2.1 Effects of Salinity on conventional biological nitrogen removal techniques
In the traditional biological nitrogen removal process, the nitrification and denitrification reactions are completed by nitrifying bacteria and denitrifying bacteria respectively. Since most nitrifying bacteria are aerobic bacteria, while most denitrifying bacteria are anaerobic bacteria, most traditional processes construct anoxic and aerobic zones respectively, forming hierarchical nitrification and denitrification processes.
High salinity has adverse effects on both nitrification and denitrification processes. Nitrification is a critical rate-limiting step in traditional nitrogen removal processes(Bassin et al. 2012). Nitrifying bacteria mainly include ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB). Nitrobacteria grow slowly and are inhibited by salinity, which has been considered an unstable factor in many wastewater treatment systems, especially in industrial environments(Moussa et al. 2006). High salt conditions can inhibit both AOB and NOB(Gonzalez-Silva et al. 2016). Because NOB is more sensitive to salinity than AOB(Chen X. et al. 2019, Le et al. 2019), a large amount of nitrite will be accumulated in the nitrification process under high salt conditions. It was found with the increase in salinity, the nitrite aggregations became more serious, and the effluent concentration reached 6.37 mg/L when the salinity was at its maximum of 6%(Glass 1999). The inhibition of salinity in the nitrification process is realized through the inhibition of enzyme activity. In particular, the activity of nitrite oxidoreductase (Nxr) will be greatly reduced in a high-salt environment, and its inhibition mechanism is shown in Figure 2.
Fig.2 Effect of salinity on nitrification enzymes
There are many kinds of denitrifying bacteria, and many heterotrophic microorganisms in nature have complete or partial denitrification functions. It was found that denitrifying bacteria had stronger salt tolerance than nitrifying bacteria, but the high salt environment still inhibited the denitrification process(Panswad T 1999). Increasing salinity will lead to decreased nitrogen removal efficiency of the system by decreasing denitrification activity(Lu et al. 2018). Some scholars have pointed out that both denitrification and nitrification in the estuary area are weakened with the increase of salinity, which is attributed to the fact that the high salinity weakens the adsorption capacity of sediment to ammonia nitrogen(Kemp 1985), which makes the nitrifying bacteria in the sediment subject to the restriction of ammonia nitrogen, thus affecting the denitrification process. In addition, the influence of salinity on denitrification may also be due to its direct physiological influence on the denitrification microbial community(Rysgaard S 1999). Salinity can reduce nitrate removal performance in the system through enzyme inhibition or electronic competition by denitrifiers, as shown in Figure 3. During denitrification, the activity of dehydrogenase is reduced by salting out, which inhibits the first step of the electron transfer chain(Zhao Wei et al. 2013).In addition, Nar, Nir, and Nos were all located in the periplasm, so the presence of a large amount of Na+ in the periplasm would adversely affect the activities of these enzymes. Among them, Na+ inhibits its activity by substituting copper ions on Nos. This inhibition is non-competitive and the involved chemicals such as metallic ions react with enzymes by complexing metallic activators of the enzyme or combining with the non-active site of the enzyme. Also, a decrease in the abundance of nitrite reductase Nir and nitrous oxide reductase NosZ was observed in high salt environments(Deng Y. et al. 2019, Pan et al. 2023).
Fig.3 Effect of salinity on denitrification enzymes
Many studies have observed the decrease of nitrogen removal effect in high salt environments. When the activated sludge method is used to treat high ammonia nitrogen and high salt wastewater, it is found that when the influent salinity exceeds 30 g/L, the nitrification efficiency drops sharply(Lema 2002). When the SBR system was used to study the nitrogen removal efficiency, the salinity increased from 2% to 3%, and the removal rate of ammonia nitrogen decreased from 95% to 56%(Zhao Y. et al. 2016). Some studies have also obtained better nitrogen removal effects under high salt conditions(Moussa et al. 2006, Yoshie et al. 2006), which may be caused by different raw water characteristics, salinity addition methods, or differences in experimental conditions (temperature, pH, etc.). However, in general, increased salinity will inhibit nitrification and denitrification processes, thus affecting the biological nitrogen removal effect.
2.2 Effects of Salinity on novel biological nitrogen removal
The traditional biological nitrogen removal technology has many shortcomings, such as high operation costs, large areas, and poor anti-impact load capacity. Thus plenty of new technologies break through the traditional theory came into being, such as shortcut nitrification-denitrification, simultaneous nitrification-denitrification, and anaerobic ammonia oxidation.
