S1

Supporting Information

Hydroxylamine Diffusion Can Enhance N2O Emissions in Nitrifying Biofilms:

A Modeling Study

Fabrizio Sabba1, Cristian Picioreanu

2, Julio Pérez

2,3, Robert Nerenberg

1*

1Department of Civil and Environmental Engineering and Earth Science, University of Notre

Dame, 156 Fitzpatrick Hall, Notre Dame, IN 46556 USA

2Department of Biotechnology, Faculty of Applied Sciences, Delft University of Technology,

Julianalaan 67, 2628 BC Delft, The Netherlands

3Department of Chemical Engineering, Universitat Autonoma de Barcelona, Engineering School,

Ed. Q, 08193, Bellaterra, Spain

*Corresponding author:

Robert Nerenberg, Phone: +1 574 631 4098; fax +1 574 631 9236; e-mail: rnerenbe@nd.edu

The following are included as supporting information for this paper:

Number of pages: 19

Number of supplementary sections: 2

Number of figures: 6

Number of tables: 4

S2

SI 1. Methods – Numerical model

The model for N2O production in biofilms developed here is based on traditional diffusion-

reaction material balances for the relevant chemical species in the biofilm and in continuous

ideally-mixed reactor, by incorporating the existing AOB kinetic model from Ni et al. (2014). A

one-dimensional stationary biofilm with fixed thickness LF was assumed in all cases, thus

without including any biomass growth, attachment or detachment. The model was constructed in

a planar geometry, except for the case evaluating N2O production in granular (spherical)

biofilms. For the standard case, only ammonia-oxidizing microorganisms (AOB) were

considered, with biomass uniformly distributed throughout the biofilm and constant

concentration CF,XAOB (kg biomass/m3 biofilm).

1. Solute balances in the biofilm

Six model solutes (index i) were included, with concentrations CF,i (mol/m3 biofilm): dissolved

oxygen (O2), ammonia (NH3), hydroxylamine (NH2OH), nitrite (NO2-), nitric oxide (NO) and

nitrous oxide (N2O). Time-dependent mole balances for all solutes in the biofilm (eq.(1))

included rates of reaction and transport by diffusion:

, 2

, ,

F i

F i F i i

CD C r

t

(1)

with 2 2 2x for planar and 2 2 2 2x x x for spherical biofilms. The effective

diffusion coefficients in the biofilm, DF,i, were chosen smaller (typically 50%) than those in

aqueous phase, Daq,i. The net rates for each soluble component, ri, result from the process

stoichiometry and kinetics (according to the AOB reaction model from Ni et al., 2014), as

provided in the supplementary information, Table S1 and Table S2. For example, the net N2O

S3

production rate was expressed as 4 60.5 0.5N2Or r r , and the net hydroxylamine rate was

1 2NH2OHr r r .

The boundary condition at the biofilm support or at the granule center (x=0) was set as

zero-flux for all solutes, eq. (2):

,

,

0

0F i

F i

x

CD

x

(2)

For simplicity of model analysis, the external mass transfer resistance (diffusion boundary layer)

was neglected for all solutes. Therefore, the concentration at the biofilm surface (x=LF) was

equal with that in the reactor bulk liquid, eq. (3):

, ,F i F B iC x L C (3)

with CB,i in mol/m3 liquid.

2. Redox mediator balances in the biofilm

The redox mediator concentrations in the biofilm, CF,Mox and CF,Mred (mol/kg biomass) were

calculated from the time-dependent balances, eq. (4). The mediator balances only include the net

reactions ri (with iMred or Mox) and no transport because these compounds are immobilized in

the stationary microbial phase:

,

,

F i i

F XAOB

C r

t C

(4)

As explained in Ni et al. (2014), results are independent of the constant value chosen for the

total mediator concentration , , ,F MedT F Mox F MredC C C because the affinity coefficients were all

scaled with this concentration.

S4

3. Solute balances in the bulk liquid

With the exception of oxygen, set by aeration at a constant value CB,O2, the solute concentrations

in bulk liquid resulted from the time-dependent balance, eq. (5) (i=NH3, NH2OH, NO2-, NO):

,

, ,

B i Fin i B i i

B B

C AQC C J

t V V

(5)

For N2O, assumed to be stripped by aeration, an additional transfer term was included in the N2O

balance in liquid, such that:

,

, , , ,

B N2O Fin N2O B N2O N2O L G N2O N2O B N2O

B B

C AQC C J k a C H C

t V V

(6)

The influent flow rate Q, influent concentrations Cin,i, liquid volume VB and biofilm surface area

AF were constant (Table S3). The flux exchanged with the biofilm was calculated at the biofilm

surface as , ,( / ) Fi F i F i x LJ D C dx , for all solutes including N2O.

