biostimulation by glycerol phosphate to precipitate

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 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


7,874 58.3 Pseudomonas




migulae KF857261.1 100%


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





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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%


270 4.1 Pseudomonas

migulae KF857261.1 100%


242 3.7 Pelosinus UFO1 CP008852.1 96%



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



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 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.

biostimulation by glycerol phosphate to precipitate

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