Supplemental Material

Anaerobic Ammonium Oxidation Coupled to Iron Reduction in Constructed

Weitao Shuai and Peter R. Jaffé

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Contents

page

Synthesis of 2-line ferrihydrite S3

Table S1. Vitamin supplement stock solution for the Feammox Enrichment Culture S4

Table S2. Trace element stock solution for the Feammox Enrichment Culture S5

Table S3. Diurnal cycle settings in the environmental growth chamber S6

Table S4. Recipe for the half-strength modified Hoagland nutrient solution S7

Table S5. Total bacteria and A6 numbers in CW mesocosm sediments at the

beginning of the experiment S8

Table S6. Relative abundance of A6 and ammonium-oxidizing bacteria (AOB)

in the high and low Fe CW mesocosms S9

Table S7. Rlative abundance of phyla Planctomycetes and Thaumarchaeota in

the high and low Fe CW mesocosms S10

Table S8. Relative abundance of Geobacter spp. and fraction of A6 to

Geobacter spp. in the high and low Fe CW mesocosms S11

Table S9. Relative abundance of Fe reducing bacteria genera in the high Fe

and low Fe CW mesocosms S12

Figure S1. Vertical profile of NH4+ in the CW mesocosms S13

Figure S2. Fe(III) concentration at different depths in high and low CW mesocosms S14

Figure S3. Total bacteria numbers in the high and low Fe CW mesocosms S15

Figure S4. Microbial community composition at the phylum level in high and

low CW mesocosms S16

Ammonium adsorption onto the high and low Fe sediments at different pH S17

Figure S5. Partition coefficients of ammonium onto the solid strata as a function of pH S17

Figure S6. pH at different depths in the high and low Fe CW mesocosms after

the injection of the A6 enrichment culture S18

Figure S7. Fe(II) concentrations at different depths S19

Ammonium oxidation in the presence of the high and low Fe strata S20

Figure S8. Ammonium removal in the presence of high and low Fe sediments S20

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Synthesis of 2-line ferrihydrite

This protocol was adapted from Schwertmann and Cornell, 2000.

Materials:

Fe(NO3)3.9H2O, KOH 1 M, deionized water, dialysis membrane

Preparation:

1. Dissolve 8 g Fe(NO3)3.9H2O in 100 mL deionized water in a 250-mL flask with a stirring

speed of 500 rpm.

2. Add 60 ml 1 M KOH while stirring to bring pH to 7.0 – 8.0. Slowly add approximately 6

ml 1 M KOH to bring the final pH to 7.0 – 7.5.

3. Stir vigorously overnight with a parafilm top.

4. *Centrifuge everything at 8000 rpm for 15 min and discard the supernatant. Re-suspend

pellet with leftover solution in tube or by adding about 5 ml of deionized water.

* Only do this step if final concentration needs to be above 10 g/L, otherwise go straight to step 6.

5. Transfer the suspension into a dialysis tube and perform a three to five days’ dialysis by

submerging the tubes in deionized water and slowly running deionized water from the

bottom of the container so the excess water overflows at the top.

6. Transfer the content of dialysis tube to a new bottle. Use deionized water to get all solids

out of the bag.

7. Stir the suspension overnight until there are no aggregated big particles.

8. Measure Fe(III) concentration using the ferrozine method.

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Table S4. Recipe for the vitamin supplement stock solution for the Feammox Enrichment

Culture. Dissolve the vitamins listed below in water and dilute to a final volume to 1 L. The

vitamin supplement stock solution is 10,000 x. A volume of 0.1 μL stock solution should be

added to one liter of Feammox enrichment culture medium.

Chemical Amount

Biotin 1000 mg

Nicotinic acid 1500 mg

Cyanocobalamin 1000 mg

ρ(4)-aminobenzoic acid 2500 mg

Thiamine 2500 mg

Pantothenic acid 2500 mg

Riboflavin 2500 mg

Folic acid 1000 mg

Pyridoxin hydrocholoride 1000 mg

Thioctic acid 1000 mg

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Table S5. Recipe for the trace element stock solution for the Feammox Enrichment Culture.

Modified from Sawayama (2006). Dissolve the following amounts of chemicals to make 1 liter

of trace element stock solution. Use 1 mL trace element stock solution in each liter of Feammox

enrichment culture medium.

