! 1!

SUPPLEMENTARY INFORMATION for 1!

2!

Orenia metallireducens sp. nov. strain Z6, a Novel Metal-reducing Firmicute from the Deep 3!

Subsurface 4!

5!

Yiran Donga,b,c#, Robert A. Sanfordb, Maxim I. Boyanovd,e, Kenneth M. Kemnerd, Theodore 6!

M. Flynnd, Edward J. O’Loughlind, Yun-juan Changa,f, Randall A. Locke IIg, Joseph R. 7!

Weberh, Sheila M. Egani, Roderick I. Mackiea,c,j, Isaac Canna,c,i,j, Bruce W. Foukea,b,c,h,j 8! 9!

Carl R. Woese Institute for Genomic Biology, University of Illinois Urbana-Champaigna; 10!

Department of Geology, University of Illinois Urbana-Champaignb, Energy Biosciences 11!

Institute, University of Illinois Urbana-Champaignc, Biosciences Division, Argonne National 12!

Laboratoryd, Institute of Chemical Engineering, Bulgarian Academy of Sciences, Bulgariae, 13!

High Performance and Research Computing, Office of Information Technology, Rutgers 14!

Universityf, Illinois State Geology Survey, University of Illinois Urbana-Champaigng, 15!

Department of Microbiology, University of Illinois Urbana-Champaignh, Department of 16!

Biochemistry, University of Illinois Urbana-Champaigni, Department of Animal Sciences, 17!

University of Illinois Urbana-Champaignj 18!

19!

Running head: Deep subsurface iron-reducing Orenia strain 20!

21!#Address correspondence to Yiran Dong: 1206 West Gregory Drive, Urbana, IL, USA, 22!

61801. Phone: +1-(217)300-1625. Fax: +1-(217)244-0877. Email: dong5600@illinois.edu 23!

24!

! 2!

MATERIAL AND METHODS 25!

Physiological Characterization. The activity of strain Z6 over pH conditions ranging from 4 26!

to 11 was evaluated via its capacity to reduce ferrihydrite in the ferrihydrite-reducing 27!

medium (FeR medium). The media at pH 4-5 were buffered with 10 mM 2-(N-morpholino)-28!

ethanesulfonic acid (MES) and equilibrated with N2:CO2 (80:20, v:v). Those at pH 6-7 were 29!

buffered with 10 mM piperazine-N,N’-bis(ethanesulfonic acid) (PIPES) and bubbled with 30!

N2:CO2 (80:20, v:v). For pH 8-9 and 10-11, the media were amended with 20 mM 3-[[1,3-31!

dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]propane-1-sulfonic acid (TAPS) and N-32!

cyclohexyl-3-aminopropanesulfonic acid (CAPS), respectively. The media were equilibrated 33!

with N2. After autoclaving and the addition of amendments, the pH was checked again and 34!

manually adjusted to the nominal values using sterile and anoxic 1M HCl or 1 M NaOH 35!

solution if necessary. 36!

The ability of strain Z6 to use other electron acceptors in addition to ferric iron was 37!

evaluated in the modified FeR media by replacing ferrihydrite with other candidate 38!

substrates. The medium contained H2 (202 µmoles/tube) as the electron donor and one of the 39!

following substrates as the alternative electron acceptor (concentrations are 5 mM unless 40!

specified): thiosulfate, sulfite, polysulfide [S(0)], MnO2, chromate (0.5 mM), fumarate, 41!

nitrate, nitrite (2.5 mM) or Co(III)-EDTA (1.5 mM). Elemental sulfur was precipitated from 42!

a 2.25 M (in terms of elemental sulfur recoverable upon acidification) alkaline polysulfide 43!

stock solution (1). MnO2 was synthesized by reacting KMnO4 with MnCl2 (2). Co(III)-EDTA 44!

was prepared according to the method of Dwyer et al. (3). Growth under these conditions was 45!

monitored by changes in color of the cultures, measuring OD600 and quantification of the 46!

substrates. Changes in concentrations for nitrate, nitrite, thiosulfate and sulfite were measured 47!

directly using ion chromatography (Metrohm Inc. USA, FL). For the experiments conducted 48!

with MnO2, Co(III)-EDTA, and chromate, activity of strain Z6 was determined by visual 49!

! 3!

monitoring of changes in the color of the cultures (e.g., brown to reddish-brown precipitates 50!

for MnO2, yellow to clear for chromate and purple to clear for Co(III)-EDTA). The 51!

concentrations of these substrates were also quantified using the methods as described in the 52!

references (4-6). To evaluate the ability of strain Z6 to reduce ferrihydrite and determine if 53!

this activity benefited this isolate, paired cultures with glucose only (fermentation) and 54!

glucose plus 10 mM ferrihydrite (fermentative iron reduction) were developed. At different 55!

time points, 0.2 mL of culture was withdrawn with a N2-flushed sterile syringe to determine 56!

the glucose concentrations using the Glucose (GO) Assay Kit (Sigma-Aldrich Corporation, 57!

