Supporting Information for:
Kinetic parameter estimation in N. europaea biofilms using a 2D reactive transport model
Ellen G. Lauchnor1*, Lewis Semprini2, Brian D. Wood2
1
Center for Biofilm Engineering, 366 EPS, Montana State University, Bozeman, MT 59717
2
School of Chemical, Biological and Environmental Engineering
102 Gleeson Hall, Oregon State University
Corvallis, OR 97331
*Corresponding Author: ellen.lauchnor@biofilm.montana.edu (406)994-2674
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Supplemental Experimental Procedures
Batch experiments
Batch experiments were performed to evaluate ammonia oxidation kinetics using batch cells
harvested after 3 days of growth. Cells were pelleted and rinsed in HEPES buffer (20 mM, pH 7.8) and
re-suspended into 150 mL bottles containing either 1 mM or 20 mM HEPES buffer solution at pH 7.8,
with an (NH4)2SO4 concentration of 0.125, 0.25, 0.5 or 2.5 mM. Rates of ammonia oxidation were
evaluated at NH4-T concentrations of 0.25 – 5 mM by monitoring NO2- production in five minute
intervals for 30 minutes. Rate experiments in low buffered medium (1 mM HEPES) and 2.5 mM
(NH4)2SO4 were conducted in order to model the change in pH and the NH3 oxidation rates during the
shift of pH.
Biofilm reactor
The DFR (BioSurface Technologies Corp., Bozeman, MT) consists of four parallel flow channels
with a glass slide in each channel as a substratum for biofilm growth. The reactors were set on an incline
so that growth medium pumped as a drip onto the coupon surface would slide down the coupon,
providing substrates under a low shear environment. The medium was pumped through the reactors at a
flow rate of 15 mL h-1. Prior to the experiments, biofilms were grown for 6-8 weeks on frosted glass slide
coupons within the DFR channels under continuous medium addition. AOB biofilm growth medium
consisted of the following: 25 mM (NH4)2SO4, 3.77 mM Na2CO3, 20 mM HEPES buffer, 730 µM
MgSO4, 200 µM CaCl2, 9.9 µM FeSO4, 16.5 µM EDTA free acid, and 0.65 µM CuSO4.
Figure S1. Side view of DFR
Microelectrode calibration and measurement
The O2 sensors were Clark-type microelectrodes described in detail elsewhere (Revsbech 1989). A twopoint calibration was performed for O2 sensors using medium saturated with dissolved O2 and sparged
with pure N2(g) for 10 minutes for a zero measurement. The pH microelectrodes were calibrated with
buffered solutions at pH 4.0, 7.0 and 10.0. Calibrations were performed at least once per day to ensure
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accuracy of measurements. A Leica S8 APO Stereozoom microscope (Leica Microsystems, Inc.,
Bannockburn, IL) was used to visualize the biofilm surface while positioning the microelectrodes.
Measuring
chamber
Microsensor
Biofilm
Air
sparging
Recirculation
Media bath
Figure S2. Schematic representation of experimental chamber and recirculating media bath for
microelectrode measurements in AOB biofilms.
Reference
Revsbech NP. 1989. An oxygen microsensor with a guard cathode. Limnology and Oceanography
34(2):474-478.
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Sensitivity analyses and additional supplementary model figures
A
B
Figure S3. Sensitivity analysis of effective diffusivity in the biofilm, using Study I biofilm scenario with
(A) 1 mM NH4-N and (B 5 mM NH4-N. The analysis of the effective diffusivity indicated that no
observable difference was found between effectiveness factors of 0.7 and 1 in the biofilm.
Figure S4. Data and model fit of oxygen in biofilm with no NH4 in the medium, indicating slight oxygen
consumption due to endogenous respiration. The biofilm surface was located at 0 µm and positive values
of depth indicate measurements taken inside biofilm. Data points show microsensor measurements and
the solid line shows the model results of calibrating the endogenous decay rate constant to a value of b =
8.2 e-5 µmol mg prot.-1 s-1.
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Figure S5. Microelectrode measurements of pH in biofilm under well buffered conditions with 5 mM
NH4 in the medium. The biofilm surface was located at 0 µm and positive values of depth indicate
measurements taken inside biofilm. The data verifies that the pH does not change more than 0.2 pH units
in the well-buffered experiments of Study I, with 10 mM HEPES buffer.
Figure S6. Two dimensional representation of modeled dissolved oxygen concentration in biofilm from
Study I with 5 mM NH4-N. The entrance effects can be observed in the first third of the fluid flow path,
but do not appear to influence the oxygen profile in the biofilm along the flow regime. DO and pH
vertical profiles were therefore obtained at least 2.5 cm from the entrance.
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A
B
Figure S7. Study I sensitivity analysis of the half-saturation constant for oxygen, Kso. (A) DO profiles in
AOB biofilm with 1 mM NH4-N and three different Kso values. (B) DO profiles in AOB biofilm with 5
mM NH4-N and three different Kso values. The sensitivity analysis for both 1 and 5 mmol L-1 NH4-T
indicates low sensitivity under these conditions to Kso. The analysis also shows that a higher value of Kso
begins to deviate from the data and does not fit the Study I results with the fitted values of kmax and Ksn.
A
B
Figure S8. Study I sensitivity analysis of the half-saturation constant for ammonia, Ksn at the two values
of 0.04 mM and 0.004 mM. (A) DO profiles in AOB biofilm with 1 mM NH4-N. (B) DO profiles in AOB
biofilm with 5 mM NH4-N. The analysis indicates that at 1 mM total NH4, a lower Ksn value used in
Study III does not fit the data. In the 5 mM NH4-T case, the model is not sensitive to Ksn, thus allowing
the kmax fitting to be independent of the Ksn value.
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