been be used for environmental remediation by immobilizing contaminants. The results obtained here complicate the picture of waste management; at least the systems studied here, show that bacteria could not be good candidates for the retarding of contamination of Cs and Sr, and support the need to study this phenomenon more thoroughly.
Effects of Microbial Fe(III) Reduction on the Sorption of Cs and Sr on Biotite and Chlorite. D. R. Brookshaw, J. R. Lloyd, D. J. Vaughan & R. A. D. Pattrick. 2016. Geomicrobiology journal (33). 206-215.
Breakthrough offers greater understanding of safe radioactive waste disposal
A group of scientists from The University of Manchester, the National Nuclear Laboratory and the UK’s synchrotron science facility, Diamond Light Source, has completed research into radioactively contaminated material to gain further understanding around the issue, crucial for the safe and more efficient completion of future decommissioning projects.
Safely decommissioning the legacy of radioactively contaminated facilities from nuclear energy and weapons production is one of the greatest challenges of the 21st Century. Current estimates suggest clean-up of the UK’s nuclear legacy will cost around £117bn and take decades to complete.
The team identified a concrete core taken from the structure of a nuclear fuel cooling pond contaminated with radioactive isotopes of caesium and strontium, located at the former Hunterston A, Magnox nuclear power station in Ayrshire. The core, which was coated and painted, was taken to the Diamond synchrotron for further analysis.
Strontium is a high yield nuclear fission product in nuclear reactors and tests showed that it was bonded to the titanium oxide found in the white pigment of the paint on the concrete core’s coating.
By identifying the specific location of the radioactive isotopes, the research makes future investigation easier and could potentially leads to more efficient decontamination, saving millions of pounds by reducing the volume of our radioactive waste.
The work also found that the painted and rubberised under layers were intact and the paint had acted as a sealant for 60 years. However, experiments were conducted to examine what would happen if the contaminated pond water had breached the coating. It showed that the strontium would be bound strongly to the materials in the cement but the caesium was absorbed by clays and iron oxides forming part of the rock fragments in the concrete.
Characterising legacy spent nuclear fuel pond materials using microfocus X-ray absorption spectroscopy. Bower W R, Morris K, Mosselmans J F W, Thompson O R, Banford A W, Law K, Pattrick, R A D, 2016. Journal of Hazardous Materials, 317, 97-107.
Iron oxyhydroxide formation during radioactive effluent treatment
Nanoparticulate iron oxyhydroxide minerals, such as ferrihydrite, are ubiquitous. For example, ferrihydrite is widespread in the environment, present in almost all living organisms within the protein ferritin, and used widely in effluent treatment as an effective sink for contaminants. Despite this, ferrihydrite formation pathways are poorly understood.
New research conducted at The University of Manchester, and in partnership with Sellafield Ltd, has characterised ferrihydrite formation during a process mimicking radioactive effluent treatment. This treatment process is used at the Enhanced Actinide Removal Plant (EARP) at Sellafield, UK which currently treats effluents from nuclear fuel reprocessing operations. In the future, EARP will be important in treating decommissioning feeds and therefore maintaining the efficiency of EARP is a high priority. The research shows that ferrihydrite formation proceeds via the initial formation of Fe13 Keggin clusters. This is followed by aggregation and precipitation of small iron hydrolysis products to form “core-shell” ferrihydrite nanoparticles which preserve the Keggin cluster. In this way, the Fe13 Keggin cluster acts a molecular building block for ferrihydrite. The work provides essential support to the growing body of evidence for cluster-mediated pathways to solid formation, which challenge the established thermodynamic view that solid formation occurs via monomeric species.
The research provides fundamental insight into the mechanism of ferrihydrite formation and delivers essential, molecular scale understanding of the EARP treatment process which is vital in enabling clean-up of the UK’s nuclear legacy.
Ferrihydrite Formation: The Role of Fe13 Keggin Clusters. Weatherill J, Morris K, Bots P, Stawski T M, Janssen A, Abrahamsen L, Blackham R, Shaw S, 2016. Environmental Science & Technology, 50 (17), 9333-9342.