MO). For the cultures with both glucose and ferrihydrite, an additional 0.2 mL of culture was 58!

sampled at each time point and promptly acidified with 0.2 mL of 1M HCl for quantification 59!

of 0.5 N HCl-extractable Fe(II). At the end of the experiment (~14 days), one milliliter of 60!

headspace gas was withdrawn and transferred into a sealed 10 mL glass serum vial to analyze 61!

gaseous fermentation products (e.g., CO2 and H2). In addition, 1.5 mL of well-mixed culture 62!

was collected: 0.5 mL was immediately injected into a 5 mL sealed glass serum vial 63!

containing 100 µL 50 % phosphoric acid to quantify dissolved inorganic carbon (i.e., CO2); 64!

the remaining 1 mL culture was centrifuged at 4000 × g for 2 min before the supernatant was 65!

carefully transferred into a new sterilized microcentrifuge tube for quantification of the 66!

aqueous fermented products (e.g., short-chain fatty acids and alcohols) and the remaining 67!

glucose. 68!

69!

Chemical analyses. The concentration of ferrous iron was measured using the ferrozine 70!

method (7, 8) and absorbance was recorded using a Genesys 20 spectrophotometer (Thermo 71!

Fisher Scientific Inc., MA) at 562 nm. Total iron was measured by reducing ferric iron in the 72!

unfiltered samples with 200 μL of 10% hydroxylamine hydrochloride before quantification 73!

with the ferrozine method (9). If necessary, an additional 0.4 mL of 2.5 M HCl was added to 74!

! 4!

the pre-acidified 0.2 mL samples amended with 0.2 mL 1 M HCl. The acidified mineral 75!

suspension was heated to completely dissolve iron oxides. Fe(III) concentrations were 76!

calculated by subtracting the amount of ferrous iron from total Fe. 77!

Fermentation products (e.g., organic acids and alcohols) were quantified with a high-78!

pressure liquid chromatograph (HPLC, Agilent Technologies 1200 series) equipped with a 79!

Rezex ROA-Organic Acid H+ (8%) column (Phenomenex Inc.). The analytes were eluted 80!

with 5 mM H2SO4 at 42 °C. The concentrations of CO2 and H2 were measured using an SRI 81!

8610C Gas Chromatograph (SRI Instruments, CA) coupled with a sequential thermal 82!

conductivity detector (TCD) discharged into a SRI Model 810C reduction gas detector 83!

(RGD) (SRI Instruments, CA). The ranges for quantification of CO2 and H2 were 1000−5000 84!

and 50−250 ppmv, respectively. The rate constants for microbial iron reduction were 85!

calculated using a pseudo-zero-order model for the initial time points, during which changes 86!

in Fe(II) concentration followed a linear relationship. 87!

88!

Membrane Lipid Extraction and Analyses. Membrane lipid composition was analyzed on 89!

the cells grown on the basal medium (4) amended with 10 mM glucose at 42 °C. The cells 90!

were harvested during exponential growth (OD600 approximately 0.5), centrifuged at 1467 × g 91!

for 5 min, and washed twice with chilled phosphate-buffered saline (PBS) (pH = 7.4). Cell 92!

metabolism was promptly quenched by adding 10 mL of pre-chilled methanol (HPLC grade, 93!

Sigma-Aldrich, MO) and metabolite-profiling analyses were performed at the Roy J. Carver 94!

Biotechnology Center of UIUC following the methods used by Singh et al. (2011) (10). 95!

! 5!

Table S1. Geochemistry of formation watera

a The results are cited from our previous study (11); bpH, electrical conductivity, electrical potential, and density were immediately determined after sample collection at atmospheric pressure in the field, while other parameters were measured on pre-treated samples in the lab; The values indicate average of the measurements on replicate samples or in different laboratories; the variation among replicates is less than 7.5 %; c TDS and TN were measured as the total solid content in freeze-dried formation water that passed through a membrane with 0.22 µm pores. dALK indicates alkalinity.

Groundwater (2.02 km)

pHb 6.40 Electrical conductivity (m/s)b 142 Electrical potential (mV)b -84.0 Density (g/mL)b 1.1375 Fe(II) (mM) 1.37±0.02 TOC (mg C/L) 56.5±1.2 TDS (g/L)c 258±7 Ions Cl- (mg/L) 120438 Br-(mg/L) 713 SO4

2-(mg/L) 291 Ca2+(mg/L) 21464! K+(mg/L) 2272 Na+(mg/L) 47156 Zn2+(mg/L) 1.933 ALKd (mg/L CaCO3) 112±22 TN (mg/L)c 19.5±1.4

! 6!