4. N2O balance in the gas

An additional balance equation, eq. (7), was solved for the gas phase concentration of N2O,

CG,N2O (mol/m3 gas).

,

, , ,0G N2O G L

B N2O L G N2O N2O B N2O

G G

C Q VC k a C H C

t V V

(7)

The gas flow rate QG, head gas volume VG, gas-liquid mass transfer coefficient kLa and Henry

coefficient HN2O were all constant. As initial values, all concentrations in biofilm, bulk liquid and

gas were chosen equal to the corresponding influent concentrations.

S5

5. Model solution

The model was implemented in COMSOL Multiphysics (v4.4, Comsol Inc., Burlington, MA).

Although the model analysis could have been entirely executed in the more familiar AQUASIM

computer package (Reichert et al, 1994), COMSOL was chosen for its flexible modeling

interface, superior high numerical capabilities, data processing and visualization. Model

equations were solved with variable time step on a biofilm domain discretized with a maximum

mesh size of 1 m. The simulation times were in the order of seconds per case. All reported

steady state results were in all conditions obtained after maximum 3 days.

SI 2. Methods – Experimental setup

The pilot plant was an airlift granular sludge reactor with a capacity of 150 L. Previous to the

N2O measuring campaign, the reactor had been operating for ca. 100 days oxidizing ca. 95% of

the influent ammonium to nitrite, with negligible production of nitrate. The hydraulic retention

time (HRT) was maintained within the range of 0.4-0.6 d and the sludge residence time (SRT)

was kept at 50 d. The nitrogen loading rate (NLR) of 0.85 g N/L/d at 30ºC, a biomass

concentration of 5 g MLVSS/L, and a mean granule size of 0.5 mm (Tora et al., 2013). More

details about the reactor, start-up and reactor operation can be found in Tora et al. (2013). The

wastewater contained TAN 726 50 mg N/L, total organic carbon (TOC) 240 – 696 mg C/L

(although only 5% was biodegradable COD), total inorganic carbon (TIC) 358 – 723 mg C/L,

total nitrite nitrogen (TNN=NO2--N + HNO2-N) 2 – 7 mg N/L, NO3

- 0 mg N/L, MLSS 122 – 239

mg/L, MLVSS 100 – 206 mg/L; pH 8.1 – 8.8.

S6

Table S1. Stoichiometry matrix of the reaction model (according to Ni et al., 2014)

Components

Reactions O2 NH3 NH2OH N2O NO NO2

- NO3

- Mred Mox Rates

1. Ammonia

oxidation (AMO) -1 -1 1 -1 1 r1

2. Hydroxylamine

oxidation (HAO) -1 1 1.5 -1.5 r2

3. Nitric oxide

oxidation -1 1 0.5 -0.5 r3

4. Nitric oxide

reduction (NOR) 0.5 -1 -0.5 0.5 r4

5. Oxygen

reduction -0.5 -1 1 r5

6. Nitrite reduction

(NirK, NOR) 0.5 -1 -1 1 r6

Table S2. Reaction rates for the model of Ni et al. (2014)

Reactions Rates

1. Ammonia

oxidation (AMO)

, , ,

1 , , ,

, , , , , , , ,

F O2 F NH3 F Mred

AOB NH3 ox F XAOB

AOB O2 NH3 F O2 AOB NH3 F NH3 AOB Mred 1 F Mred

C C Cr k C

K C K C K C

2. Hydroxylamine

oxidation (HAO)

, ,

2 , , ,

, , , ,

F NH2OH F Mox

AOB NH2OH ox F XAOB

AOB NH2OH F NH2OH AOB Mox F Mox

C Cr k C

K C K C

3. Nitric oxide

oxidation

, ,

3 , , ,

, , , , ,

F NO F Mox

AOB NO ox F XAOB

AOB NO ox F NO AOB Mox F Mox

C Cr k C

K C K C

4. Nitric oxide

reduction (NOR)

, ,

4 , , ,

, , , , , ,

F NO F Mred

AOB NO red F XAOB

AOB NO red F NO AOB Mred 2 F Mred

C Cr k C

K C K C

5. Oxygen

reduction

, ,

5 , , ,

, , , , , ,

F O2 F Mred

AOB O2 red F XAOB

AOB O2 red F O2 AOB Mred 3 F Mred

C Cr k C

K C K C

6. Nitrite reduction

(NirK, NOR)

