A robust nitrifying community in a bioreactor at 50°C opens up the path for thermophilic nitrogen removal

Emilie N. P. Courtens1, Eva Spieck², Ramiro Vilchez-Vargas1, Samuel Bodé3, Pascal Boeckx3, Stefan Schouten4, Ruy Jauregui5, Dietmar H. Pieper5, Siegfried E. Vlaeminck1,6* and Nico Boon1*

1Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, Coupure Links 653, 9000 Gent, Belgium

2Biocenter Klein Flottbek, University of Hamburg, Microbiology & Biotechnology, Ohnhorststrasse 18, D-22609 Hamburg, Germany

3Isotope Bioscience Laboratory, Ghent University, Coupure Links 653, 9000 Gent, Belgium

4Royal Netherlands Institute for Sea Research (NIOZ), Landsdiep 4, 1797 SZ, ‘t Horntje, Texel, Netherlands

5Microbial Interactions and Processes Research Group, Helmholtz Centre for Infection Research, Braunschweig, Germany

6Research Group of Sustainable Energy, Air and Water Technology, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerpen, Belgium

* These authors contributed equally and are both senior authors for this work

The authors declare no conflict of interest

 Corresponding author: Nico Boon
Tel.: +32-9-2645976
Fax: +32-9-2646248
E-mail:

Table S1. Overview of the operational parameters during different ex-situ batch activity measurements enabling the separation of the effect of pH, temperature, ammonium and free ammonia (FA) on ammonia oxidation. =: not-varying, ≠: varying.

Investigated parameter / Conditions applied during batch test / Figure
Temperature / pH / Ammonium / FA
FA / = / = / ≠ / ≠ / 4A
Ammonium / = / = / ≠ / = (*) / 4A
pH / = / ≠ / = (**) / = (*) / 5A
Temperature / ≠ / = / = / = (*) / 5B

*: Concentrations below 2.4 mg NH3-N L-1 were considered as not-varying, as Figure 4A showed no inhibition of ammonia oxidation up to this concentration

**: Concentrations with a corresponding FA below 2.4 mg NH3-N L-1 were considered as not-varying, as Figure 4A showed no influence of ammonium concentration on ammonia oxidation.

Table S2. Overview of the operational parameters during different ex-situ batch activity measurements enabling the separation of the effect of pH, temperature, nitrite and free nitrous acid (FNA) on nitrite oxidation. =: not-varying, ≠: varying.

Investigated parameter / Conditions applied during batch test / Figure
Temperature / pH / Nitrite / FNA
FNA / = / = / ≠ / ≠ (*) / 4D
Nitrite / = / = / ≠ / ≠ (*) / 4D
pH / = / ≠ / = / ≠ (**) / 5C
Temperature / ≠ / = / = / = (***) / 5D

* To distinguish between FNA and nitrite inhibition effect two tests were performed with the same nitrite concentrations but different FNA concentrations. Different FNA concentrations were obtained by varying pH, as Figure 6 showed no significant differences between pH 7 and pH 8.

** Different pH led to different FNA concentrations. This test was performed at two different initial nitrite concentrations to differentiate between the ‘high’ and the ‘low’ FNA.

*** FNA variation due to varying temperature was considered negligible in the tested range (0.0019 ± 0.0003 mg HNO2-N L-1)

Table S3. Inorganic nitrogen compound distribution in the compost solution samples originating from composting facilities.

Sample / NH4+
(mg N L-1) / NO2-
(mg N L-1) / NO3-
(mg N L-1)
A / Green waste / 0 / 2 / 18
B / Rabbit manure/green waste / 0 / 4 / 50
C / Digested organic waste / 114 / 0 / 1
D / Cow manure / 26 / 0 / 1

Table S4. Transmission electron micrographs of ultrathin sections of the thermophilic reactor biofilm. (A) putative single “Candidatus Nitrososphaera gargensis”–like AOA cell(s). (B) Single Nitrospira cells. (C) co-occurrence of single Nitrospira and putative AOA cells loosely associated with each other.

A
/
/
B

/
C
/
/

Figure S1. Enchained ammonia/nitrite oxidizing (A,B) and nitrite oxidizing (C,D) thermophilic batch enrichments (second dilution series) originally inoculated with samples from different composting facilities. (A) Green waste, (B) rabbit manure/green waste mixture, (C) digested organic manure waste and (D) cow manure.

Figure S2. Total number of reads of the different Nitrospira-related sequences in the thermophilic nitrifying reactor biomass over 6 months of operation.

Figure S3. Copies of the functional amoA gene and the 16S rRNA of Nitrospira spp. ng-1 DNA, determined with qPCR in the thermophilic nitrifying reactor community during the second start-up period (days 245-425).

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Figure S4. Heat map showing the evolution of the members in the community with a relative abundance higher than 1% at least at one sampling point. Color code is in % of relative abundance per sampling time.

Figure S5: Effect of free ammonia (FA) concentration on the ammonia oxidation activity. Data points represent the average replicate tests (n=6), error bars represent the standard error.

Figure S6. Effect of nitrate concentration on the nitrite oxidation activity. Data points represent the average replicate tests (n=6), error bars represent the standard error.

Figure S7. Inhibitory effect of the ammonia oxidation inhibitor allythiourea (ATU) and the AOA specific inhibitor carboxy-PTIO (2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) (n=6). Data points represent the average replicate tests (n=6), error bars represent the standard error.