Chemical Amount

ZnSO4•7H2O                     0.430 g

CoCl2•6H2O 0.240 g

MnCl2 0.629 g

CuSO4•5H2O 0.250 g

Na2MoO4•2H2O 0.220 g

NiCl2•6H2O 0.190 g

Na2SeO4•10H2O 0.210 g

H3BO3 0.014 g

NaWO4•2H2O 0.050 g

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Table S3. Diurnal cycle settings in the environmental growth chamber*

Time Temperature (C) Humidity (%)

06:00 15.0 90

07:00 16.0 90

09:00 21.0 72

10:00 24.0 58

16:00 28.0 39

17:00 28.0 37

19:00 25.0 47

20:00 23.0 55

* Lights were on from 6:00 to 20:00.

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Table S4. Recipe for the half-strength modified Hoagland nutrient solution with 100 mg/L NH4+.

Stock solutionVolume added to

per liter of water

1.00 M NH4H2PO4 0.5 mL

0.50 M K2SO4 3.0 mL

1.00 M CaCl2 2.0 mL

1.00 M MgSO4 1.0 mL

Micronutrient stock 0.5 mL

Iron stock 0.125 mL

1.00 M NH4Cl 6.64 mL

This recipe is modified from Hoagland and Arnon (1950). To avoid introducing nitrate to the

CW mesocosms, NO3- was substituted with other anions such as SO4

- and Cl-. The form of iron

added was changed as described below.

To make the micronutrient stock, combine the following amount of salts in 1 L deionized water:

2.86 g H3BO3

1.81 g MnCl2·4H2O

0.22 g ZnSO4·7H2O

0.08 g CuSO4·5H2O

0.02 g H2MoO4·H2O

To make the iron stock, dissolve 26.1 g EDTA in 286 mL water that has about 19 g KOH.

Dissolve 24.9 g FeSO4·7H2O in 500 mL water. Add the FeSO4 solution slowly to the potassium

EDTA solution and aerate the mixture overnight while stirring. Make the final volume 1 L and

store in an opaque bottle.

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Table S5. Total bacteria and A6 numbers in CW mesocosm sediments at the beginning of the

experiment. Copies of gene were obtained using qPCR with 1055F-1392R primer for total

bacteria and V5 (forward: 5’-GTBCCAGCAGCCGCGGKAA-3’/ reverse: 5’-

AGGGTTRCGCTCGTTG-3’; B: G, C or T; K: G or T/U; R: G or A) primer for A6.

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Total bacteria

(copies of gene / g dry sediment)

A6

(copies of gene / g dry sediment)

High Fe CW 1.14 × 106 ± 3.00 × 104 6.96 × 105 ± 7.41 × 103

Low Fe CW 1.10 × 106 ± 4.79 × 104 7.12 × 105 ± 3.67 × 104

Table S6. Relative abundance of A6 and ammonium-oxidizing bacteria (AOB) in the high and

low Fe CW mesocosms at different depths at the end of the experiment. AOB relative abundance

is the sum of the relative abundance of Nitrosospira and Nitrosococcus.

Depth

(cm)

A6

(fraction in total

sequences)

AOB

(fraction in total

sequences)

A6 to AOB A6 in AOB + A6

High Fe Low Fe High Fe Low Fe High Fe Low Fe High Fe Low Fe

0-3 4.25 × 10-4 3.01 × 10-4 3.62 × 10-4 1.34 × 10-3 1.17 0.22 0.54 0.18

6-9 4.42 × 10-4 1.95 × 10-4 2.53 × 10-3 8.84× 10-5 0.18 2.20 0.15 0.69

12-15 4.83 × 10-4 5.33 × 10-4 1.76 × 10-3 3.90 × 10-4 0.27 1.37 0.22 0.58

18-21 9.88 × 10-4 4.08 × 10-4 2.54 × 10-4 5.03 × 10-4 3.89 0.81 0.80 0.45

24-27 6.68 × 10-4 4.76 × 10-4 5.43 × 10-4 9.12 × 10-4 1.23 0.52 0.55 0.34

30-33 4.92 × 10-4 2.73 × 10-4 9.84 × 10-4 6.56 × 10-4 0.50 0.42 0.33 0.29

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Table S7. Relative abundance of phyla Planctomycetes and Thaumarchaeota in the high Fe and

low Fe CW mesocosms at different depths. Two-tail paired two sample t-test for means were

conducted to compare the microorganisms’ relative abundances in the high Fe and low Fe CW

mesocosms.

