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S1
Supporting Information for:
Biostimulation by glycerol phosphate to
precipitate recalcitrant uranium(IV) phosphate
Laura Newsome1*
, Katherine Morris1, Divyesh Trivedi
2, Alastair Bewsher
1 and Jonathan R
Lloyd,1,2
1 Williamson Research Centre and Research Centre for Radwaste Disposal, School of Earth,
Atmospheric and Environmental Sciences, Williamson Building, Oxford Road, Manchester,
M13 9PL, UK
2 National Nuclear Laboratory, Chadwick House, Birchwood, Warrington, WA3 6AE, UK
* Email [email protected]
Submitted to Environmental Science & Technology
Number of Pages: 13
Number of Tables: 3 (Table S1 – S3)
Number of Figures: 9 (Figure S1 – S9)
S2
Table S1. Details of molecular ecology sequences
Sample Number of reads
Number after quality control, chimera
check & denoising
Number of identified
OTUs
Simpson diversity
Glycerol phosphate Day 0 12,484 11,131 981 0.987
Glycerol phosphate Day 4 14,657 13,509 480 0.649
Glycerol phosphate Day 14 9,059 6,615 200 0.809
Glycerol phosphate Day 92 11,944 10,446 511 0.958
Glycerol Day 92 5,979 4,848 625 0.985
Table S2. Details of EXAFS fit parameters for the reoxidised U(IV)-phosphate biomineral
Sample Path Co-ordination
number Atomic
distance (Å) Debye-Waller factor σ2 (Å2)
Confidence level of
adding shell (α)^
U(IV) phosphate biomineral
O eq 4 2.27 (2) 0.006 (2) -
O eq 4 2.42 (2) 0.006 (2) 0.91*
P bidentate 2 3.12 (2) 0.005 (2) 1.00
P monodentate 4 3.64 (5) 0.018 (6) 0.91
Amplitude factor (S02) was fixed at 1.0 for each sample. Numbers in parentheses are the SD on the last decimal place(s).
Energy shift ∆E0 from calculated Fermi level (eV) = 2.32 ± 1.8. Reduced χ2 = 1,750. R “goodness of fit factor”
= 0.038. Number of variables = 9. Number of independent points = 23.
^ f-test results, α > 0.95 statistically improves the fit with 2 sigma confidence, α > 0.68 with 1 sigma confidence.
* This value was for splitting the shell of 8 equatorial oxygen atoms into two shells each containing 4 O atoms, after adding the P shells
S3
Table S3. Closest phylogenetic relatives of the five most abundant OTUs from glycerol
phosphate stimulated sediments after 0, 4, 14 and 92 days, and glycerol stimulated sediment
after 92 days
Number of clones
% of clones
Closest phylogenetic
relative
Accession number
ID similarity Environment
Glycerol phosphate Day 0
708 6.4 Pseudomonas
mandelii CP005960.1 100%
P. mandelii is a cold adapted strain. Closely related species from wetlands, CaCO3-resistant, roots, cold desert soil, rice paddy soil, rhizosphere
512 4.6 Pseudomonas sp.
10424 EF422198.1 100%
P. sp. 10424 is a nitrate reducer from a permeable reactive barrier. Closely related species from anaerobic benzene degrading community, alkali tolerant benzene degrader, phenol degrader, nitrifying enrichment culture
327 2.9 Pseudomonas
migulae KF857261.1 100%
P. migulae is a groundwater cryophile capable of aerobic denitrification. Closely related species are a cyrophilic nitrogen fixer, arsenite oxidiser, phenol degrader or from Antarctic soils, Arctic cyanobacterial mat, Arctic rhizosphere, Atacama desert, tufa
327 2.9 Polaromonas sp.
