Biostimulation by Glycerol Phosphate to Precipitate Recalcitrant

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Biostimulation by glycerol phosphate to precipitate recalcitrant uranium(IV) phosphate Laura Newsome, Katherine Morris, Divyesh Trivedi, Alastair Bewsher, and Jonathan R. Lloyd Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b02042 • Publication Date (Web): 20 Aug 2015 Downloaded from http://pubs.acs.org on August 29, 2015

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Environmental Science & Technology

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Biostimulation by glycerol phosphate to precipitate

2

recalcitrant uranium(IV) phosphate

3

Laura Newsome1*, Katherine Morris1, Divyesh Trivedi2, Alastair Bewsher1 and Jonathan R

4

Lloyd,1,2

5

1

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Atmospheric and Environmental Sciences, Williamson Building, Oxford Road, Manchester,

7

M13 9PL, UK

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Williamson Research Centre and Research Centre for Radwaste Disposal, School of Earth,

2

National Nuclear Laboratory, Chadwick House, Birchwood, Warrington, WA3 6AE, UK * L. Newsome. Email: [email protected]. Phone: +44 (0)161 275 0309.

10 11

Uranium; Bioremediation; Ningyoite; Nuclear

12

Stimulating the microbial reduction of aqueous uranium(VI) to insoluble U(IV) via electron

13

donor addition has been proposed as a strategy to remediate uranium-contaminated groundwater

14

in situ. However, concerns have been raised regarding the longevity of microbially-precipitated

15

U(IV) in the subsurface, particularly as it may become remobilised if the conditions change to

16

become oxidising. An alternative mechanism is to stimulate the precipitation of poorly soluble

17

uranium phosphates via the addition of an organophosphate and promote the development of

18

reducing conditions. Here we selected a sediment sample from a UK nuclear site and stimulated ACS Paragon Plus Environment

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the microbial community with glycerol phosphate under anaerobic conditions to assess whether

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uranium phosphate precipitation was a viable bioremediation strategy. Results showed that

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U(VI) was rapidly removed from solution and precipitated as a reduced crystalline U(IV)-

22

phosphate mineral similar to ningyoite. This mineral was considerably more recalcitrant to

23

oxidative remobilisation than the products of microbial U(VI) reduction. Bacteria closely related

24

to Pelosinus species may have played a key role in uranium removal in these experiments. This

25

work has implications for the stewardship of uranium-contaminated groundwater, with the

26

formation of U(IV)-phosphates potentially offering a more effective strategy for maintaining low

27

concentrations of uranium in groundwater over long time periods.

28

INTRODUCTION

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Uranium contamination in groundwater poses a significant environmental problem at current

30

and former uranium mining and nuclear facilities. Bacterially-mediated precipitation of uranium

31

minerals can be used as a strategy to remove aqueous uranium from groundwater in situ, and

32

therefore prevent its uncontrolled migration and dispersal.1–4 Potential approaches to stimulate

33

microbial activity include the addition of an electron donor to stimulate microbial U(VI)

34

reduction leading to the precipitation of insoluble U(IV) minerals,5 or the use of an

35

organophosphate compound which bacteria break down to release orthophosphate and

36

consequently cause the precipitation of insoluble uranium phosphate minerals.6 To date most

37

research has focused on microbial U(VI) reduction,1,2 however, biogenic U(IV) has been found

38

to be susceptible to oxidative remobilisation after exposure to oxygen and/or nitrate7–10 and

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therefore may not be an ideal end-product for a long-term in situ remediation strategy. The

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precipitation of uranium phosphate minerals is a promising alternative as U(VI) phosphates are

41

largely insoluble, not sensitive to oxidative changes, and their longevity has been demonstrated

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in natural analogue sites.11–14 Indeed, the presence of phosphate has been found to limit the

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mobility of U(VI) in uranium contaminated systems.15,16 Most work on uranium phosphate

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biomineralisation has focused on aerobic systems that form U(VI) phosphates. Interestingly,

45

strategies to produce reduced uranium(IV) phosphate minerals, for example ningyoite

46

[CaU(PO4)2.2H2O], have not been studied in detail although reduction coupled to phosphate

47

biomineralisation has significant merit when considering treatment for radionuclide

48

contaminated groundwaters. Uranium(IV) phosphate would be a desirable end product because

49

of its very low solubility even compared to U(VI) phosphates.17 Microbial precipitation of

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U(IV)-phosphate has only been observed in one previous study18 with putative identification of

51

either monomeric U(IV) complexed to phosphate19 or U(IV)-phosphate19,20 also reported.