PhylumPlanctomycetes

(fraction of total sequences)

Thaumarchaeota

(fraction of total sequences)

Depth (cm) High Fe Low Fe High Fe Low Fe

0-3 3.48 × 10-3 3.21 × 10-3 7.37 × 10-5 0.00

6-9 3.04 × 10-3 9.22 × 10-4 0.00 0.00

12-15 2.51 × 10-3 4.55 × 10-3 0.00 0.00

18-21 4.53 × 10-3 4.37 × 10-3 1.84 × 10-5 1.84 × 10-5

24-27 5.00 × 10-3 3.28 × 10-3 0.00 3.69 × 10-5

30-33 4.70 × 10-3 4.63 × 10-3 7.37 × 10-5 0.00

p-value 0.551 0.363

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Table S8. Relative abundance of Geobacter spp. and fraction of A6 to Geobacter spp. in the

high and low Fe CW mesocosms at different depth. Two-tail paired two sample t-test for means

were conducted to compare the microorganism’s relative abundances in high Fe and low Fe CW

mesocosms.

Geobacter spp.

(fraction of total sequences)

Fraction of A6

to Geobacter spp.

Depth (cm) High Fe Low Fe High Fe Low Fe

0-3 5.11 × 10-3 4.50 × 10-3 0.083 0.067

6-9 5.31 × 10-3 3.32 × 10-3 0.083 0.059

12-15 5.63 × 10-3 2.07 × 10-2 0.086 0.026

18-21 1.38× 10-2 3.71 × 10-2 0.072 0.011

24-27 5.20× 10-3 2.81 × 10-2 0.128 0.017

30-33 2.22 × 10-2 2.84 × 10-2 0.022 0.010

p-value 0.066 0.027*

* relative abundance in high Fe CW mesocosm > relative abundance in low Fe CW mesocosm, p

<0.05

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Table S9. Relative abundance of Fe reducing bacteria genera in the high Fe and low Fe CW

mesocosms at different depth. Two-tail paired two sample t-test for overall means were

conducted to compare the microorganism’s relative abundances between the high and low Fe

CW mesocosms.

Acidobacterium Acidocella Rhizomicrobium GeothrixDepth (cm) High Fe Low Fe High Fe Low Fe High Fe Low Fe High Fe Low Fe

0-3 1.11E-01 9.59E-02 1.12E-02 7.19E-03 6.05E-03 5.10E-03 8.45E-03 4.95E-03

6-9 4.17E-02 1.51E-02 3.46E-02 6.49E-02 5.81E-03 6.26E-03 4.85E-03 4.12E-03

12-15 1.14E-02 9.91E-03 5.14E-03 7.54E-03 1.03E-02 1.50E-02 2.70E-02 4.59E-03

18-21 9.52E-03 9.55E-03 2.75E-03 2.48E-03 1.06E-02 1.39E-02 1.88E-02 4.73E-03

24-27 8.07E-03 9.28E-03 2.76E-03 2.90E-03 1.10E-02 1.44E-02 1.17E-02 4.63E-03

30-33 8.95E-03 8.46E-03 2.82E-03 2.65E-03 9.71E-03 1.21E-02 1.69E-02 4.89E-03

p-value 0.186 0.404 0.049* 0.027**

Bacillus Rhodobacter Paenibacillus AcidibacterDepth (cm) High Fe Low Fe High Fe Low Fe High Fe Low Fe High Fe Low Fe

0-3 1.00E-02 1.24E-02 2.63E-03 2.32E-03 2.98E-03 3.20E-03 8.65E-03 8.01E-03

6-9 2.65E-02 2.86E-03 2.71E-03 3.48E-03 3.72E-03 1.49E-03 2.86E-03 2.18E-03

12-15 6.46E-03 2.85E-03 2.78E-03 3.74E-03 2.24E-03 1.61E-03 2.23E-03 2.01E-03

18-21 4.33E-03 3.82E-03 5.73E-03 1.44E-02 4.66E-03 1.59E-03 1.83E-03 1.65E-03

24-27 3.52E-03 1.40E-02 4.10E-03 1.74E-02 7.11E-03 3.54E-03 2.00E-03 1.46E-03

30-33 7.28E-03 2.60E-03 3.86E-03 5.52E-03 5.97E-03 1.69E-03 2.22E-03 1.38E-03

p-value 0.514 0.123 0.025** 0.005**

Cellulomonas Desulfobulbus AnaeromyxobacterDepth (cm) High Fe Low Fe High Fe Low Fe High Fe Low Fe

