José M. Carvajal-Arroyo

Inhibition of anaerobic ammonium oxidizing (anammox) enrichment cultures by substrates, metabolites and common wastewater constituents

Anaerobic ammonium oxidation (anammox) is an emerging technology for nitrogen removal that provides a more environmentally sustainable and cost effective alternative compared to conventional biological treatment methods. The objective of this study was to investigate the inhibitory impact of anammox substrates, metabolites and common wastewater constituents on the microbial activity of two different anammox enrichment cultures (suspended and granular), both dominated by bacteria from the genus Brocadia. Inhibition was evaluated in batch assays by comparing the N(2) production rates in the absence or presence of each compound supplied in a range of concentrations. The optimal pH was 7.5 and 7.3 for the suspended and granular enrichment cultures, respectively. Among the substrates or products, ammonium and nitrate caused low to moderate inhibition, whereas nitrite caused almost complete inhibition at concentrations higher than 15 mM. The intermediate, hydrazine, either stimulated or caused low inhibition of anammox activity up to 3mM. Of the common constituents in wastewater, hydrogen sulfide was the most severe inhibitor, with 50% inhibitory concentrations (IC(50)) as low as 0.03 mM undissociated H(2)S. Dissolved O(2) showed moderate inhibition (IC(50)=2.3-3.8 mg L(-1)). In contrast, phosphate and salinity (NaCl) posed very low inhibition. The suspended- and granular anammox enrichment cultures had similar patterns of response to the various inhibitory stresses with the exception of phosphate. The findings of this study provide comprehensive insights on the tolerance of the anammox process to a wide variety of potential inhibiting compounds.

Pre-exposure to nitrite in the absence of ammonium strongly inhibits anammox.

Anaerobic ammonium oxidizing bacteria (Anammox) are known to be inhibited by their substrate, nitrite. However, the mechanism of inhibition and the physiological conditions under which nitrite impacts the performance of anammox bioreactors are still unknown. This study investigates the role of pre-exposing anammox bacteria to nitrite alone on their subsequent activity and metabolism after ammonium has been added. Batch experiments were carried out with anammox granular biofilm pre-exposed to nitrite over a range of concentrations and durations in the absence of ammonium. The effect of pre-exposure to nitrite alone compared to nitrite simultaneously fed with ammonium was evaluated by measuring the anammox activity and the accumulation of the intermediate, nitric oxide. The results show that the inhibitory effect was more dramatic when bacteria were pre-exposed to nitrite in absence of ammonium, as revealed by the lower activity and the higher accumulation of nitric oxide. The nitrite concentration causing 50% inhibition was 53 and 384 mg N L(-1) in the absence or the presence of ammonium, respectively. The nitrite inhibition was thus 7.2-fold more severe in the absence of ammonium. Biomass exposure to nitrite (25 mg N L(-1)), in absence of ammonium, led to accumulation of nitric oxide. On the other hand when the biomass was exposed to nitrite in presence of ammonium, accumulation of nitric oxide was only observed at much higher nitrite concentrations (500 mg N L(-1)). The inhibitory effect of nitrite in the absence of ammonium was very rapid. The rate of decay of the anammox activity was equivalent to the diffusion rate of nitrite up to 46% of activity loss. The results taken as a whole suggest that nitrite inhibition is more acute when anammox cells are not actively metabolizing. Accumulation of nitric oxide in the headspace most likely indicates disruption of the anammox biochemistry by nitrite inhibition, caused by an interruption of the hydrazine synthesis step.

Kinetic characterization of Brocadia spp.-dominated anammox cultures.

In this study, kinetic analyses were conducted for two Brocadia-dominated enrichment cultures, granular and flocculent, retrieved from lab-scale anaerobic ammonium oxidation (anammox) reactors. Substrate KS ranged from 0.35 to 0.69 mMN and VSmax ranged from 0.67 to 0.88 mmol Ng(-1)VSSh(-1). The model respected the experimentally measured stoichiometry of N-compounds, serving as an independent validation. Growth kinetics of the flocculent sludge was also studied, which indicates a μmax of 0.0984 d(-1) and a YX/S of 0.105 mol C-biomass mol(-1)NH4(+). The flocculent enrichment culture was used to determine the stoichiometric equation. The kinetic parameters of the Brocadia spp. cultures measured here can be used for optimizing applications of the anammox process.

