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Approach 3: Model including within-guild diversity (2 AOB and 2 NOB) for whole period
As Period A and Period B could be described individually by a single AOB and a single NOB species (see Table 6 for their characteristics), these two AOB and two NOB populations, , were integrated in a single model also containing 1 heterotrophic population.
However, it was not possible to calibrate the resulting model in terms of the bulk liquid
concentrations of ammonium, nitrite and nitrate over the complete experimental period of 592 days (Supplementary Information, Figure S.3). The model predicted nitrate accumulation for the whole experimental period; the overall fit of the simulation results for bulk liquid nitrogen concentrations was very low (χ²tot = 3204); the model efficiencies for total ammonium (ETNH = -0.44), total nitrite (ETNO2 = -1.26) and especially for nitrate (ENO3 = -5.19) were negative.
As for the microbial community composition, the AOB and NOB species of Period A remained dominant also during Period B, while the AOB and NOB species of Period B should become dominant after 100 days. The species of period B did not survive the severe DO drop from day 50 onwards (see Figure S.2 in the Supplementary Information), due to their lower affinity for oxygen than the species from Period A. In the real system, the species of Period B could have invaded the system by attachment from the bulk liquid, while in the model attachment was neglected and the influent was assumed to contain no bacterial species. Alternatively, the populations of Period A could have been acclimated to the conditions in Period B, which also can result in changed
parameter values. Furthermore, as calibrated parameters such as the affinity constants may describe apparent features, lumping other phenomena such as diffusion (Arnaldos et al. 2015), these
phenomena may have been changing over the experimental period.
C o n c l u s i o n s
The influence of process dynamics on the nitrifying microbial community in a biofilm reactor controlled for nitrite accumulation, was analysed.
The correlation analysis confirmed that temperature, pH, bulk liquid oxygen concentration and nitrogen loading rate all constitute suitable control handles for process operation in
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terms of nitrogen conversion.
The experimental observations could only be described with a model considering a single (lumped) AOB and a single NOB, besides a single heterotrophic population, when the microbial parameters for AOB and NOB were calibrated separately for two operating periods resulting from an increased nitrogen loading rate and associated drop in the bulk liquid oxygen concentration. This indicated a change of the AOB and NOB guild.
The diversity of the total bacterial community resembled the diversity of the NOB, while the diversity of the AOB was much lower. A large number of small, subdominant NOB
populations was presumably present in the biofilm. The calibrated AOB parameter values for the two different periods correspond with two different AOB species (Nitrosomonas halophila and Nitrosomonas europaea), while the NOB parameters represent two different NOB guilds.
As revealed through CE-SSCP analysis, the two different control strategies for nitrite accumulation influenced the microbial diversity. As modelling microbial diversity was unnecessary to reproduce the reactor performance during the period in which both control strategies were applied, the changes in diversity did not influence the reactor performance instantaneously. However, one cannot exclude that such changes in diversity influence the reactor performance and process stability in the long term.
A c k n o w l e d g e m e n t s
Thomas Vannecke was supported by the Research Foundation-Flanders (FWO) through a Ph.D.
fellowship. The collaboration between the Biosystems Engineering Department (UGent, Belgium) and the LBE-INRA (France) was supported by the Tournesol Project T2013.03). Mari Winkler was funded by a Marie Curie Intra-European Fellowship (PIEF-GA-2012-329328).
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L i s t o f T a b l e sTable 1 Standard curve parameters for the qPCR-analysis.
Table 2 Overview of the simulation set-up. In Approach 3, the two AOB and NOB populations are based on the calibrated values for period A and B from Approach 2.
Table 3 Periods distinguished during continuous operation (592 days) of the nitrifying reactor (Bougard et al. 2006b). NLR: nitrogen loading rate and Qin: liquid flow rate.
Table 4 Correlation matrix for the chemical and physical data (291 observations, DO 290
observations), including correlation coefficients r and p-values. Correlations with a p-value smaller than 0.05 are in bold.
Table 5 Correlation matrix between data on the process conditions and the microbial community (n=28), including correlation coefficients r and p-values. Bold values refer to significant
correlations (p-value<0.05).
Table 6 Constraints and calibrated microbial parameters values for AOB and NOB at 30 °C and pH 7.5 when two periods were calibrated, i.e., Period A: day 0-100 and Period B: day 100-592 (Approach 2). The calibrated values should be carefully interpreted, as the uncertainty of the estimated values was not verified.