, ,

6 , , , 2

, , , , , , , ,/

F NO2 F Mred

AOB NO2 red F XAOB

AOB NO2 F NO2 F NO2 AOB I NO2 AOB Mred 4 F Mred

C Cr k C

K C C K K C

S7

Table S3. Model parameters in the base case

Parameter Symbol Value Units Source

Maximum rate coefficients

- NH3 oxidation, AOB kAOB,NH3,ox 14.75 mmol g-1

h-1

Ni et al. (2014), Law et al. (2012)

- NH2OH oxidation, AOB kAOB,NH2OH,ox 22.86 mmol g-1

h-1

Ni et al. (2014), Law et al. (2012)

- NO oxidation, AOB kAOB,NO,ox 22.86 mmol g-1

h-1

Ni et al. (2014), Law et al. (2012)

- O2 reduction, AOB kAOB,O2,red 48.02 mmol g-1

h-1

Ni et al. (2014)

- NO2- reduction, AOB kAOB,NO2,red 3.06 mmol g

-1 h

-1 Ni et al. (2014)

- NO reduction, AOB kAOB,NO,red 0.016 mmol g-1

h-1

Ni et al. (2014)

Half-saturation rate coefficients

- O2 in NH3 oxidation, AOB KAOB,O2,NH3 0.019 mmol L-1

Ni et al. (2014), Law et al. (2012)

- NH3 oxidation, AOB KAOB,NH3 0.17 mmol L-1

Ni et al. (2014), Law et al. (2012)

- NH2OH oxidation, AOB KAOB,NH2OH 0.05 mmol L-1

Ni et al. (2014), Law et al. (2012)

- NO oxidation, AOB KAOB,NO,ox 0.0006 mmol L-1

Ni et al. (2014), Law et al. (2012)

- O2 reduction, AOB KAOB,O2,red 0.0019 mmol L-1

Ni et al. (2014), Law et al. (2012)

- NO2- reduction, AOB KAOB,NO2 0.01 mmol L

-1 Ni et al. (2014), Law et al. (2012)

- NO reduction, AOB KAOB,NO,red 0.0006 mmol L-1

Ni et al. (2014), Law et al. (2012)

- Mox in NO oxidation, AOB KAOB,Mox 0.01CT,Med mmol L-1

Ni et al. (2014), Pan et al. (2013)

- Mred in NH3 oxidation, AOB KAOB,Mred,1 0.001CT,Med mmol L-1

Ni et al. (2014)

- Mred in NO reduction, AOB KAOB, Mred,2 0.001CT,Med mmol L-1

Ni et al. (2014), Pan et al. (2013)

- Mred in O2 reduction, AOB KAOB,Mred,3 0.069CT,Med mmol L-1

Ni et al. (2014)

- Mred in NO2- reduction, AOB KAOB,Mred,4 0.19CT,Med mmol L

-1 Ni et al. (2014)

- NO2- inhibition, AOB KAOB,I,NO2 3.45 mmol L

-1 Ni et al. (2014)

Diffusion coefficients in water

at 30 C

- oxygen Daq,O2 2.6710-9

m2 s

-1 CRC Handbook (2014) (b)

- ammonia Daq,NH3 2.1610-9

m2 s

-1 CRC Handbook (2014) (a)

- hydroxylamine Daq,NH2OH 2.3810-9

m2 s

-1 Zare et al. (2007)

- nitrous oxide Daq,N2O 2.8410-9

m2 s

-1 CRC Handbook (2014) (b)

- nitric oxide Daq,NO 2.4410-9

m2 s

-1 Zacharia and Deen (2005)

- nitrite Daq,NO2 2.1910-9

m2 s

-1 CRC Handbook (2014) (a)

Reduction factor diffusion

coefficients in biofilm fdiff 0.5 -

various

Horn and Morgenroth (2006)

Oxygen concentration in

influent and bulk liquid Cin,O2, CO2

0.001 to 5

(varied) mg L

-1 typical range

Ammonia concentration in

influent

Cin,NH3

80

(base case)

40, 160

(varied)

mgN L-1 typical values

main stream treatment

Hydroxylamine, nitrous oxide,

nitric oxide, nitrite, nitrate in

influent

Cin,i 0 mg L-1

chosen

Initial concentrations

C0,i Cin,i mg L-1

chosen

S8

Biomass concentration in the

biofilm CF,XAOB

50

(base case)

g L-1

typical value, Wanner et al (2006)

