Efficient management of the nitritation-anammox microbiome through intermittent aeration: absence of the NOB guild and expansion and diversity of the NOx reducing guild suggests a highly reticulated nitrogen cycle.

Obtaining efficient autotrophic ammonia removal (aka partial nitritation-anammox, or PNA) requires a balanced microbiome with abundant aerobic and anaerobic ammonia oxidizing bacteria and scarce nitrite oxidizing bacteria. Here, we analyzed the microbiome of an efficient PNA process that was obtained by sequential feeding and periodic aeration. The genomes of the dominant community members were inferred from metagenomes obtained over a 6 month period. Three Brocadia spp. genomes and three Nitrosomonas spp. genomes dominated the autotrophic community; no NOB genomes were retrieved. Two of the Brocadia spp. genomes lacked the genomic potential for nitrite reduction. A diverse set of heterotrophic genomes was retrieved, each with genomic potential for only a fraction of the denitrification pathway. A mutual dependency in amino acid and vitamin synthesis was noted between autotrophic and heterotrophic community members. Our analysis suggests a highly-reticulated nitrogen cycle in the examined PNA microbiome with nitric oxide exchange between the heterotrophs and the anammox guild.

Aeration strategies to mitigate nitrous oxide emissions from single-stage nitritation/anammox reactors.

Autotrophic nitrogen removal is regarded as a resource efficient process to manage nitrogen-rich residual streams. However, nitrous oxide emissions of these processes are poorly documented and strategies to mitigate emissions unknown. In this study, two sequencing batch reactors performing single-stage nitritation/anammox were operated under different aeration strategies, gradually adjusted over six months. At constant but limiting oxygen loading, synthetic reject water was fed (0.75 g-N/L · d) and high nitrogen removal efficiencies (83 ± 5 and 88 ± 2%) obtained. Dynamics of liquid phase nitrous (N2O) and nitric oxide (NO) concentrations were monitored and N2O emissions calculated. Significant decreases in N2O emissions were obtained when the frequency of aeration was increased while maintaining a constant air flow rate (from >6 to 1.7% ΔN2O/ΔTN). However, no significant effect on the emissions was noted when the duration of aeration was increased while decreasing air flow rate (10.9 ± 3.2% ΔN2O/ΔTN). The extant ammonium oxidation activity (mgNH4(+)-N/gVSS · min) positively correlated with the specific N2O production rate (mgN2O-N/gVSS · min) of the systems. Operating under conditions where anaerobic exceeds aerobic ammonium oxidation activity is proposed to minimize N2O emissions from single-stage nitritation/anammox reactors; increasing the frequency of aeration cycling is an efficient way of obtaining those conditions.

Critical assessment of extracellular polymeric substances extraction methods from mixed culture biomass.

Extracellular polymeric substances (EPS) have a presumed determinant role in the structure, architecture, strength, filterability, and settling behaviour of microbial solids in biological wastewater treatment processes. Consequently, numerous EPS extraction protocols have recently been published that aim to optimize the trade off between high EPS recovery and low cell lysis. Despite extensive efforts, the obtained results are often contradictory, even when analysing similar biomass samples and using similar experimental conditions, which greatly complicates the selection of an extraction protocol. This study presents a rigorous and critical assessment of existing physical and chemical EPS extraction methods applied to mixed-culture biomass samples (nitrifying, nitritation-anammox, and activated sludge biomass). A novel fluorescence-based method was developed and calibrated to quantify the lysis potential of different EPS extraction protocols. We concluded that commonly used methods to assess cell lysis (DNA concentrations or G6PDH activities in EPS extracts) do not correlate with cell viability. Furthermore, we discovered that the presence of certain chemicals in EPS extracts results in severe underestimation of protein and carbohydrate concentrations by using standard analytical methods. Keeping both maximum EPS extraction yields and minimal biomass lysis as criteria, it was identified a sonication-based extraction method as the best to determine and compare tightly-bound EPS fractions in different biomass samples. Protein was consistently the main EPS component in all analysed samples. However, EPS from nitrifying enrichments was richer in DNA, the activated sludge EPS had a higher content in humic acids and carbohydrates, and the nitritation-anammox EPS, while similar in composition to the nitrifier EPS, had a lower fraction of hydrophobic biopolymers. In general, the easily-extractable EPS fraction was more abundant in carbohydrates and humic substances, while DNA could only be found in tightly bound EPS fractions. In conclusion, the methodology presented herein supports the rational selection of analytical tools and EPS extraction protocols in further EPS characterization studies.