Biological nitrogen removal through the nitrite pathway (NH → NO → N) is favorable for wastewater treatment plants without sufficient carbon sources. This study demonstrates an innovative approach for attaining the nitrite pathway based on sludge treatment using free ammonia (FA, i.e., NH). This approach is based on our innovative discovery in this study that FA at 210 mg NH-N/L is far less biocidal to ammonium-oxidizing bacteria (AOB) than to nitrite-oxidizing bacteria (NOB). A total of 22% of the activated sludge from the sequencing batch reactor (SBR) receiving synthetic domestic wastewater was treated in an FA treatment unit at 210 mg NH-N/L for 1 day. The FA-treated sludge was afterward recirculated back to the SBR. A nitrite accumulation ratio of above 90% was quickly achieved (in 40 days) and maintained stably in the SBR, indicating the establishment of the nitrite pathway. The NOB population and activity after implementing FA treatment was less than 5% of those without FA treatment, suggesting the washout of NOB. In contrast, the AOB population and activity in the SBR were not affected. The nitrogen-removal performance was significantly improved after incorporating the FA approach. The FA approach is a closed-loop approach and is economically and environmentally attractive.

Stable nitritation is a critical bottleneck for achieving autotrophic nitrogen removal using the energy-saving mainstream deammonification process. Herein we report a new strategy to wash out both the Nitrospira sp. and Nitrobacter sp. from the treatment of domestic-strength wastewater. The strategy combines sludge treatment using free nitrous acid (FNA) with dissolved oxygen (DO) control in the nitritation reactor. Initially, the nitrifying reactor achieved full conversion of NH4+ to NO3−. Then, nitrite accumulation at ~60% was achieved in the reactor when 1/4 of the sludge was treated daily with FNA at 1.82 mg N/L in a side-stream unit for 24 h. Fluorescence in-situ hybridization (FISH) revealed FNA treatment substantially reduced the abundance of nitrite oxidizing bacteria (NOB) (from 23.0 ± 4.3 to 5.3 ± 1.9%), especially that of Nitrospira sp. (from 15.7 ± 3.9 to 0.4 ± 0.1%). Nitrite accumulation increased to ~80% when the DO concentration in the mainstream reactor was reduced from 2.5–3.0 to 0.3–0.8 mg/L. FISH revealed the DO limitation further reduced the abundance of NOB (to 2.1 ± 1.0%), especially that of Nitrobacter sp. (from 4.9 ± 1.2 to 1.8 ± 0.8%). The strategy developed removes a major barrier for deammonification in low-strength domestic wastewater.

Free nitrous acid (FNA) exerts a broad range of antimicrobial effects on bacteria, although susceptibility varies considerably among microorganisms. Among nitrifiers found in activated sludge of wastewater treatment processes (WWTPs), nitrite-oxidizing bacteria (NOB) are more susceptible to FNA compared to ammonia-oxidizing bacteria (AOB). This selective inhibition of NOB over AOB in WWTPs bypasses nitrate production and improves the efficiency and costs of the nitrogen removal process in both the activated sludge and anaerobic ammonium oxidation (Anammox) system. However, the molecular mechanisms governing this atypical tolerance of AOB to FNA have yet to be understood. Herein we investigate the varying effects of the antimicrobial FNA on activated sludge containing AOB and NOB using an integrated metagenomics and label-free quantitative sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) metaproteomic approach. The Nitrosomonas genus of AOB, on exposure to FNA, maintains internal homeostasis by upregulating a number of known oxidative stress enzymes, such as pteridine reductase and dihydrolipoyl dehydrogenase. Denitrifying enzymes were upregulated on exposure to FNA, suggesting the detoxification of nitrite to nitric oxide. Interestingly, proteins involved in stress response mechanisms, such as DNA and protein repair enzymes, phage prevention proteins, and iron transport proteins, were upregulated on exposure to FNA. In addition enzymes involved in energy generation were also upregulated on exposure to FNA. The total proteins specifically derived from the NOB genus Nitrobacter was low and, as such, did not allow for the elucidation of the response mechanism to FNA exposure. These findings give us an understanding of the adaptive mechanisms of tolerance within the AOB Nitrosomonas to the biocidal agent FNA.