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Indeed, it has been proposed that some ningyoite ore deposits are of biogenic origin, suggesting

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some precedent for microbially-mediated ningyoite formation in the natural environment.21

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Stimulating the precipitation of U-containing phosphate minerals in the subsurface is not

55

straightforward, as adding inorganic phosphate can lead metal phosphate precipitation and

56

localised clogging at injection wells.22

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compounds that are slowly hydrolysed or biodegraded may be used, such as glycerol phosphate,

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long-chain polyphosphates, or phytate from plant matter.6,23,24 Typically glycerol phosphate has

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been used as both a carbon and phosphate source for aerobic microorganisms.6,25 It should also

60

be noted that under phosphate-limited conditions, it is possible that bacteria can “mine” uranyl

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phosphates, releasing uranium to solution,19,26,27 which may potentially affect their longevity in

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the subsurface.

To prevent this, phosphorus-containing organic

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A number of bacterial species have been shown to precipitate uranyl phosphates when supplied

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with glycerol phosphate including: Serratia species,6,28 Caulobacter crescentus,29 environmental

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isolates from the US Department of Energy (DOE) Oak Ridge site closely related to Bacillus and


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Rahnella species25,30 and Aeromonas hydrophila,31 as well as sediment microbial consortia.32,33

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The mechanism of glycerol phosphate biodegradation involves the expression of a phosphatase

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enzyme, which catalyses the hydrolytic cleavage of the C-P bond and so releases inorganic

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phosphate to solution. The released phosphate rapidly precipitates abiotically with U(VI) that is

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present in solution as uranyl complexes, forming uranyl phosphate minerals.6,34 Phosphatase

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activity is a characteristic common to many microorganisms as it ensures that they are able to

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obtain phosphorus, an essential nutrient, in its preferred form (orthophosphate). This suggests

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that many more species may be capable of uranium-phosphate biomineralisation via this

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mechanism. The composition of the microbial community of uranium-contaminated sediments

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from the US DOE Oak Ridge site following stimulation with glycerol phosphate under oxic

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conditions has been investigated using the PhyloChip array.35 Over 2,000 archaeal and bacterial

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taxa were detected; the authors highlighted increases in the relative abundances of hydrogen-

78

dependent methanogens, and also bacteria closely related to species known to denitrify, exhibit

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phytase activity or accumulate polyphosphate. This suggests that glycerol phosphate amendment

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not only stimulated microbes involved in phosphorus cycling, but also many other species,

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leading to complex changes in microbial community dynamics.

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Precipitation of uranium phosphates using extant microbial communities has been investigated

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previously using sediments from the US DOE Oak Ridge site.

Stimulation with glycerol

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phosphate lead to the precipitation of uranyl phosphates over a range of conditions, although

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uranium removal was dominated by sorption effects.32,33

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between glycerol phosphate biostimulation and U-phosphate biomineralisation, we conducted

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microcosm experiments using sediment from a UK nuclear site under anaerobic conditions.

88

Throughout our focus has been on uranium speciation and fate coupled to sediment microbial

To further explore the interplay


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community dynamics to gain insight into the potential for targeted U(IV)-phosphate

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biomineralisation in shallow subsurface environments.

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MATERIALS AND METHODS

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Solubility tests were carried out to assess whether uranium(VI) remained in solution in

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artificial groundwater containing the organic phosphorus containing compounds glycerol

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phosphate, phytic acid and sodium phytate. U(VI) as uranyl chloride at 0.05, 0.1 and 0.2 mM

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was added to a sterile artificial groundwater10 (containing the following in mM: K+ 0.089; Na+

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3.37; Ca2+ 1.69; Mg2+ 0.795; Cl- 1.06; HCO3- 2.88; NO3- 0.332; CO32- 1.69; SO42- 0.39), with 1

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or 10 mM glycerol phosphate or phytic acid. U(VI) in solution was determined over a 12 day

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period by separating the supernatant by centrifugation (16,200 g for five minutes) then

99

measuring aqueous U(VI) via spectrophotometry.36 As the addition of phytic acid caused the pH

100

to decrease from 7.8 to less than 3, a neutral solution of 50 mM sodium phytate was prepared by

101

adding sodium hydroxide to phytic acid. Sodium phytate (1 or 5 mM) was added to the artificial

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groundwater with 0.05 or 0.5 mM U(VI), and the solubility was monitored over 26 hours.