Concentration total redox

mediators CT,Med 0.01 mmol g

-1 Ni et al. (2014), Pan et al. (2013)

Biofilm thickness LF

100

(base case)

2, 20, 50, 200

(varied)

m typical values

Liquid flow rate Q 11 mL min-1

reactor F. Sabba

Liquid volume in the reactor VB 4 L reactor F. Sabba

Biofilm surface area AF 0.5 m2 reactor F. Sabba

Gas volume VG 3.5 L reactor F. Sabba

Gas flow rate QG 2 L min-1

reactor F. Sabba

Gas-liquid mass transfer coeff. kLa 100 h-1

chosen

Henry gas-liquid coefficient

N2O (30 ºC) HN2O 0.621 mol/mol CRC Handbook (2014) (c)

S9

Table S4. Model parameters changed for comparison with experimental data from

Pijuan et al. (2014)

Parameter Symbol Value Units Source

Oxygen concentration in

influent and bulk liquid

Cin,O2, CO2 0.5 to 8 mg L-1

experimental

Ammonia concentration in

influent

Cin,NH3 730 mgN L-1 experimental as for

side stream treatment

Set ammonia concentration in

bulk liquid

(controlled by flow rate)

CNH3

40

mgN L-1

experimental

AOB biomass concentration in

the biofilm

CF,XAOB 100 g L-1

experimental

Average granule radius LF 250 m experimental

Liquid flow rate Q variable L min-1

varied to control bulk NH3

concentration

Liquid volume in the reactor VB 135 L experimental

Biofilm surface area AF 90 m2 experimental

Gas volume VG 10 L experimental

Gas flow rate QG 50 L min-1

experimental

S10

Figure S1. Comparison of N2O production rates, per unit reactor volume and time, as a function

of bulk DO for planar and spherical biofilms of different thicknesses. (a) A spherical biofilm

(e.g. aerobic granular sludge) with the same thickness and total biomass volume as an equivalent

planar biofilm would have a 3-times larger surface area, thus allowing for more NH3 conversion,

less NH3 in the bulk liquid and narrower N2O production peak. However, if the spherical biofilm

has the same total volume and area as the planar one (i.e., by increasing 3 times the granule

radius), the N2O production would be comparable with the planar case (inner regions are inactive

in both planar and spherical biofilms). (b) Spherical biofilms of variable size were analyzed

here, while keeping same total biomass in the reactor by changing the number of granules.

Because the smaller the granule size the more area would result when reaching the same biomass

amount, more NH3 conversion is possible leading to NH3 limitation below a certain biofilm

thickness (20 m here). No N2O production peak can be observed at the limit (2 m)

corresponding to suspended biomass. Results for a similar planar biofilm case are presented in

Figure 2a.

Afs=3Afp Lfs=3Lfp

(a) (b)

S11

(a)

(b)

(c)

(d)

(e)

3

3

S12

Figure S2. (a-c) Effect of the set dissolved oxygen concentration at different influent ammonia

concentrations (40, 80 and 160 mg N-NH3/L). (a) N2O production rate (per L reactor volume),

(b) % N-N2O produced per N-NH3 converted, (c) NH3 concentration in the bulk liquid. (d-e)

Effect of the set dissolved oxygen concentration at different controlled NH3 concentrations in

bulk liquid (2.5, 5 and 10 mg N-NH3/L, from 80 mg N-NH3/L in influent). (d) N2O production

rate (per L reactor volume), (e) % N-N2O produced per N-NH3 converted. The NH3

concentration in bulk cb,NH3 was controlled by varying the flow rate Q following the method

proposed in Jemaat et al. (2013): 0 , , ,1 /set NH3 b NH3 set NH3Q Q a c c c . The set NH3

concentrations cs,NH3 were 2.5, 5 and 10 mg N-NH3/L, the reference flow rate Q0 was 1 mL/min

and a sufficiently high value of a was chosen for a fast control action, a=10000.

S13

Figure S3. The net rate of N2O production is effectively determined by the availability of

reduced mediators in the biofilm with not limiting ammonia and nitrite. (a) ammonia

concentration; (b) nitrite concentration; (c) N2O net rate; (d) Mred concentration; (e) O2

concentration; (f) NH2OH concentration. All results were obtained in the standard case

conditions.

NO2-

NH3(a)

(b)

MredrN2O(c) (d)

O2 NH2OH(e) (f)

S14

Figure S4. Solute concentrations and rates in the biofilm. (a) Component concentrations, (b)

net component rates, (c) reaction rates, and (d) electron rates over the biofilm depth. Results are

for the standard case conditions at a bulk DO of 2 mg/L. Compare these results with those

obtained at DO 1 mg/L in Figure 4.

