The PI, Michele Laureni, conducted the research at TU Delft in the Environmental Biotechnology group (prof. Mark van Loosdrecht), with secondments at Aalborg University in the Section for Biotechnology of the Chemistry and Bioscience Department (prof. Jeppe Lund Nielsen).
Five retentostats (2 L), equipped with custom-made membrane filtration modules (pore size of 0.1 μm), were operated in parallel at 30°C under anoxic conditions. Highly-enriched planktonic cultures of Candidatus Kuenenia and Ca. Brocadia were used to assess the long-term impact of different organics and availability of nitrogen substrates. Continuous exposure to different organic substrates provided key insights on the ecological niche of the two anammox genera. Ca. Brocadia stably co-existed with the developed heterotrophic denitrifiers (family Rhodocyclaceae), while Ca. Kuenenia was outcompeted under all tested conditions. Differential proteomics revealed that Ca. Kuenenia consistently up-regulated one HAO-like protein – a candidate nitrite reductase – likely in response to the increased competition for nitrite.
The same experimental set-up was used at 20°C for the continuous cultivation of denitrifying communities with acetate and N2O as sole electron donor and acceptor, respectively. Under the imposed conditions, the enrichments were also dominated by members of the family Rhodocyclaceae. Importantly, at relatively long sludge retention times (7d), meta-proteomics revealed that both acetate and N2O limiting conditions enriched for clade II N2O-reducers, usually displaying higher substrate affinities and thus potentially acting as more efficient N2O-sinks. Yet, specialist N2O-reducers were not enriched for.
Ultimately, a one-dimensional biofilm model was developed to elucidate the impact of process conditions on the competition between DNRA, anammox, and denitrification, and evaluate implications for process performance. Owing to the limited kinetic and stoichiometric information on DNRA performing bacteria, Monte Carlo simulations were used to cover the space of potential parameter values. The growth yield of DNRA was identified as key parameter determining the biofilm microbial composition, and defining the nice for anammox in particular at high influent ratios of organics over nitrogen.
The work resulted in one published paper, and in eight more manuscripts submitted or in preparation. The results were presented at eight international conferences, and during three invited talks. Also, one international workshop was organized on the topic of the action. In terms of outreach activities, besides an invited talk at a scientific event for children, Michele Laureni organized an Improvisation Theatre show on the topic of bacteria and toilets to present the topic of the project and the different EU funding opportunities to the general public.