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To determine whether the microbial community present in Sellafield sediment could

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precipitate uranium phosphate biominerals, microcosm experiments were set up containing 10 g

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of gravelly sand sediment (previously characterised as “RB27”37) 100 ml of artificial

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groundwater, 10 mM glycerol phosphate (Sigma G6501) and 0.05 mM U(VI) in glass serum

107

bottles, in triplicate.

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incubated in the dark at room temperature, with periodic shaking. Electron donor free controls

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had no added glycerol phosphate. To investigate how the presence of phosphate may affect

110

U(VI) removal an additional set of microcosm experiments were set up containing 10 mM

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glycerol as the electron donor.

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sacrificially sampled.

The headspace was degassed with argon and the microcosms were

Sterile controls were created by autoclaving and were


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Aliquots

from

each

biostimulation

experiment

were

periodically

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withdrawn

for

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biogeochemical monitoring. Sediment slurry was added to 0.5 N HCl or 0.5 N hydroxylamine-

115

HCl for digestion before analysis for Fe(II) and total bioavailable Fe by the ferrozine assay.38

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Supernatant was separated by centrifugation (16,200 g, 5 minutes) and analysed for U(VI)36 and

117

nitrite39 by spectrophotometry, and for Eh and pH. Surplus supernatant was frozen (-20°C) and

118

stored before ion chromatography analysis for phosphate and nitrate (Dionex ICS 5000 with an

119

AS18 2 mm ion exchange column at 0.25 ml/min), and glycerol phosphate and volatile fatty

120

acids by ion chromatography (Dionex ICS 5000 with an AS11HC 0.4 mm high capacity ion

121

exchange column at 0.015 ml/min).

122

Sediment composition was analysed using X-ray diffraction before and after biostimulation

123

with glycerol phosphate. Scans were performed using a Bruker D8 Advance fitted with a Göbel

124

Mirror and a Lynxeye detector. Electron microscopy was carried out on microbially-reduced

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sediment using a Phillips XL30 ESEM-FEG operated under low vacuum conditions (0.5 torr

126

chamber pressure) at 20 kV accelerating voltage. Elemental distributions were estimated using

127

an EDAX Genesis energy dispersive spectroscopy system.

128

An additional batch of microcosms was set up to allow for analysis of uranium speciation and

129

co-ordination via X-ray absorption spectroscopy (XAS). These contained 0.2 mM U(VI), to

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generate approximately 500 ppm U on sediments after reaction. After confirming that U(VI) had

131

been removed from solution, sediment was stored at -80°C prior to analysis. The sample was

132

prepared under anaerobic conditions by separating the sediment from slurry by centrifugation

133

(5,000 g, 20 minutes), then adding the wet paste to a cryovial which was stored at -80°C under

134

argon. XAS was carried out at the Diamond Light Source, Harwell, UK on Beamline B18 using

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a liquid nitrogen cryostat; uranium LIII-edge spectra were collected in fluorescence mode using a

136

9 or 36 element Ge detector.40 ATHENA41 was used to calibrate, background subtract and


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normalise X-ray absorption near edge structure (XANES) spectra. ARTEMIS41 was used to fit

138

extended X-ray absorption fine structure (EXAFS) spectra, and shells were included in the final

139

fit if they made a statistically significant change to the model as confirmed by the f-test.42

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The long-term stability of the precipitated uranium phosphate was assessed via exposure to

141

oxidants to represent potential changes to the ambient subsurface conditions. Nitrate, a common

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contaminant at nuclear sites, was added initially at 3 mM to represent the highest concentration

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reported in Sellafield groundwater,43 and the bottles were periodically agitated throughout the

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course of the experiment. After 43 days, an additional 30 mM nitrate was added to provide

145

excess oxidant to the system. To represent oxygen ingress, sediment slurry was transferred into

146

conical flasks at a 1 : 3 slurry to headspace ratio and aerated periodically by gentle mixing under

147

ambient atmospheric conditions. Geochemical monitoring of U, Fe, pH and Eh was carried out

148

throughout these reoxidation experiments.

149

A pyrosequencing methodology was used to determine changes in the microbial community

150

during the glycerol phosphate biostimulation experiment at selected time points. Full details are

151

documented previously.37 In brief, DNA was extracted from sediment pellets collected on Day

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0, Day 4, Day 14 and Day 92 using a PowerSoil DNA Isolation Kit (MO BIO Laboratories INC,

153

Carlsbad, CA, USA). The 16S-23S rRNA intergenic spacer region was amplified using primers

154

ITSF and ITSReub, then electrophoresis (in Tris-acetate-EDTA gel) was used to separate the

155

polymerase chain reaction (PCR) products. PCR products were then cleaned up and quantified

156

before sequencing of the 16S rRNA gene (Roche 454 Life Sciences GS Junior system). Details

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of the sequence processing are provided in Table S1. The 454 pyrosequencing reads were

158

analysed using Qiime 1.6.0,44 taxonomic classification performed using the Ribosomal Database

159

Project (http://rdp.cme.msu.edu), and the closest GenBank matches identified by Blastn


7


160

nucleotide search (http://blast.ncbi.nlm.nih.gov).