BAC104 EU130986.1 99%
P. sp. BAC104 is from a water treatment activated carbon filter. Closely related to species from glacial meltwater & oligotrophic denitrifyers from upland soil
304 2.7 Pseudomonas stutzeri RCH2
CP003071 99%
P.stutzeri from Arctic sea ice. Closely related to species known to denitrify, emit nitrous oxide emitter, be resistant to heavy metals, solubilise phosphate, or degrade humics and organics, or isolated from the rhizosphere or Alaskan soil
Glycerol phosphate Day 4
7,874 58.3 Pseudomonas
mandelii CP005960.1 100%
P. mandelii is a cold adapted strain. Closely related species from wetlands, CaCO3-resistant, roots, cold desert soil, rice paddy soil, rhizosphere
1,112 8.2 Pseudomonas
migulae KF857261.1 100%
P. migulae is a groundwater cryophile capable of aerobic denitrification. Closely related species are a cyrophilic nitrogen fixer, arsenite oxidiser, phenol degrader or from Antarctic soils, Arctic cyanobacterial mat, Arctic rhizosphere, Atacama desert, tufa
577 4.3 Pseudomonas
koreensis LK391522.1 100%
P.koreensis from a study on biofilm induction in culturable bacteria. Closely related species from a wetland, cold desert soil, rhizosphere, Arctic cyanobacterial mat, snow pit, tufa
302 2.2 Pseudomonas stutzeri RCH2
CP003071 99%
P.stutzeri from Arctic sea ice. Closely related to species known to denitrify, emit nitrous oxide emitter, be resistant to heavy metals, solubilise phosphate, or degrade humics and organics, or from the rhizosphere or Alaskan soil
229 1.7 Hydrogenophaga
sp. 7B-224 KF441666.1 99%
H. sp. 7B-224 from a uranium-contaminated mine. Closely related species known to transform arsenic and metabolise N, facultative autotrophic hydrogen oxidiser, or isolated from a carbonate cave, tufa, magnetite mine drainage, activated sludge
Glycerol phosphate Day 14
2,623 39.7 Pseudomonas
mandelii CP005960.1 100%
P. mandelii is a cold adapted strain. Closely related species from wetlands, CaCO3-resistant, roots, cold desert soil, rice paddy soil, rhizosphere
S4
Number of clones
% of clones
Closest phylogenetic
relative
Accession number
ID similarity Environment
981 14.8 Pelosinus UFO1 CP008852.1 95%
P.UFO1 isolated from pristine US DOE Oak Ridge sediments. Closely related species from uranium & heavy metal contaminated soils, an Fe(III)-reducing microbial community in U(VI) contaminated soils, subsurface response to acetate amendment, uranium mine
527 8.0 Pelosinus UFO1 CP008852.1 95%
P.UFO1 isolated from pristine US DOE Oak Ridge sediments. Closely related species from uranium & heavy metal contaminated soils, an Fe(III)-reducing microbial community in U(VI) contaminated soils, subsurface response to acetate amendment, uranium mine
270 4.1 Pseudomonas
migulae KF857261.1 100%
P. migulae is a groundwater cryophile capable of aerobic denitrification. Closely related species are a cyrophilic nitrogen fixer, arsenite oxidiser, phenol degrader or from Antarctic soils, Arctic cyanobacterial mat, Arctic rhizosphere, Atacama desert, tufa
242 3.7 Pelosinus UFO1 CP008852.1 96%
P.UFO1 isolated from pristine US DOE Oak Ridge sediments. Closely related species from uranium & heavy metal contaminated soils, an Fe(III)-reducing microbial community in U(VI) contaminated soils, subsurface response to acetate amendment, uranium mine
Glycerol phosphate Day 92
1,215 11.6 Bacteriodales
bacterium PB90-2 AJ229236.1 93%
PB90-2 is from rice paddy soil. Nearest identified species is Suxiuginia faeciviva (88% similarity) from organic and methane rich sediment
964 9.2 Rhizobium gallicum
AY972457.1 100% R. gallicum from activated sludge. Closely related species are from rhizosphere, denitrifying bacterial community
837 8.0 Aztobacter
chroococcum CP010415.1 99%
A. Chroococcum is a nitrogen fixing species from the rhizosphere
567 5.4 Magnetospirillum
bellicus NR_116009.
1 95%
M. bellicus from a bioelectrical reactor. Closely related species from succinate assimilating population in denitrifying rice paddy soil, chlorinated solvent remediation site, straw decomposition, rice paddy soil, rhizosphere
552 5.3 Magnetospirillum
magneticum AP007255.1 97%
M. magneticum is a facultative anaerobic magnetotatic bacterium. Closely related to aromatic degrader, phenol degrader, aerobic magnetic bacterium
Glycerol Day 92
315 6.5 Hydrogenophaga
defluvii NR_029024.
1 99%
H. defluvii is from activated sludge. Closely related to uncultured species from petroleum contaminated Arctic soils, tufa, water treatment activated carbon filter
295 6.1 Pseudomonas
mandelii CP005960.1 100%
P. mandelii is a cold adapted strain. Closely related species from wetlands, CaCO3-resistant, roots, cold desert soil, rice paddy soil, rhizosphere
206 4.2 Bacteriodales
bacterium PB90-2 AJ229236.1 93%
PB90-2 is from rice paddy soil. Nearest identified species is Suxiuginia faeciviva (88% similarity) from organic and methane rich sediment
109 2.2 Uncultured
bacterium clone B72_H09
FJ458030.1 97%
Clone B72_H09 is from a reactive Fe barrier enabling microbial dehalorespiration of 1,2-DCE. Closely related to uncultured species from the US DOE Hanford aquifer, rice paddy soil, anaerobic digestor. Nearest identified species is Lutispora thermophila (87% similarity) from a thermophilic methanogenic bioreactor
106 2.2 Uncultured
bacterium clone N2_5_446
JQ146511.1 97%
Clone N2_5_446 is from an anaerobic digestor. Closely related to uncultured species from lake sediment, rich paddy soil, PAH degrading community, anaerobic dechlorinator. Nearest identified species is Butryicimonas virosa (84% similarity) from rat faeces
S5
Figure S1. Volatile fatty acids generated during the biodegradation of 10 mM glycerol
phosphate and 10 mM glycerol in sediment microcosms containing 0.05 mM U(VI)
S6
Figure S2. Comparison of rates of U(VI) removal and Fe(III)-reduction following addition of
different electron donors
S7
Figure S3. ESEM images of biostimulated sediment. Upper left - GSE image showing
general sediment material containing Si, O, Al, Ca, C, Na, Mg, P, S, K, Fe. Upper right - BSE
image of quartz grain, composition dominated by Si and O, but also containing C, Al, K, Ca,
Ti, Fe. Lower left - BSE image of the same region as the upper left image showing a bright
spot rich in Fe. Lower right - BSE image of general sediment showing micron-sized cell
shaped objects (highlighted with red arrows) rich in Fe
S8
Figure S4. ESEM elemental mapping of an area rich in uranium showing correlation with P
and co-location with Fe and Ti rich areas.