Table S2. Conditions for the physiological characterization of strain Z6a

a10 mM ferrihydrite and 202 µmol/tube H2 were used as the electron acceptor and electron donor, respectively; b The actual pH values determined after pH adjustment.

Investigated variable Range

Other conditions

pH 4.3, 5.2, 6.1, 7.0, 7.9, 8.8, 9.6, 10.6b 0.4 M NaCl, 37 °C Salinity (M NaCl) 0, 0.4, 1.2, 2.0, 2.7, 3.5, 4.3, 5.0, 5.8 pH 7.2, 37 °C

Temperature (°C) 4, 20, 30, 40, 50, 60, 70 pH 7.2, 0.4 M

! 7!

Table S3. Capacity to reduce alternative electron acceptors by strain Z6 and other Orenia speciesa

a The substrates were measured using an ion chromatograph (Metrohm USA, FL) or colorimetric methods at Day 21. H2 (202 µmol/tube) was used as the electron donor and acetate (5 mM) was used as the carbon source; b+, - and +/- indicate positive, negative and marginal response for the physiological properties or activity; cNA: not available or not studied.

Character Strain Z6 Orenia

chitinitropha (60)

Orenia marismortui

(39, 50)

Orenia salinaria

(39)

Orenia sivashensis

(59) Fumarate -b NAc NA NA NA MnO2 + NA NA NA NA

Nitrate - - - - -

Nitrite - - NA NA NA Fe(III)-citrate + NA NA NA NA Ferrihydrite + NA NA NA NA Lepidocrocite + NA NA NA NA Goethite + NA NA NA NA Hematite + NA NA NA NA S(0) - - NA NA + Thiosulfate - +/- +/- - -

Sulfate +/- - - - -

Co(III)-EDTA + NA NA NA NA

K2CrO4 + NA NA NA NA

! 8!

Table S4. Glucose consumption and product formation during fermentation in the absence or presence of ferrihydrite by strain Z6a

Fermentation

alone Fermentation with

ferrihydriteb

Glucose (Initial, mM) 4.23±0.32 4.55±0.03 Glucose (end, mM) 1.18±0.07 -c Formate (mM) 3.00±0.44 2.65±0.42 Acetate (mM) 1.90±0.26 2.61±0.20 Ethanol (mM) 4.21±0.21 5.53±0.16 Lactate (mM) 0.15±0.06 - HCO3

- (mM) 3.00±0.44 6.07±0.30 H2 (µmoles/bottle) 135.7±3.5 88.6±9.2 Fe(II) (mM)d - 5.1

a The results showed the concentrations at the 14th day; b10 mmol/L ferrihydrite was amended; c-: below detection limit (100 µM for glucose and 50 µM for lactate); d 0.5 N HCl extractable Fe(II).

! 9!

Figure S1. Active and control samples indicate that iron reduction was carried out biologically by strain Z6 and not by abiotic reactions. For the cultures in (a), 10 mM ferrihydrite was amended as the electron acceptor; 202 µmol/tube H2 was used as the electron donor. In (b), the cultures were pre-grown under fermenting conditions in the presence of 5 mM glucose for ~72 hours before they were autoclaved and amended with one of the ferric iron oxides (~10 mmol/L). The samples were prepared in duplicate and the error bars indicate standard deviation of the replicates.

0

1

2

3

4

5

0 5 10 15 20 25

Fe(II),(mM)

Time,(Days)

Active

Abiotic,control

Inoculation,control

Heat@killed,control

(a)

0

1

2

3

4

5

0 5 10 15 20 25

Fe(II),(mM)

Time,(Days)

Ferrihydrite

Hematite

Goethite

Lepidocrocite

(b)

! 10!

Figure S2. Glucose consumption (a) and Fe(II) generation (b) by fermentative iron reduction by strain Z6 grown on 5 mM glucose in the presence or absence of 6.8 mM ferrihydrite (AFH). Duplicate samples were prepared and the error bars indicate standard deviation of the replicates.

0

20

40

60

80

100

0 5 10 15

C/C

0 (%

)

Time (days)

Glucose

Glucose+AFH

Control Glucose

Control Glucose+AFH

0

20

40

60

80

0 5 10 15

Fe(II

)/Fe(

III) in

itial

(%)

Time (days)

(a)

(b)

! 11!

Figure S3. Sensitivity to antibiotics by strain Z6 under iron-reducing condition. The control indicated the iron-reducing culture without amendment of antibiotics. In the cultures, 10 mM ferrihydrite and 202 µmol/tube H2 was used as the electron acceptor and electron donor, respectively. 5 mM acetate was amended as the carbon source. Error bars illustrate standard deviation of duplicate samples.