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amended experiment was also analysed using the same methodology.

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A sample from Day 92 of the glycerol-

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RESULTS AND DISCUSSION

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Uranium solubility: To help determine the appropriate biostimulation method for the

164

Sellafield sediment, a range of phosphate delivery compounds were added to artificial

165

groundwater containing uranium, in order to assess its solubility. Uranium at 0.2 mM was

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soluble in artificial groundwater in the presence of 1 mM and 10 mM glycerol phosphate.

167

However, it was not soluble in artificial groundwater containing phytic acid or sodium phytate at

168

any of the concentrations tested. This rapid interaction between U(VI) and phytic acid/phytate,

169

suggests that in situ application of phytate is unlikely to be suitable for bioremediation due to a

170

lack of dispersivity into a contaminated aquifer.

171

Biostimulation geochemistry: On the basis of the initial screening experiments, glycerol

172

phosphate and glycerol were added to sediment microcosm experiments to stimulate microbial

173

uranium precipitation. With glycerol phosphate, U(VI) was removed rapidly from solution, with

174

near complete removal by Day 14 (Figure 1). Phosphate concentrations in solution initially

175

increased rapidly from 0 to 1.4 mM after 1 hour incubation (Figure 1), but then decreased until

176

Day 4, presumably when the rate of metal phosphate precipitation exceeded the rate of

177

biodegradation of glycerol phosphate. The concentrations of U(VI) and phosphate both remained

178

constant between Days 4 and 7, perhaps suggesting that in this early stage, the rates of

179

removal/production may be controlled by phosphate geochemistry rather than the redox

180

geochemistry of iron or other related processes.

181

Additions of both glycerol phosphate and glycerol stimulated a cascade of terminal electron

182

accepting processes (Figure 1), with very rapid nitrate reduction, followed by Fe(III)-reduction

183

and sulfate removal. With glycerol phosphate approximately half of the U(VI) had already been


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removed from solution before Fe(III)-reduction commenced while with glycerol, uranium

185

removal occurred concomitantly with Fe(III)-reduction (Day 7). These enhanced removal rates

186

in the glycerol phosphate system are presumably due to interactions with the phosphate that was

187

released following the microbial metabolism of glycerol phosphate. Glycerol can be oxidised as

188

an electron donor or fermented and consequently may generate a wide variety of breakdown

189

products. The concentrations of volatile fatty acids (VFAs) detected using ion chromatography

190

showed that with both electron donors, acetate, propionate and formate were generated (Figure

191

S1, Supporting Information) although their proportions were different in each system. Propionate

192

was the dominant VFA breakdown product from glycerol phosphate, while formate

193

predominated following glycerol stimulation. This highlights the complexity of glycerol

194

substrate biodegradation in these systems and suggests that the presence of phosphate may

195

stimulate different microbial processes. Interestingly, glycerol phosphate (and glycerol) addition

196

stimulated the removal of sulfate from solution, possibly due to sulfate reduction. To the best of

197

our knowledge this is the first time this has been observed for these electron donor systems, and

198

for example is in contrast to studies at the US DOE Oak Ridge site,33 although further research

199

would be required to confirm the precise mechanism of sulfate removal.

200

Comparison of the rates of uranium removal and Fe(III)-reduction between experiments

201

containing the same sediment and artificial groundwater stimulated with glycerol phosphate,

202

glycerol or an acetate/lactate mix37 revealed that U(VI) removal was fastest with glycerol

203

phosphate (Figure S2), while rates with glycerol were remarkably similar to those with

204

acetate/lactate, perhaps suggesting the involvement of similar metabolic pathways for these

205

bioreduction-only stimulations.

206

acetate/lactate and slowest with glycerol (Figure S2), likely due to the different substrates

207

stimulating different microorganisms with different metabolic pathways.