S9
Figure S5. Crystal structure of ningyoite (Dusausoy et al. 1996 and Rui et al. 2013). The
Inorganic Crystal Structure Database (http://icsd.cds.rsc.org) was used to calculate U-U bond
distances, via the Jmol function (Copyright © FIZ Karlsruhe and A. Hewat, 2009). This gave
estimates of 5.22 Å and 5.74 Å; we did not observe peaks above background at these atomic
distances in our EXAFS spectra.
S10
Figure S6. Comparison of EXAFS spectra for the glycerol phosphate stimulated sediment
(BLUE) and for sediment where monomeric U(IV) dominates (GREY) (Newsome et al.
2015). Data presented are k3 weighted EXAFS and non-phase shift corrected Fourier
transform of EXAFS. Both experiments were conducted using the same sediment sample and
experimental conditions, and each sample was analysed after approximately 90 days. The
ningyoite like spectrum has clear features in the Fourier transform at 2.7 and 3.2 Å which are
also present in published spectra for chemogenic U(IV) phosphate (Alessi et al. 2014), but are
absent in the monomeric U(IV) spectrum and also in other monomeric U(IV)-phosphate
spectra in the literature (Bernier-Latmani et al. 2010, Alessi et al. 2014).
S11
Figure S7. Comparison of EXAFS spectra for the U(IV) phosphate mineral generated via
glycerol phosphate biostimulation (BLUE), and post-oxygen reoxidation (GREEN). Data
presented are k3 weighted EXAFS and non-phase shift corrected Fourier transform of
EXAFS. The features of both spectra were fitted using the ningyoite crystal structure. Data
were collected during different beamtime allocations.
S12
Figure S8. Bacterial phylogenetic diversity within Sellafield sediments after stimulation with
glycerol phosphate or glycerol. Phyla/classes detected at greater than 1 % of the bacterial
community are illustrated
Figure S9. Rarefaction curves showing sample diversity at n = 4,800 reads
0
100
200
300
400
500
600
700
800
900
0 1000 2000 3000 4000 5000
Nu
mb
er o
f O
TU
s
Number of reads
Glycerol phosphate Day 0
Glycerol phosphate Day 4
Glycerol phosphate Day 14
Glycerol phosphate Day 92
Glycerol Day 92
S13
SUPPORTING INFORMATION REFERENCES
Alessi, D. S.; Lezama-Pacheco, J. S.; Stubbs, J. E.; Janousch, M.; Bargar, J. R.; Persson, P.;
Bernier-Latmani, R. The product of microbial uranium reduction includes multiple species
with U(IV)–phosphate coordination. Geochim. Cosmochim. Acta 2014, 131, 115–127.
Bernier-Latmani, R.; Veeramani, H.; Dalla Vecchia, E.; Junier, P.; Lezama-Pacheco, J. S.;
Suvorova, E. I.; Sharp, J. O.; Wigginton, N. S.; Bargar, J. R. Non-uraninite products of
microbial U(VI) reduction. Environ. Sci. Technol. 2010, 44, 9456–9462.
Dusausoy, Y.; Ghermani, N.-E.; Podor, R.; Cuney, M. Low-temperature ordered phase of
CaU(PO4)2: synthesis and crystal structure. Eur. J. Mineral. 1996, 8, 667–674.
Newsome, L.; Morris, K.; Shaw, S.; Trivedi, D.; Lloyd, J. R. The stability of microbially
reduced U(IV); impact of residual electron donor and mineral ageing. 2015, 409, 125–135.
Rui, X.; Kwon, M. J.; O’Loughlin, E. J.; Dunham-Cheatham, S.; Fein, J. B.; Bunker, B. A.;
Kemner, K. M.; Boyanov, M. I. Bioreduction of hydrogen uranyl phosphate: Mechanisms
and U(IV) products. Environ. Sci. Technol. 2013, 47, 5668–5678.