Starved anammox cells are less resistant to NO2- inhibition

Anaerobic ammonium oxidizing (anammox) bacteria are be inhibited by their terminal electron acceptor, nitrite. Serious nitrite inhibition of the anammox bacteria occurs if the exposure coincides with the absence of the electron donating substrate, ammonium and pH < 7.2. Starvation of biomass occurs during underloading of bioreactors or biomass storage. This work investigated the effect of starvation on the sensitivity of anammox bacteria to nitrite exposure. Batch activity tests were carried out evaluating the response of anammox biomass subjected to different levels of starvation upon exposure to nitrite in the presence and absence of ammonium (active- and resting-cells, respectively). The response of the bacteria was evaluated by measuring the specific anammox activity and the evolution of the ATP content in the biomass over time. The 50% inhibitory concentrations of nitrite in starved- and fresh-resting-cells was 7 mg N L(-1) and 52 mg N L(-1), respectively. By contrast, only moderate nitrite inhibition occurred to starved anammox biomass when exposed to nitrite and ammonium simultaneously. Maximum ATP levels were observed in fresh cells. The ATP content in starved resting cells peaked 2-3 h after addition of NO2(-)(-). The response was hindered in cells starved for long periods. These findings agreed with a bioreactor study in which underloading of anammox biomass (0.10 g N L(-1) d(-1)) decreased its tolerance to a nitrite (only) exposure (101 mg NO2(-)-N L(-1)) and completely disrupted the N removal capacity of the biomass. A similar accumulation of 108 mg NO2(-)-N L(-1) after operation at 0.95 g N L(-1) d(-1) did not cause observable inhibition of the bacteria. The results taken as a whole demonstrate that starved anammox biomass is highly sensitive to nitrite toxicity. An explanation is proposed based on energy requirements to translocate nitrite in the cell.

The role of pH on the resistance of resting‐ and active anammox bacteria to NO2− inhibition

The anaerobic oxidation of ammonium (anammox) uses nitrite as terminal electron acceptor. The nitrite can cause inhibition to the bacteria that catalyze the anammox reaction. The literature shows a great divergence on the levels of NO2− causing inhibition. Moreover, the conditions influencing the resistance of anammox bacteria to NO2− inhibitory effect are not well understood. This work investigated the effect of the pH and the concentration of nitrite on the activity and metabolism of anammox granular sludge under different physiological conditions. Batch activity tests in a range of pH values were carried out in which either actively metabolizing cells or resting cells were exposed to nitrite in the presence or absence of the electron donating substrate ammonium, respectively. The response of the bacteria was evaluated by analyzing the specific anammox activity, the accumulation of nitric oxide, and the evolution of the ATP content in the biomass. Additionally, the effect of the pH on the tolerance of the biomass to single substrate feeding interruptions was evaluated in continuous anammox bioreactors. The results show that the influence of the pH on the NO2− inhibition of anammox bacteria is greater under non‐metabolizing conditions than during active metabolism. The exposure of resting cells to NO2− (100 mg N L−1) at pH values below 7.2 caused complete inhibition of the anammox activity. The inhibition was accompanied by accumulation of the intermediate, nitric oxide, in the gas phase. In contrast, just mild inhibition was observed for resting cells exposed to the same NO2− concentration at pH values higher than 7.5 or any of the pH values tested in assays with actively metabolizing cells. ATP initially increased and subsequently decreased in time after resting cells were exposed to NO2− suggesting an active response of the cells to nitrite stress. Furthermore, bioreactors operated at pH lower than 6.8 had greater sensitivity to NO2− during an ammonium feed interruption than a bioreactor operated at pH 7.1. The results suggest that the ability of resting cells to tolerate NO2− inhibition is seriously impeded at mildly acidic pH values; whereas actively metabolizing biomass is resistant to NO2− toxicity over a wide range of pH values. Biotechnol. Bioeng. 2014;111: 1949–1956.

Exogenous nitrate attenuates nitrite toxicity to anaerobic ammonium oxidizing (anammox) bacteria.