Fe(III)-reduction was most rapid in the presence of


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208

Clearly adding glycerol phosphate stimulated microbial activity, leading to the precipitation of

209

uranium from solution. As U(VI) and phosphate concentrations show similar trends during the

210

early stages of this experiment (Figure 1), this suggests that microbial phosphatase activity may

211

have played a role in U(VI) removal, mediating the release of phosphate from glycerol

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phosphate. Furthermore, 50 % of U(VI) was removed from solution before Fe(II) concentrations

213

increased (Figure 1); this implies that a biogeochemical process other than microbial U(VI)

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reduction by Fe(III)-reducing bacteria was partly responsible for U(VI) removal in these

215

experiments. This theory is also supported by the faster rate of U(VI) removal with glycerol

216

phosphate compared to experiments that solely stimulated microbial U(VI) reduction e.g. with

217

glycerol and acetate/lactate (Figure S2).

218

Mineralogy:

XRD analysis found little difference in the bulk sediment pre- and post-

219

biostimulation with glycerol phosphate.

Sediment mineralogy comprised mostly of silicate

220

minerals including quartz, feldspar, mica and chlorite, with some calcite also present. Pyrite

221

made a minor contribution to the post-biostimulation spectrum. Uranium concentrations were

222

too low to be detected with XRD, even in the “high” uranium XAS sediment which contained up

223

to 0.05% wt. U.

224

Imaging of biostimulated sediment using ESEM (Figure S3), revealed a heterogeneous

225

substrate composed of predominantly silicon, oxygen, calcium, aluminum and carbon with some

226

contribution from Na, Mg, P, S and K; elements which are common in silicate minerals.

227

Numerous bright spots were identified in backscatter mode, indicative of elements of higher

228

atomic weight. EDAX spectra confirmed the majority of these areas were rich in iron, although

229

titanium and uranium were also observed at selected sites. An area rich in uranium was identified

230

in backscatter mode, of approximately 1 µm2 in size (Figure S4). Elemental mapping of this

231

localised region revealed the uranium hot spot to be closely correlated with phosphorus and


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located in an area generally high in iron and titanium, although uranium did not appear to be

233

correlated with these elements.

234

X-ray absorption spectroscopy: To identify the speciation of uranium in sediments

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stimulated with glycerol phosphate, XANES spectra were collected and compared to those of

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U(IV) as uraninite and U(VI) as uranyl phosphate as standards.28 Both the edge position of the

237

sample and the shape of the spectra were most similar to the U(IV) standard (Figure 2). The

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position of the first derivative at 17170 eV, was the same as that of uraninite, and lower than that

239

of uranyl phosphate (17173 eV). Linear combination fitting of XANES spectra indicated that

240

100 % of the uranium present in the glycerol phosphate biostimulated sediment was present as

241

U(IV).

242

The local co-ordination environment of U(IV) in the glycerol phosphate stimulated system was

243

investigated by examining the EXAFS spectra (Figure 3). Given the presence of U(IV) and the

244

likely presence of phosphate, we used the crystal structure of the U(IV) phosphate mineral

245

ningyoite to fit the spectra (Figure S5).45 An excellent fit was obtained with the U(IV) in a

246

ningyoite type environment, with the central U atom in 8-fold co-ordination with oxygen (with

247

four O atoms at 2.28 Å and four O atoms at 2.44 Å), and with two bidentate P atoms at 3.14 Å

248

and four monodentate P atoms at 3.69 Å (Figure 3, Table 1). Given the presence of Ca in the

249

ningyoite crystal structure45 and that calcite was detected in XRD analysis of this sediment37, we

250

attempted to include Ca in the EXAFS fit. Adding a shell of Ca atoms at 3.85 Å improved the

251

EXAFS fitting parameters but not with statistical significance and it also lead to relative large

252

errors on the Debye-Waller factor (which is not surprising given the distance of Ca from the

253

central absorber and that Ca is a relatively weak scatterer), and therefore it was not included in

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the final fit. This U(IV)-phosphate co-ordination environment is very similar to the ningyoite

255

crystal structure, and with only small variations (0.01 – 0.06 Å) compared to the published bond


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distances. By contrast, fitting to a model monomeric U(IV) phosphate phase from our past

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work46 on this sediment (Figure S6) resulted in a poor fit with no resolution of key features in the

258

Fourier transform between 2.5 and 3.5 Å (Figure S6). Additionally, our spectrum did not match

259

published monomeric U(IV)-phosphate spectra47 and overall this suggested that the

260

biostimulation product was a U(IV)-phosphate with a ningyoite-like crystal structure.

261

Comparison of our EXAFS data with those for chemogenic crystalline U(IV) phosphate

262

previously published47 revealed remarkable similarities; similar oscillations were observed in the

263

EXAFS and the main peaks in the Fourier transforms for both datasets plotted at around 1.7 Å,

264

2.7 Å, 3.2 Å and 4.0 Å.