0

2

4

6

8

Control

Ampici

llin

Anisom

ycin

Chlora

mphen

icol

Eryth

rom

ycin

Kanam

ycin

Tetra

cylin

e

Fe(II

) Con

c. (m

M)

! 12!

Figure S4. Ferrihydrite reduction by strain Z6 cultured in the synthetic Orenia medium. Triplicate samples were prepared for each condition and the plots showed average of the replicates and error bars indicated standard deviation of the replicates. In the cultures, 10 mM ferrihydrite and 202 µmol/tube H2 was used as the electron acceptor and electron donor, respectively. 5 mM acetate was amended as the carbon source. The abiotic controls were prepared under the same condition except without cell inoculation.

0

10

20

30

40

50

60

70

0 10 20 30

Con

c. (m

M)

Time (days)

Ferrihydrite

Abiotic control

Fe(II)/Fe(Tot)*(%)

! 13!

!Figure S5. Fe K-edge XANES spectra collected from the reactor solids compared to the corresponding oxidized and reduced iron standards. Blue lines show data from the controls and red lines show data from the inoculated reactors. The shift of the edge position to lower energy indicates the presence of reduced Fe in the culture bottles. The spectrum in green symbols is from siderite (FeCO3) and is used here as the reduced Fe(II) standard. Linear combination fit results are listed in Table 1.

7110 7120 7130 7140 7150-3

-2

-1

0

1

Z6

Hematite

Goethite

Lepidocrocite

Ferrihydrite

N

orm

aliz

ed a

bsor

ptio

n

Incident photon energy (eV)7110 7120 7130 7140 7150

Z9

Lepidocrocite

Ferrihydrite

Ferric oxide std Vivianite Control Reacted

Incident Photon Energy (ev)

! 14!

References

1. Moser DP, Nealson KH. 1996. Growth of the facultative anaerobe Shewanella

putrefaciens by elemental sulfur reduction. Appl Environ Microbiol 62:2100-2105.

2. Murray JW. 1974. The Surface Chemistry of Hydrous Manganese Dioxide. Journal

of Colloid and Interface Science 46:357-371.

3. Dwyer FP, Gyarfas EC, Mellor DP. 1955. The resolution and racemization of

potassium ethylenediaminetetraacetatocobaltate(III). J Phys Chem 59:296-297.

4. Roh Y, Liu SV, Li G, Huang H, Phelps TJ, Zhou J. 2002. Isolation and

characterization of metal-reducing Thermoanaerobacter strains from deep subsurface

environments of the Piceance Basin, Colorado. Appl Environ Microbiol 68:6013-

6020.

5. Zhang CL, Liu S, Logan J, Mazumder R, Phelps TJ. 1996. Enhancement of

Fe(III), Co(III), and Cr(VI) reduction at elevated temperatures and by a thermophilic

bacterium. Appl Biochem Biotechnol 57-8:923-932.

6. Brewer PG, Spencer DW. 1971. Colorimetric determination of manganese in anoxic

waters. Limnol Oceanogr 16:107-110.

7. Gibbs CR. 1976. Characterization and application of ferrozine iron reagent as a

ferrous iron indicator. Anal Chem 48:1197-1200.

8. Stookey LL. 1970. Ferrozine - a new spectrophotometric reagent for iron. Anal Chem

42:779.

9. Lovley DR, Phillips EJ. 1987. Rapid assay for microbially reducible ferric iron in

aquatic sediments. Appl Environ Microbiol 53:1536-1540.

10. Singh AK, Ulanov AV, Li Z, Jayaswal RK, Wilkinson BJ. 2011. Metabolomes of

the psychrotolerant bacterium Listeria monocytogenes 10403S grown at 37 degrees C

and 8 degrees C. Int J Food Microbiol 148:107-114.

! 15!

11. Dong Y, Sanford RA, Locke RA, Cann IK, Mackie RI, Fouke BW. 2014. Fe-oxide

grain coatings support bacterial Fe-reducing metabolisms in 1.7-2.0 km-deep

subsurface quartz arenite sandstone reservoirs of the Illinois Basin (USA). Front

Microbiol 5:511.

12. Moune S, Eatock C, Matheron R, Willison JC, Hirschler A, Herbert R, Caumette

P. 2000. Orenia salinaria sp. nov., a fermentative bacterium isolated from anaerobic

sediments of Mediterranean salterns. Int J Syst Evol Microbiol 50 Pt 2:721-729.

13. Oren A, Pohla H, Stackebrandt E. 1987. Transfer of Clostridium-Lortetii to a New

Genus Sporohalobacter Gen-Nov as Sporohalobacter-Lortetii Comb-Nov, and

Description of Sporohalobacter-Marismortui Sp-Nov. Systematic and Applied

Microbiology 9:239-246.

14. Sorokin DY, Kolganova TV. 2014. Bacterial chitin utilization at halophilic

conditions. Extremophiles 18:243-248.

!