Anaerobic ammonium oxidizing bacteria (anammox) can be severely inhibited by one of its main substrates, nitrite (NO2(-)). At present, there is limited information on the processes by which anammox bacteria are able to tolerate toxic NO2(-). Intracellular consumption or electrochemically driven (transmembrane proton motive force) NO2(-) export are considered the main mechanisms of NO2(-) detoxification. In this work, we evaluated the potential of exogenous nitrate (NO3(-)) on relieving NO2(-) toxicity, putatively facilitated by NarK, a NO3(-)/NO2(-) transporter encoded in the anammox genome. The relative contribution of NO3(-) to NO2(-) detoxification was found to be pH dependent. Exposure of anammox cells to NO2(-) in absence of their electron donating substrate, ammonium (NH4(+)), causes NO2(-) stress. At pH 6.7 and 7.0, the activity of NO2(-) stressed cells was respectively 0 and 27% of the non-stressed control activity (NO2(-) and NH4(+) fed simultaneously). Exogenous NO3(-) addition caused the recovery to 42% and 80% of the control activity at pH 6.7 and 7.0, respectively. The recovery of the activity of NO2(-) stressed cells improved with increasing NO3(-) concentration, the maximum recovery being achieved at 0.85 mM. The NO3(-) pre-incubation time is less significant at pH 7.0 than at pH 6.7 due to a more severe NO2(-) toxicity at lower pH. Additionally, NO3(-) caused almost complete attenuation of NO2(-) toxicity in cells exposed to the proton gradient disruptor carbonyl cyanide m-chlorophenyl hydrazone at pH 7.5, providing evidence that the NO3(-) attenuation is independent of the proton motive force. The absence of a measurable NO3(-) consumption (or NO3(-) dependent N2 production) during the batch tests leaves NO3(-) dependent active transport of NO2(-) as the only plausible explanation for the relief of NO2(-) inhibition. We suggest that anammox cells can use a secondary transport system facilitated by exogenous NO3(-) to alleviate NO2(-) toxicity.

Mechanisms and control of NO- 2 inhibition of anaerobic ammonium oxidation (anammox)

Nitrite (NO2-), one of the main substrates in the anaerobic ammonium oxidation (anammox) process, has the potential to inhibit anammox bacteria. The sensitivity of anammox cells with different energy status to NO2- was evaluated, and addition of nitrate (NO3-) inhibition on the basis of narK gene with the putative function of facilitating NO3-/NO2- antiporter. The results showed that the resistance of anammox bacteria to NO2- inhibition follows the order: active-cells > starved-cells > resting-cells > starved-/resting-cells. Anammmox resting cells have increasing tolerance to NO2- in the pH range from 7.0 to 7.5. Dissipating the proton gradient by using carbonyl cyanide m-chlorophenyl hydrazine (CCCP) caused severe inhibition at all pH values including pH = 7.5. Addition of NO3- enabled activity recovery of NO2--inhibited anammox bacteria regardless of whether the proton gradient was disrupted or not, supporting the hypothesis of NO3--dependent detoxification via a secondary transport system.

Kinetics and thermodynamics of anaerobic ammonium oxidation process using Brocadia spp. dominated mixed cultures

Anaerobic ammonium oxidation (anammox) is a recently discovered microbial process commonly applied to treat ammonium pollution in effluents with low organic carbon content. Modeling anammox processes is important for simulating and controlling full-scale plants. In this study, the anammox process was simulated using three models, and substrate and growth parameters obtained by different research groups. Two Brocadia spp.-dominated mixed cultures, one granular and the other flocculent, were used for this purpose. A very good correlation between experimental data using both sludges and model predictions was achieved by one of the models, obtaining correlation coefficients higher than 0.997. Other models and stoichiometric equations tested were unable to predict the anammox kinetics and stoichiometry. Furthermore, the thermodynamic behavior of the two mixed cultures was compared through the determination of the energy of activation of the anammox conversion at temperatures ranging from 9 to 40 °C. Optimum temperature for anammox activity was established at 30-35 °C in both cases. The energy of activation values calculated for granular sludge and flocculent sludge were 64 and 124 kJ mol(-1), respectively.