265 266

Table 1. Details of EXAFS fit parameters for the U(IV)-phosphate biomineral.

Sample

U(IV) phosphate biomineral

Path

Coordination number

Atomic distance (Å)

Confidence Debyelevel of Waller factor adding shell 2 2 σ (Å ) (α)^

O eq

4

2.28 (1)

0.004 (1)

-

O eq

4

2.44 (1)

0.004 (1)

1.00*

P bidentate

2

3.14 (1)

0.006 (1)

0.99

P monodentate

4

3.69 (2)

0.013 (3)

0.96

267 268

Amplitude factor (S02) was fixed at 1.0 for each sample. Numbers in parentheses are the SD on the last decimal place.

269 270

Energy shift ∆E0 from calculated Fermi level (eV) = 5.14 ± 0.86. Reduced χ2 = 54.6. R “goodness of fit factor” = 0.025. Number of variables = 9. Number of independent points = 20.

271 272 273

^ f-test results, α > 0.95 statistically improves the fit with 2 sigma confidence. * This value is for splitting the shell of 8 equatorial oxygen atoms into two shells each containing 4 O atoms, after adding the P shells.

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Our results differ from a previous study33 where U(VI) phosphates were formed in anaerobic

276

sediment microcosms stimulated by glycerol phosphate. A key difference is that U(VI) rapidly

277

sorbed to the sediments used in this earlier study, with just 0.7 % (2 µM) of the added U(VI)

278

remaining in solution. This suggests that the observed formation of U(IV) phosphate in the

279

current study could be linked to the concomitant reductive precipitation of uranium as U(IV) and

280

the release of inorganic phosphate. Supporting evidence for this theory may be found in the

281

formation of a ningyoite-like mineral during U(VI)aq reduction experiments with Shewanella

282

putrefaciens CN32 when the medium contained sodium orthophosphate.20 Another study

283

observed the formation of ningyoite-like U(IV) from hydrogen uranyl phosphate (HUP) in the

284

presence of Geobacter sulfurreducens PCA, linked to the dissolution of the HUP.19 This again

285

supports the theory that the reduction of aqueous U(VI) is integral to the formation of U(IV)

286

phosphate. If this were the case, it could explain the lack of formation of U(IV) phosphate in the

287

previous study, as the U(VI) had rapidly sorbed to sediments, well before phosphate release to

288

solution was measured (after 14 days) and Fe(III)-reducing conditions developed (after 5 days).33

289

Clearly additional work is further required to elucidate the mechanism of U(IV) phosphate

290

formation in sediment systems. The potential for stimulating microbial precipitation of

291

recalcitrant actinide phosphate minerals3 in situ has broader implications for the management of

292

radioactive legacy materials.

293

Recalcitrance of the U(IV) phosphate biomineral: The long-term stability of the U(IV)

294

phosphate biomineral was assessed in the context of reoxidation caused by nitrate and oxygen

295

ingress into the shallow subsurface at a nuclear site. Addition of 3 mM nitrate did not cause

296

significant quantities of uranium to be released to solution (Figure 4). Some Fe(II) was initially

297

oxidised to Fe(III) (data not shown), likely due to the microbial community using residual

298

electron donor to reduce nitrate to nitrite, which could then oxidise Fe(II) to Fe(III).48 This


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increase in Fe(III) was transient; by Day 14 it had been reduced back to Fe(II), probably as the

300

added nitrate had been metabolised by the microbial community, as observed previously.46

301

Consequently to provide a stoichiometric excess of oxidant, 30 mM nitrate was added on Day

302

43. This reoxidised all of the Fe(II) to Fe(III), but just 3 % of the uranium was released to

303

solution (Figure 4).

304

phosphate and monomeric U(IV)46 (Figure 4) clearly shows the recalcitrance of U(IV) phosphate

305

to remobilisation under oxidising conditions. Other previous experiments where the products of

306

microbial U(VI) reduction were reoxidised during nitrate additions showed variable results, from

307

3 %8 to 97 %49, with the presence of residual electron donor potentially an important factor in

308

determining levels of U(IV) reoxidation.46

A comparison of the results of nitrate-mediated reoxidation of U(IV)

309

Exposure of the glycerol phosphate stimulated sediment to air caused around 40 % of the

310

uranium to be remobilised after 90 days (Figure 4). Compared to previous data under similar

311

experimental conditions, but stimulated with acetate/lactate and containing monomeric U(IV)46

312

and where 100 % was remobilised after 60 days, the U(IV) phosphate biomineral was

313

considerably more resistant to oxidative remobilisation. Previous data published for the products

314

of microbial U(IV) reduction generally also showed U(IV) reoxidation following exposure to

315

oxygen or oxygenated groundwater.8,49,50

316

ordination of uranium was possible by XAS in a parallel reoxidised sample, for which the

317

geochemical data indicated that ~ 20 % of the U(IV) had been reoxidised (Figure 5).