It is feasible to use partial nitrification and denitrification to treat high salt nitrogen wastewater in a certain salinity range. In the process of nitrification, NOB is more sensitive to salinity than AOB, so as the salinity increases, the reaction will shift from full nitrification and denitrification to short denitrification and denitrification. When shortcut nitrification and denitrification technology were used to treat ammonia nitrogen in high-salt wastewater(Li J. et al. 2018), although AOB activity was inhibited at the initial stage, it quickly adapted to the salinity of wastewater and recovered the denitrification performance, with the removal rate of ammonia nitrogen reaching 98.7%. When the salinity increases further, the growth and metabolic activity of AOB will be inhibited, leading to the obstruction of the ammonia oxidation process and the deterioration of the nitrification effect. Most studies have shown that the salt tolerance of AOB can reach 30 g/L NaCl or higher(Ge et al. 2019), and the highest salinity of NOB that is not inhibited is around 20 g/L NaCl(Pronk et al. 2014).
In the process of simultaneous nitrification-denitrification, nitrifying bacteria, and denitrifying bacteria are mostly added to a reactor for mixed culture. Some researchers adopted simultaneous nitrification-denitrification to treat high-salt nitrogen-containing wastewater and achieved good treatment results. For example, Xia et al. (2019) obtained an ammonia nitrogen removal rate of more than 90% at a salinity of 2.4%. The discovery of heterotrophic nitrification-aerobic denitrification bacteria (HN-AD) provides a possible explanation for the simultaneous nitrification and denitrification process. In 1983, Robertson (1983) successfully isolated the first heterotrophic nitrification-aerobic denitrifying bacterium-Thiosphaera pantotropha. Figure 4 shows the bacterium observed under the microscope. So far, more than 20 HN-AD strains have been isolated, including Alcaligenes(Chen J. et al. 2021, G 1992), Pseudomonas(Deng M. et al. 2021, Yang L. et al. 2019, Yi et al. 2010, Zhang et al. 2011), Acinetobacter(Zhao B. et al. 2010, Zhu et al. 2012), Thauera(Chang et al. 2011), Bacillus(Rout et al. 2017, Yang X. P. et al. 2011b). Many HN-AD bacteria showed both salt tolerance and nitrogen removal. A halophilic strain Vibrio diabolicus sp.SF16 isolated from Marine sediments was inoculated into a biological aerated filter to treat high-salt wastewater. The removal rate of ammonia nitrogen reached 90.86% at a salinity of 3%(Duan et al. 2015). Most of the reported HN-AD bacteria belong to moderate halophilic bacteria and can tolerate the salinity range of 3%-15%, which can overcome the restriction of high salt on traditional biological nitrogen removal process to a certain extent, and has a good application prospect.
Fig.4 Thiosphaera pantotropha under the microscope(the bar marker represents 0.5µm)(Robertson 1983)
The main reason why the anaerobic ammonium oxidation process is used to treat high salt nitrogen wastewater is that AnAOB showed a relatively stable salinity tolerance(Li Wenyu et al. 2023). Under salinity stress, AnAOB can maintain osmotic pressure by reducing the permeability of the anammox membrane to water. Low salinity even has a positive stimulating effect on improving the nitrogen removal performance of AnAOB. When anaerobic anammox is used to treat saline wastewater, it is found that the reactor runs stably and the denitrification efficiency exceeds 80% when the salinity is 0-20 g/L. However, when the salinity is higher than 30 g/L, the denitrification efficiency of the system decreases, and the granular sludge becomes loose and hard(Wang G. et al. 2020). NaCl affects the activity of AnAOB by reducing the ammonia transportation rate and inhibiting enzyme activity (including hydrazine dehydrogenase Hzs and hydrazine synthase Hdh)(Lin et al. 2021). Different AnAOB has different degrees of salinity tolerance, and many studies generally believed that fresh anammox bacteria could adapt to the salinity of 30 g/L(Jin et al. 2011, Kartal et al. 2006, Ma et al. 2012, Wang G. et al. 2020). In addition, the denitrification efficiency of AnAOB is also affected by the amplitude of salinity change. The denitrification efficiency will also decrease when the salinity is increased greatly, such as when the salinity is suddenly increased from 14 g/L to 20 g/L(Yang J. et al. 2011a).