Discovering the potential of bacteria in nuclear waste disposal
Sarah Smith’s new paper looks at the proposed scenario of intermediate level waste disposal in underground geological disposal facilities. Concrete used to encase the waste would react with water ingress to form high pH zones in the surrounding rocks. A shift in pH could cause changes in the types of bacteria found in these zones, which in turn can change the flow dynamics of the groundwater and potentially alter the mobility of radioactive chemicals.
Sarah set up some lab experiments to mimic this environment using columns of sandstone which she could flow water through to test different scenarios. It was important to find some bacterial communities accustomed to survival in a high pH environment, and for this Sarah used cultures from a hyper-alkaline spring in Buxton, near Manchester. When the bacteria and organic carbon were added to the sandstone, dominant types of bacteria could be identified and fermentation processes occurred.
The paper showed that biofilm formation was possible on grain surfaces at high pH values normally thought to stop growth of conventional groundwater bacteria. Gas generation as a result of organic carbon utilization was also noted, which may impact on the physical properties of the host rock, potentially influencing radionuclide migration through the geosphere. Blocking flow paths by microbial growth in the subsurface could help prevent the release and migration of radionuclides from nuclear waste disposed of into the deep subsurface.
The impact of bioreduction on iodate in sediment microcosms
As part of the Minmag special edition from November 2015, Fabiola from the Geomicro group published a paper studying the effects of bioreduction on iodate. This work has particular relevance to radioactive waste disposal and contaminated land as a key long-lived radionuclide is Iodine-129; a fission product with a half-life of 15.7 million years.
The work found that under oxic conditions, iodate (IO3–) may be partially stable as sorbing to sediments is favoured. Upon reduction to iodide (I–) under mildly reducing conditions, iodine can be released into solution, increasing the environmental mobility.
In the microbially active experiments performed, a net release of iodide from the sediments can be observed during bioreduction (see inserted pictured below), suggesting that an additional source of naturally sediment bound iodine is released under metal- or sulfate-reducing conditions.
The results from this paper are particularly interesting as bioreduction is often thought to retard radionuclide migration (for example, with uranium and technetium). However, the increase in iodine detected under anoxic conditions suggests that bioreduction causes indirect reductive solubilisation of iodine in the form of iodide.
If you’d like to read more, the paper is available via open access at the mineralogical magazine website here.
New technique discovered to clean up uranium-contaminated groundwater
Groundwater contaminated with uranium is a problem at many current and former nuclear sites worldwide. Now a new method has been found to treat this contamination, harnessing the power of the natural subsurface microbial community.
Sediment samples were collected from the Sellafield nuclear site and added to laboratory “microcosms” containing artificial groundwater and uranium added as aqueous uranium(VI), its highly soluble oxidised form common in oxic waters. The natural sediment bacteria were supplied with glycerol phosphate, to stimulate the development of anaerobic conditions, and to investigate whether the bacteria could produce uranium phosphate biominerals.
Results showed that the uranium(VI) was rapidly removed from groundwater, in conjunction with the development of reducing conditions and the release of phosphate to solution. Geochemical analyses revealed that the sediment bacteria had used the glycerol phosphate and produced a chemically reduced form of uranium, present as insoluble uranium(IV) phosphate biominerals, while DNA sequencing showed the presence of bacteria closely related to those known to be involved with uranium biocycling.
Additional experiments to investigate the long-term stability of the insoluble uranium(IV) phosphate biominerals found that they were not affected by nitrate, a common co-contaminant at nuclear sites which can often oxidise and mobilise U(IV) minerals; and they were only partially remobilised when they were stirred vigorously with air to generate highly oxidising conditions.
Although the use of organic carbon compounds to stimulate the subsurface microbial community to generate uranium(IV) biominerals has long been considered a potential bioremediation technique, this approach can lead to problems with reoxidation and remobilisation of the uranium under oxic conditions. Our approach to include phosphate with the organic carbon supplement could enhance the longevity of the uranium(IV) biominerals in the subsurface, and therefore offers promise as a better long-term solution for treating uranium-contaminated groundwater at nuclear sites.
New research shows how uranium can be immobilised by iron containing minerals
New research from the Geomicro group shows how uranium can be incorporated into iron containing minerals found in the ground to reduce the radionuclide’s mobility in the environment. The study shows that, under conditions similar to those of a geological disposal facility, U(VI) can be reduced and incorporated into the iron containing mineral, magnetite, as it crystallises from ferrihydrite.