318

spectra appeared remarkably similar to the original U(IV) phosphate mineral (Figure S7), and the

319

best fit was achieved using the same fitting parameters (Table S2), indicating the presence of a

320

refractory U(IV) phosphate phase.

In this study, analysis of the speciation and co-

The

321

In summary biogenic U(IV) phosphate was considerably more recalcitrant to oxidative

322

remobilisation via exposure to 30 mM nitrate or oxygen, when compared to sediments where


14

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323

bioreduction was stimulated with just an electron donor and specifically in similar sediment

324

systems stimulated by acetate/lactate where monomeric U(IV) had formed. These experimental

325

results suggest that targeted phosphate precipitation may be a long-term treatment option for

326

uranium-contaminated groundwaters.

327

Molecular ecology: We performed 16S rRNA pyrosequencing to investigate the changes in

328

the composition of the microbial community during glycerol phosphate biostimulation, and to

329

compare the changes to those following glycerol biostimulation. At the phylum/class level the

330

results were dominated by Proteobacteria (alpha, beta and gamma) and Firmicutes (Figure S8).

331

At the start of the experiment (Day 0, samples taken 1 hour after the experiment was set up), a

332

relatively diverse microbial community was present (Table S1, Figure S9), with Pseudomonas

333

species making up four of the five most abundant operational taxonomic units (OTUs, Figure 6,

334

Table S3).

335

After 4 days, species diversity had decreased (Table S1, Figure S9) and the microbial

336

community was dominated by those affiliated with Pseudomonas species (Table S3); members

337

of the order Pseudomondales comprised 76% of the microbial community (Figure 6). The most

338

abundant OTU was closely related to Pseudomonas mandelii (100 % ID similarity) and

339

comprised 58 % of all clones. P. mandelli is a facultative anaerobe known to denitrify, and has

340

been previously used to study the effects of denitrification on nitrate-rich radioactive wastes in a

341

geological disposal facility.51 Other bacteria in the Day 4 microbial community were closely

342

related to Pseudomonas migulae, known to be able to denitrify and fix nitrogen, and a

343

Hydrogenophaga species that had been isolated from a uranium-contaminated mine and related

344

to hydrogen-oxidising and nitrogen-metabolising species (Table S3). These results are similar to

345

those of a previous study using sediments stimulated with glycerol phosphate, which also

346

observed increases in species involved in hydrogen metabolism and denitrification.52


15


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347

A significant shift in the relative proportions of species had occurred in the glycerol phosphate

348

stimulated microbial community between Day 4 and Day 14 (Figure 6), with large increases in

349

bacteria closely related to Pelosinus UFO1 (95 % ID similarity), comprising 33 % of bacteria

350

identified at the genus level. Pelosinus species are fermentative bacteria and are known to be

351

able to reduce metals such as Cr, Fe and U,53,54 and it is noteworthy that this increase coincides

352

with the start of Fe(III) reduction in our experiments. The presence of Pelosinus UFO1 is

353

particularly of interest as it was originally isolated from pristine sediments at the US DOE Oak

354

Ridge site and has been shown to be able to remove uranium from solution by multiple

355

mechanisms, including by reduction to U(IV) and via precipitation as U(VI) phosphates.52

356

Additional support for its potential role in uranium bioremediation comes from closely related

357

species also being present in uranium and heavy metal contaminated sediments and found in

358

soils amended with acetate to stimulate U(VI) bioremediation (Table S3).

359

By Day 92 a more diverse microbial community was detected following glycerol phosphate

360

biostimulation (Table S1, Figure S9), with the most abundant OTUs including species closely

361

related to Rhizobium, Aztobacter, Magnetospirillum and a Bacteriodales species (Table S3). In

362

comparison, 92 days after glycerol biostimulation the most abundant OTUs comprised species

363

closely related to Hydrogenophaga, a Bacteriodales species and other uncultured species from

364

Bacteriodales and the family Gracilibacteraceae (Table S3). The presence of phosphate in the

365

glycerol phosphate biostimulated sediments must account for these differences in the microbial

366

community composition, given no other variables were changed. Following from this, a different

367

microbial community with different metabolic characteristics may explain why propionate was

368

the dominant VFA detected following glycerol phosphate biostimulation and formate

369

predominated with glycerol (Figure S1).