1

23

4

5

6

(c) (d)

1

2

3

5

4, 6

total e-production

total e-consumption

NH3

O2

NO2-

NH2OH

N2O

O2

NO

NH2OH

N2O

(a) (b)

Component rates

Component concentrations

Electron rates

Reactionrates

S15

Figure S5. Effect of effective diffusivity on the N2O production rates at different dissolved

oxygen concentrations. (a) Reduced diffusion coefficients for all components in the biofilm (Df),

compared to diffusivities in water (Daq), would allow less oxygen penetration, thus widen the DO

interval with large N2O production. (b) A 10-times decrease in the NH2OH diffusion coefficient

only, eliminates the peak of N2O production at low DO. In this case, which would be possible if

NH2OH did not diffuse outside cells, the NH2OH does not reach into the anoxic biofilm zone.

Hydroxylamine diffusion in the biofilm appears to be essential for a surge in N2O production.

(a) (b)

S16

a)

b)

Figure S6a,b: Sensitivity analyses for the kinetic parameters in the AOB rate expressions r1 (a)

and r2 (b). The parameters was either left as in the base case, increased two-fold, or reduced by

one half.

S17

c)

d)

Figure S6c,d: Sensitivity analyses for the kinetic parameters in the AOB rate expressions r3 (c)

and r4 (d). The parameters was either left as in the base case, increased two-fold, or reduced by

one half.

S18

e)

f)

Figure S6e,f: Sensitivity analyses for the kinetic parameters in the AOB rate expressions r5 (e)

and r6 (f). The parameters was either left as in the base case, increased two-fold, or reduced by

one half.

S19

REFERENCES

CRC Handbook of Chemistry and Physics, 95th

Edition, 2014. (a) Section 5: Ionic Conductivity

and Diffusion at Infinite Dilution, (b) Section 6: Diffusion of gases in water, (c) Section 5:

Solubility of selected gases in water. Online at http://www.hbcpnetbase.com/

Horn, H.; Morgenroth, E. Transport of oxygen, sodium chloride, and sodium nitrate in biofilms.

Chemical Engineering Science. 2006, 61(5):1347-1356.

Jemaat, Z.; Bartoli, A.; Isanta, E.; Carrera, J.; Suarez-Ojeda, M.E.; Perez, J. Closed-loop control

of ammonium concentration in nitritation: Convenient for reactor operation but also for

modeling. Bioresource Technology. 2013, 128, 655-663.

Law, Y.; Ni, B. J.; Lant, P.; Yuan, Z. Nitrous oxide (N2O) production by an enriched culture of

ammonia oxidizing bacteria depends on its ammonia oxidation rate. Water Research. 2012,

46(10) 3409-3419.

Ni, B.; Peng, L.; Law, Y.; Guo, J.; Yuan, Z. Modeling of nitrous oxide production by autotrophic

ammonia-oxidizing bacteria with multiple production pathways. Environmental Science and

Technology. 2014, 48 (7), 3916-3924.

Pan, Y.; Ni, B.J.; Yuan, Z. Modeling electron competition among nitrogen oxides reduction and

N2O accumulation in denitrification. Environmental Science and Technology. 2013, 47(19),

11083-91.

Reichert, P. AQUASIM, a tool for simulation and data analysis of aquatic systems. Water

Science and Technology. 1994, 30(2):21-30.

Tora, J.A.; Moline, E.; Carrera, J.; Perez, J. Efficient and automated start-up of a pilot reactor

for nitritation of reject water: From batch granulation to high rate continuous operation.

Chemical Engineering Journal. 2013, 226:319-325.

Wanner, O.; Eberl, H.J.; Morgenroth, E; Noguera, D.; Picioreanu, C.; Rittmann, B.E.; Van

Loosdrecht, M.C.M. IWA Task Group on Biofilm Modeling. Mathematical Modeling of

Biofilms. IWA Scientific and Technical Report No.18, IWA Publishing, 2006. ISBN

1843390876.

Zacharia, I.G.; Deen, W.M. Diffusivity and solubility of nitric oxide in water and saline. Annals

of Biomedical Engineering 2005, 33, 214–222.

Zare, H.R.; Sobhani, Z.; Mazloum-Ardakani, M. Electrocatalytic oxidation of hydroxylamine at

a rutin multi-wall carbon nanotubes modified glassy carbon electrode: Improvement of the

catalytic activity. Sensors and Actuators B, 2007, 126(2), 641-647.