The work uses techniques such as XANES, EXAFS, chemical extraction and XRD to determine that U is reductively incorporated into magnetite and may be present in the oxidation state U(V). Further to this, the paper finds that the U(V) may be present as either mixed valence U in multiple sites (U(IV) and U(VI)), or as U(V) stabilised by the magnetite structure.
The work was published as an open source material in Minmag as part of a special issue by our very own Kath Morris. The issue is part of the ‘Implementing Geological Disposal – Technology Platform’ (IGD-TP) Conference and includes a lot of other work by the Geomicro group, as well as other research groups from the University of Manchester. If you’d like to know more, follow the link here.
New research on how microbes restrict the movement of radioactive chemicals in the environment
New research by members of the Manchester Geomicro group published in Environmental Science and Technology, highlights the critical role that bacteria play in the cycling of metals in the Earth. The complex interactions between bacteria and minerals results in immobilization of radionuclides in contaminated land and around radioactive disposal sites. Here the bacterium Geobacter sulfurreducens mediated the reduction of Fe(III) in the minerals biotite and chlorite, generating pools of reactive Fe(II). This reaction primed the minerals for reductive scavenging of the radionuclides Tc(VII), U(VI) and Np(V). These minerals are common components of rocks and soils and if carbonate, a common component of groundwater, was present it inhibited the U(VI)-mineral reaction and the reduction of U(VI).
This study presents in-depth EXAFS interpretations of the radionuclide reaction products, and is among the first to attempt this for Np(IV) reacted with biotite and chlorite. The reduction behaviour of the three radionuclides and the variety of their reaction products shows that these microbial processes offer a promising, and only recently recognized route to limit the solubility of a range of radionuclides in environmental systems.
Can uranium bioremediation be effective over long time periods?
A new paper describes the results of experiments to simulate the long-term behaviour of uranium in the subsurface, post-bioremediation. Uranium-contaminated groundwater is a problem at many nuclear sites; stimulating the microbial reduction of aqueous U(VI) to insoluble U(IV) via the application of an electron donor has been proposed as a mechanism to remediate this contamination. However, questions remain regarding the long-term fate of biogenic U(IV) phases in the subsurface, particularly should conditions change to become oxidising. Sediment microcosm experiments were biostimulated with an electron donor to generate microbially-reduced U(IV) phases. The sensitivity of these U(IV) phases to oxidising conditions was assessed by exposure to air and nitrate, including after periods of ageing for up to 15 months.
The results showed that uranium was initially precipitated from solution as a non-crystalline “monomeric” U(IV) phase, which partially transformed to nanocrystalline uraninite after 15 months of ageing. However, this increase in crystallinity did not affect the susceptibility of the U(IV) minerals to oxidative remobilisation, with full reoxidation to aqueous U(VI) observed after 90 days aeration and partial reoxidation observed after exposure to an excess of nitrate. The presence of residual electron donor was found to control U(IV) reoxidation kinetics, and therefore could be key in maintaining low concentrations of uranium in groundwater post-bioremediation.
Sellafield bacterium shown to precipitate uranium from solution
Recent work published in PLOS ONE has shown that a Serratia species isolated from Sellafield sediment was able to precipitate uranium from solution using a variety of different mechanisms. The bacterium was able to metabolise glycerol phosphate, which lead to the precipitation of U(VI) as uranyl(VI) phosphate minerals of the autunite group. This occurred under both anaerobic and fermentative conditions. When the Serratia species was supplemented with glycerol under phosphate-limited non-growth conditions, it caused U(VI) to be removed from solution via reduction to U(IV) as nanocrystalline uraninite. This work has implications for the bioremediation of uranium contaminated groundwater at nuclear sites
Radiation tolerant bacteria could be even more effective at clearing up nuclear waste through natural processes than previously thought.
Studies from the group have shown that land contaminated with radioactive waste can also be cleaned up by bacteria that convert soluble forms of radionuclides, such as uranium, to insoluble forms that are less hazardous and mobile. However, for this to be useful, a critical question has needed addressing for some time; whether these naturally occurring activities are killed off by radiation associated with the radioactive waste.
