pH-Dependent Hydrogenotrophic Denitratation Based on Self-Alkalization.

Producing stable nitrite is a necessity for anaerobic ammonium oxidation (anammox) but remains a huge challenge. Here, we describe the design and operation of a hydrogenotrophic denitratation system that stably reduced >90% nitrate to nitrite under self-alkaline conditions of pH up to 10.80. Manually lowering the pH to a range of 9.00-10.00 dramatically decreased the nitrate-to-nitrite transformation ratio to <20%, showing a significant role of high pH in denitratation. Metagenomics combined with metatranscriptomics indicated that six microorganisms, including a Thauera member, dominated the community and encoded the various genes responsible for hydrogen oxidation and the complete denitrification process. During denitratation at high pH, transcription of periplasmic genes napA, nirS, and nirK, whose products perform nitrate and nitrite reduction, decreased sharply compared to that under neutral conditions, while narG, encoding a membrane-associated nitrate reductase, remained transcriptionally active, as were genes involved in intracellular proton homeostasis. Together with no reduction in only nitrite-amended samples, these results disproved the electron competition between reductions of nitrate and nitrite but highlighted a lack of protons outside cells constraining biological nitrite reduction. Overall, our study presents a stably efficient strategy for nitrite production and provides a major advance in the understanding of denitratation.

Effect of self-alkalization on nitrite accumulation in a high-rate denitrification system: Performance, microflora and enzymatic activities.

The self-alkalization of denitrifying automatic circulation (DAC) reactor resulted in a large increase of pH up to 9.20 and caused a tremendous accumulation of nitrite up to 451.1 ± 49.0 mgN L(-1) at nitrate loading rate (NLR) from 35 kgN m(-3) d(-1) to 55 kgN m(-3) d(-1). The nitrite accumulation was greatly relieved even at the same NLR once the pH was maintained at 7.6 ± 0.2 in the system. Enzymatic assays indicated that the long-term bacterial exposure to high pH significantly inhibited the activity of copper type nitrite reductase (NirK) rather than the cytochrome cd1 type nitrite reductase (NirS). The terminal restriction fragment length polymorphism (T-RFLP) analysis revealed that the dominant denitrifying bacteria shifted from the NirS-containing Thauear sp. 27 to the NirK-containing Hyphomicrobium nitrativorans strain NL23 during the self-alkalization. The significant nitrite accumulation in the high-rate denitrification system could be therefore, due to the inhibition of Cu-containing NirK by high pH from the self-alkalization. The results suggest that the NirK-containing H. nitrativorans strain NL23 could be an ideal functional bacterium for the conversion of nitrate to nitrite, i.e. denitritation, which could be combined with anaerobic ammonium oxidation (Anammox) to develop a new process for nitrogen removal from wastewater.

Effects of operation mode on self-alkalization of high-load denitrifying reactor.

To study the alkalization issue and its potential effects on high-load denitrifying system, the effects of operation mode on self-alkalization of high-load denitrifying reactor were investigated. The results showed that both the increase of substrate concentration and decrease of hydraulic retention time (HRT) can induce notable self-alkalization of high-load denitrification reactor (with the nitrogen loading rate (NLR) higher than 25kg Nm(-3)d(-1)). The effluent pH surpassed the 9.20 when the influent pH value was 7.0±0.1. The self-alkalization of denitrification process originated from the nitrate reduction, while the methanol oxidation could alleviate the self-alkalization by neutralizing OH(-) and setting up a buffering system of HCO3(-)/CO3(2-). At the same NLR, the self-alkalization induced by increase of substrate concentration was remarkably stronger than that induced by decrease of HRT. Keeping the nitrate concentration below inhibition concentration improved the performance of high-load reactor and alleviated the self-alkalization.

Characteristics of self-alkalization in high-rate denitrifying automatic circulation (DAC) reactor fed with methanol and sodium acetate.

Denitrification is a self-alkalization process. In this experiment, the characteristics of self-alkalization in high-rate heterotrophic denitrifying automatic circulation (DAC) reactor fed with methanol and sodium acetate were investigated, respectively. The results showed that, (1) The self-alkalization of high-rate denitrifying reactors was remarkably strong both with methanol and sodium acetate as carbon sources, while the effluent pH was much lower than the stoichiometric values and the malfunction from self-alkalization of denitrification was far less serious than expected. (2) The self-adaptation of the reactors was attributed to the neutralization of carbon dioxide (oxidization product of organic matter) and the self-adaptation of denitrifying sludge. The formation and discharge of calcium carbonate precipitates gave rise to lower effluent pH and alkalinity than the stoichiometric values.