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L i s t o f F i g u r e sFigure 1 Macroscopic and microscopic dynamic behaviour of the inverse turbulent bed reactor: (A) Bulk liquid concentrations of total ammonium (TNH), total nitrite (TNO2) and nitrate, taken from Bougard et al. (2006b), (B) the corresponding free ammonia (FA) and free nitrous acid (FNA) concentrations, calculated based on reactor temperature and pH (Anthonisen et al. 1976), (C) the percentage of AOB and NOB in the microbial community, based on qPCR-analysis and (D) the diversity of the microbial community, analysed using CE-SSCP and expressed as the negative logarithm of the Simpson index, DSSCP. The roman numbers denote the different periods distinguished (Table 3). Note the difference scales of the y-axes in plot B and C.
Figure 2 Boxplots representing the reported ranges for inhibition constants for FA (KI,FA, left) and FNA (KI,FNA, right) of AOB (top) and NOB (bottom) found in literature. Max = maximum value found in literature, Q3= third quartile, M = median, Q1= first quartile and min = minimum value.
The raw data of the literature review can be found in the Supplementary Information (Table S.4). It should be noted that FA inhibition of AOB and FNA inhibition of NOB is generally described by a Haldane term, while FNA inhibition of AOB and FA inhibition of NOB is described with a non-competitive inhibition term.
Figure 3 Simulation of the macroscopic reactor behaviour observed during reactor operation using the calibrated 1-dimensional biofilm model considering growth and decay of 1 AOB and 1 NOB species, besides heterotrophs: (A) Approach 1, (B) Approach 2, Period A and (C) Approach 2, Period B. The roman numbers denote the different periods distinguished (Table 3).
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Table 1 Standard curve parameters for the qPCR-analysis.
Total bacterial
community AOB NOB
Fluorophore Yakima Yellow FAM SybrGreen
Slope -3.342 -3.369 -3.310
Y-intercept 42.93 43.16 35.27
Efficiency 0.99 0.98 1.00
R2 0.970 0.998 0.993
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Table 2 Overview of the simulation set-up. In Approach 3, the two AOB and NOB populations are based on the calibrated values for period A and B from Approach 2.
Approach Period calibrated (days) Nitrifying community (calibrated)
1 0-592 1 AOB + 1 NOB
2 Period A: 0-100
Period B: 100-592 1 AOB + 1 NOB
3 0-592 2 AOB + 2 NOB
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Table 3 Periods distinguished during continuous operation (592 days) of the nitrifying reactor (Bougard et al. 2006b). NLR: nitrogen loading rate and Qin: liquid flow rate.
Period Days Dominant AOB
Temperature (°C)
Control strategies
Process performance I
0-50 N. halophila
30 - NO3
-accumulation 51-113 N. halophila
N. europaea
II 114-230 N. europaea 30 35 Temperature control
NO2
-accumulation III 231-491 N. europaea 30 Lowering Qin
Shift from NO2- to NO3
-accumulation
IV 492-592 N. europaea 30
Fuzzy logic DO/ammonium
control by adjusting Qin
(and thus NLR)
NO2
-accumulation
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Table 5 Correlation matrix between data on the process conditions and the microbial community (n=28), including correlation coefficients r and p-values. Bold values refer to significant correlations (p-value<0.05).
Fraction AOB Fraction NOB Diversity total
community AOB diversity NOB diversity
(%) (%) (D') (D') (D')
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Table 6 Constraints and calibrated microbial parameters values for AOB and NOB at 30 °C and pH 7.5 when two periods were calibrated, i.e., Period A: day 0-100 and Period B: day 100-592 (Approach 2). The calibrated values should be carefully interpreted, as the uncertainty of the estimated values was not verified.Starting
(median) value Minimum value Maximum value
Calibrated value Period A
Calibrated value Period B
g FA-N.m-3 64.25 5.80 3000 490.51 489.05
g FA-N.m-3 2.52 0.43 20 12.93 10.06
g FNA-N.m-3 0.21 0.05 2.80 0.42 0.21
g FNA-N.m-3 0.12 0.02 2.80 0.27 0.13
g FA-N.m-3 0.23 0.0018 1.29 1.25 1.29
g FNA-N.m-3 1.04e-004 3.12e-006 2.41e-003 0.0015 0.0024
g O2.m-3 0.40 0.07 3 0.078 0.71
g O2.m-3 0.97 0.04 4.01 0.049 0.06
d-1 1.34 0.33 3.40 0.65 0.71
d-1 1 0.24 3.54 1.43 0.30
bAOB d-1 0.067 0.017 0.17 0.073 0.061
bNOB d-1 0.05 0.012 0.18 0.13 0.051
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Figure 1
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Figure 2
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Figure 3