Now, in a new paper published in Applied and Environmental Microbiology, we have shown that radiation could actually allow certain microbes to thrive, rather than killing them all. Despite gamma radiation doses relevant to the bioremediation of radionuclide contaminated land and the geodisposal of radioactive waste, biogeochemical processes in sediment microcosms were not restricted as expected. Rather, significant and surprising shifts in phylogenetic composition of microbial communities were observed, including the emergence of Geobacter sp. associated with enhanced levels of Fe(III)-reduction.
The study, highlighted by the journal as an ‘article of significant interest’, suggests that low doses of radiation could provide the basis of novel ecosystems in engineered environments and the deep biosphere. Indeed, processes such as this could make microbial communities more effective in the cleaning up of contaminated land or in contributing to the safety of radioactive waste disposal in the long-term.
Our very own Prof. Jon Lloyd said: “This could provide a new, and very useful extra layer of protection when we are trying to dispose of nuclear waste. There are advanced plans on how this can be done safely, often involving the use of concrete and steel barriers, but there is recognition that at some point in the distant future these barriers will be breached.
“But by assessing the ability of these useful microbes to survive radiation stress, we can be more confident that the waste will remain locked-up for very long periods of time (many thousands of years), helped by a naturally evolving “biobarrier”. Before this research, the assumption was that the radiation would probably kill off the bacteria that we are studying, but it seems that is not the case. It is potentially a very important finding for the nuclear industry, and illustrates how resilient biology can be!”
Brown AR, Boothman C, Pimblott SM, Lloyd JR. 2015. The impact of gamma radiation on sediment microbial processes. Applied and Environmental Microbiology, 81(12): 4014–4025; doi:10.1128/AEM.00590-15.
New paper published on the biodegradation of ISA under conditions relevant to a geological disposal facility
The bacterial degradation of isosaccharinic acid (ISA) under various biogeochemical conditions at high pH has been demonstrated in an article that will be published in print in the February 2015 issue of the ISME Journal (http://dx.doi.org/10.1038/ismej.2014.125).
This work shows for the first time that bacteria living at high pH in a lime-kiln pond in the Peak District were able to degrade ISA under anaerobic conditions. This is important for the nuclear industry since ISA, which will be produced from intermediate-level waste that will be disposed of in the geological disposal facility, has the potential to solubilise radionuclides and may potentially enhance their transport to the surface environment (biosphere). Given that these bacteria took only decades to evolve the machinery to utilise ISA as a carbon source for growth at high pH, we can assume that similar bacteria will evolve in and around the geological disposal facility during its thousands of year’s life-time.
This article has gained lots of national and international media attention since its publication because of the interest of the general public and scientists alike in this topic. Ongoing work includes isolation of some of these alkaliphilic bacteria and looking at their DNA to know what makes them tick.
This work was conducted by Naji Bassil, and for this he was awarded the University of Manchester School of Earth, Atmospheric and Environmental Sciences Postgraduate Research Student Best Outstanding Output Award for 2014. Additionally Naji has been nominated for the Manchester Doctoral College Excellence Award in the Faculty of Engineering and Physical Sciences category. Congratulations Naji!
Evidence for the formation of uranium(VI) colloidal nanoparticles in GDF relevant conditions
The paper details the investigation of uranium behaviour under high pH, reducing conditions – i.e. the conditions expected to dominate in a cement backfilled GDF post-closure. The results show that colloidal uranium(VI) nanoparticles with a clarkeite-type crystallographic structure can form within hours and remain stable for several years. These nanoparticles are a potential new mechanism by which U(VI) may migrate from a cementitious GDF to the wider environment. Integral to the study was the use of synchrotron based in situ and ex situ X-ray techniques (small-angle X-ray scattering (SAXS) and X-ray adsorption spectroscopy (XAS)) which were performed at the Diamond Light Source.
Mighty microbes in the Sellafield subsurface can unload uranium
Laura Newsome et al’s new paper “Microbial reduction of uranium(VI) in sediments of different lithologies collected from Sellafield” has just been published Open Access in Applied Geochemistry (http://dx.doi.org/10.1016/j.apgeochem.2014.09.008).
Here she presents data showing how uranium(VI) mobility can be controlled by stimulating biogeochemical interactions, as indigenous microorganisms in a variety of different lithology sediments could reduce aqueous U(VI) to insoluble U(IV). However, sediment cell numbers and the amount of bioavailable iron(III) present in the soil could be limiting factors in its application in the subsurface.