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An increase of nitrite in the domestic-strength range is generally recognized to stimulate nitrous oxide (N2O) production by ammonia-oxidizing bacteria (AOB). It was found in this study, however, that N2O emission from a mainstream nitritation system (cyclic nitrite = 25-45 mg of N/L) that was established by free nitrous acid (FNA)-based sludge treatment was not higher but much lower than that from the initial nitrifying system with full conversion of NH4(+)-N to NO3(-)-N. Under dissolved oxygen (DO) levels of 2.5-3.0 mg/L, N2O emission from the nitritation stage was 76% lower than that from the initial stage. Even when the DO level was reduced to 0.3-0.8 mg/L, N2O emission from the nitritation stage was still 40% lower. An investigation of the mechanism showed that FNA treatment caused a shift of the stimulation threshold of nitrite on N2O emission. At the nitritation stage, the maximal N2O emission factor occurred at ∼16 mg of N/(L of nitrite). However, it increased with increasing nitrite in the range of 0-56 mg of N/L at the initial stage. FNA treatment decreased the biomass-specific N2O production rate, suggesting that the enzymes relevant to nitrifier denitrification were inhibited. Microbial analysis revealed that FNA treatment decreased the microbial community diversity but increased the abundances of AOB and denitrifiers.

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Interactions between microorganisms in mixed communities are highly complex, being either syntrophic, neutral, predatory, or competitive. Evolutionary changes can occur in the interaction dynamics between community members as they adapt to coexistence. Here, we report that the syntrophic interaction between Geobacter sulfurreducens and Pseudomonas aeruginosa coculture change in their dynamics over evolutionary time. Specifically, Geobacter sp. dominance increases with adaptation within the cocultures, as determined through quantitative PCR and fluorescence in situ hybridization. This suggests a transition from syntrophy to competition and demonstrates the rapid adaptive capacity of Geobacter spp. to dominate in cocultures with P. aeruginosa. Early in coculture establishment, two single-nucleotide variants in the G. sulfurreducens fabI and tetR genes emerged that were strongly selected for throughout coculture evolution with P. aeruginosa phenazine wild-type and phenazine-deficient mutants. Sequential window acquisition of all theoretical spectra-mass spectrometry (SWATH-MS) proteomics revealed that the tetR variant cooccurred with the upregulation of an adenylate cyclase transporter, CyaE, and a resistance-nodulation-division (RND) efflux pump notably known for antibiotic efflux. To determine whether antibiotic production was driving the increased expression of the multidrug efflux pump, we tested Pseudomonas-derived phenazine-1-carboxylic acid (PHZ-1-CA) for its potential to inhibit Geobacter growth and drive selection of the tetR and fabI genetic variants. Despite its inhibitory properties, PHZ-1-CA did not drive variant selection, indicating that other antibiotics may drive overexpression of the efflux pump and CyaE or that a novel role exists for these proteins in the context of this interaction. IMPORTANCE Geobacter and Pseudomonas spp. cohabit many of the same environments, where Geobacter spp. often dominate. Both bacteria are capable of extracellular electron transfer (EET) and play important roles in biogeochemical cycling. Although they recently in 2017 were demonstrated to undergo direct interspecies electron transfer (DIET) with one another, the genetic evolution of this syntrophic interaction has not been examined. Here, we use whole-genome sequencing of the cocultures before and after adaptive evolution to determine whether genetic selection is occurring. We also probe their interaction on a temporal level and determine whether their interaction dynamics change over the course of adaptive evolution. This study brings to light the multifaceted nature of interactions between just two microorganisms within a controlled environment and will aid in improving metabolic models of microbial communities comprising these two bacteria.