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370

These results highlight the dynamic changes that occur following biostimulation, which are not

371

observed when just the microbial community present at the end of an experiment is examined.

372

The large increase in bacteria closely related to Pelosinus around Day 14 could account for some

373

uranium removal. Questions remain about the role played by Pseudomonas species in these

374

anaerobic systems; although they are known to denitrify, our results showed that nitrate

375

reduction was essentially complete after 24 hours, and nitrite reduction ended between Day 1 and

376

Day 4. At Day 14 Pseudomonadales species comprised 50 % of the microbial community at the

377

order level (Figure 6), so it is unclear how and whether they were functioning under these

378

anaerobic conditions and it would be of interest to investigate this further.

379

In conclusion, stimulating sediments with glycerol phosphate lead to the formation of U(IV)

380

phosphate biominerals, which were more recalcitrant to oxidative remobilisation than the

381

products of microbial U(IV) reduction alone. Phosphate played a key role in the formation of a

382

genetically distinct microbial community which generated different organic breakdown products.

383

This work has implications for the long-term management of uranium-contaminated groundwater

384

where targeted bioprecipitation of phosphate phases coupled to bioreduction has the potential to

385

treat a wide range of radionuclides in the subsurface.

386

ASSOCIATED CONTENT

387

Supporting Information. Additional results including tables of EXAFS fits and phylogenetic

388

results, and figures showing additional geochemical results, ESEM images, EXAFS data and

389

microbial ecology are available free of charge via the Internet at http://pubs.acs.org.

390

AUTHOR INFORMATION

391

Corresponding Author

392

* L. Newsome. Phone: +44 (0)161 275 0309; Email: [email protected].


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393

Notes

394

The authors declare no competing financial interest.

395

Acknowledgements

396

We thank Christopher Boothman and Athanasios Rizoulis (University of Manchester) for

397

assistance with sample preparation and processing of pyrosequencing data, and Jon Fellowes

398

(University of Manchester) for help with ESEM and elemental mapping. Beamtime at beamline

399

B18 was funded by grants SP8941-2 and SP10163-2 from the Diamond Light Source. We

400

acknowledge financial support from the Nuclear Decommissioning Authority via a PhD student

401

bursary, managed by the National Nuclear Laboratory. JRL acknowledges the support of the

402

Royal Society via an Industrial Fellowship. We also acknowledge financial support from NERC

403

via the BIGRAD consortium (NE/H007768/1).

404

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46x25mm (300 x 300 DPI)



Figure 1. Geochemical changes observed following biostimulation of Sellafield sediments with glycerol phosphate (black lines) or glycerol (grey lines). Controls contained no added donor (black dashed lines) or were sterilised by autoclaving (grey dashed lines). 94x50mm (300 x 300 DPI)


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Figure 2. Uranium LIII-edge XANES spectra for sediment biostimulated with glycerol phosphate (GP) compared to reference spectra for uranium(IV) and uranium(VI) minerals.28 61x44mm (300 x 300 DPI)



Figure 3. k3 weighted EXAFS data, non-phase shift corrected Fourier transform of EXAFS data for glycerol phosphate stimulated sediments. 114x155mm (300 x 300 DPI)


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Figure 4. Comparison of U(VI) remobilisation following exposure of U(IV) phosphate and monomeric U(IV) to nitrate and air. Reoxidation experiments were conducted under exactly the same conditions with the same setup, 90 days post-biostimulation with either glycerol phosphate to precipitate U(IV) phosphate, or acetate and lactate to stimulate monomeric U(IV) precipitation.46 125x187mm (300 x 300 DPI)



Figure 5. k3 weighted EXAFS data and non-phase shift corrected Fourier transform of EXAFS data for glycerol phosphate stimulated sediments post-oxygen reoxidation. A good fit was achieved for the reoxidised sample using the same model as for the U(IV) phosphate biomineral 116x161mm (300 x 300 DPI)


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Figure 6. Changes in microbial ecology during glycerol phosphate biostimulation, plotted at the order level. The microbial community rapidly became dominated by Pseudomonadales after biostimulation. Following this, Clostridales (Pelosinus) increased, then after 92 days a more diverse community was detected. The glycerol stimulated sediments (right column) appeared markedly different to glycerol phosphate stimulated sediments after 92 days, suggesting the presence of phosphate may influence the microbial community composition. 47x27mm (300 x 300 DPI)


Biostimulation by Glycerol Phosphate to Precipitate Recalcitrant

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