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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: [email protected]
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
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