0-3 2.32E-03 3.31E-03 1.64E-03 5.41E-03 4.57E-04 4.21E-04

6-9 9.77E-04 1.22E-03 7.74E-04 1.04E-03 3.69E-04 2.30E-04

12-15 1.69E-03 1.44E-03 2.42E-03 1.78E-03 1.54E-03 9.88E-04

18-21 2.09E-03 2.34E-03 7.34E-04 1.38E-03 7.62E-04 9.27E-04

24-27 2.10E-03 5.44E-03 9.63E-04 5.01E-03 2.79E-04 5.85E-04

30-33 3.08E-03 2.90E-03 9.70E-04 4.00E-03 3.23E-04 2.60E-04

p-value 0.242 0.072 0.677

* relative abundance in the high Fe CW mesocosm < relative abundance in the low Fe CW mesocosm, p <0.05.

** relative abundance in the high Fe CW mesocosm > relative abundance in the low Fe CW mesocosm, p <0.05.

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Figure S1. Vertical profile of NH4+ in the CW mesocosms.

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Figure S2. Fe(III) concentration at different depths in high and low CW mesocosms at the end of

the experiment.

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Figure S3. Total bacteria numbers in the high and low Fe CW mesocosms at different depths at

the end of the experiment. Copies of 16s gene were quantified by qPCR using primer set 1055F-

1392R. Error bars denote standard deviation of measurements of triplicated sediment samples

taken at the same depth.

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Figure S4. Microbial community composition at the phylum level at the end of the experiment.

“H” and “L” denote samples from CW mesocosms with high Fe and low Fe respectively.

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Ammonium adsorption onto high and low Fe sediments at different pH

To investigate the effect of pH on the amount of NH4+ sorbed on the two CW mesocosm

sediments, 1 g high or low Fe sediments was mixed with 10 mL 100 mg/L NH 4+ solution in 15

mL centrifuge tubes. The initial pH was adjusted by adding the appropriate amount of 1 N HCl

or 1N NaOH. The range of pH was determined by measurements in the CW mesocosms, which

corresponds to pH 3 to 8. The mixtures were shaken at 200 rpm at room temperature for 1 h, and

then were centrifuged for 10 minutes and filtered before analyzing the NH4+ concentration in

solution. Sorbed mass of NH4+ was calculated as the difference between the initial and final

concentration multiplied by the liquid volume. Sorption experiments at each pH were done in

triplicate.

Figure S5. Partition coefficients of ammonium onto the solid strata as a function of pH. Dotted

lines are logarithmic fit, with R2 = 0.72 and 0.81 for high and low Fe CW mesocosms

respectively.

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Figure S6. pH at different depths in the high and low Fe CW mesocosms after the injection of

the A6 enrichment culture. (A) Depth = 0 cm; (B) depth = 6 cm; (C) depth = 12 cm; (D) depth =

18 cm; (E) depth =24 cm; (F) depth = 30 cm.

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Figure S7. Fe(II) concentrations at different depths in the high Fe (A) and low Fe (B) CW

mesocosms after the injection of the A6 enrichment culture.

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Ammonium oxidation in the presence of the high and low Fe strata

A sludge with high aerobic NH4+ removal efficiency was obtained from a bioreactor which can

oxidize 50 mg/L NH4+-N in 180 mins. A total of 10 g of either high or low Fe CW mesocosm

sediments was added to 100 ml serum vials and mixed with 40 mL nutrient solution (100 mg /L

NH4+-N) and 10 mL bioreactor sludge. Autoclaved control vials with low Fe CW mesocosm

sediments were also prepared. All vials were prepared in triplicate. The vials were capped with

silicone sponge closure to allow air exchange for aerobic ammonium oxidation. The vials were

then incubated at room temperature and shaken at 200 rpm. Samples were taken at different time

to track NH4+concentrations.

The results show that the quantity of ammonium removal and kinetics are the same in the high

and low Fe level CW mesocosm sediments under aerobic conditions. Statistical test shows no

significant difference (two tailed t-test, p > 0.1) in ammonium concentrations between high and

low Fe slurries at any time of the incubation. In the control vials, in which autoclaved sludge was

added, no ammonium removal was detected. Therefore, the aerobic ammonium removal activity

does not seem to be affected by the Fe levels of CW mesocosm sediments.

Figure S8. Ammonium removal in the presence of high and low Fe sediments.

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