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Understanding nitrogen metabolic interactions in mixed anammox-based microbial communities

Periodic Reporting for period 1 - MixAmox (Understanding nitrogen metabolic interactions in mixed anammox-based microbial communities)

Reporting period: 2017-09-01 to 2019-08-31

Biological treatment of municipal wastewaters plays a fundamental role in our society by reducing the associated public-health risks and avoiding the pollution of natural resources and ecosystems. Improving current treatment solutions towards more reliable and resource-efficient processes at increasingly stringent discharge limits is a priority of the EU. Successful biological treatments rely on the concerted activity of microorganisms belonging to different functional guilds. As a result, optimized microbial community engineering strategies are required to achieve this goal. Recent years witnessed tremendous advances in our knowledge of the natural nitrogen cycle, challenging our traditional approach to wastewater treatment plants (WWTP) design and operation. In particular, the discovery of the anammox metabolism, catalysing the autotrophic oxidation of ammonium to N2 via nitrite, holds promise to turn the energy balance of WWTPs to neutral or even positive.

The overarching goal of the MixAmox project was to advance our fundamental understanding of the principles of microbial selection in anammox-based communities. Specifically, the action focused on the impact of easily degradable organic substrates, a key challenge towards the implementation of anammox processes. Three main objectives were pursued, namely i) characterize the eco-physiological response of different anammox species to easily degradable organic carbon, ii) identify the conditions selecting for high-affinity accompanying N2O reducing guilds as a mean to counteract N2O emissions, and iii) elucidate the role of dissimilatory nitrite reduction to ammonium (DNRA) in biofilm-based anammox processes. To this end, continuous enrichments were combined with meta-proteomics and mathematical modelling, leading to the following conclusions: i) shot-gun meta-proteomics is an appropriate tool for the simultaneous taxonomic and functional characterization of complex microbial communities; ii) among the two tested anammox genera, Candidatus Kuenenia and Ca. Brocadia, the latter has a clear competitive advantage in the presence of organics; iii) long sludge retention times allow for the selective enrichment of clade II N2O reducers; iv) numerical simulations suggest that DNRA defines the nice for anammox in biofilms at high influent organics concentrations.
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.
The results of the MixAmox project broaden our understanding of metabolic interactions in mixed anammox-based microbial communities towards the development and wide-adoption of more resource- and energy-efficient WWTP. A deeper knowledge of the impact of organic substrates on the anammox physiology is paramount to improve the design of anammox reactors and the preceding treatment units, predict potential process imbalances, and ultimately maximize carbon valorisation. At the same time, minor N2O emissions could completely offset the expected benefits of anammox processes in term of CO2 savings. Elucidating the microbiology underlying N2O turnover is a prerequisite for the design of new process schemes where the biological N2O reducing potential is harnessed as a mean to control emissions. Also, new metabolisms such as DNRA need to be included in WWTP mathematical models to discern their potential role, and possibly disclose unforeseen treatment opportunities. Ultimately, methodological approaches that allow for the simultaneous taxonomic and functional characterization, such as meta-proteomics, are expected to change our understanding of labour division in complex microbial communities, and how we control it to the benefit of society at large.