Isolation and identification of a salt-tolerant aerobic denitrifying bacterial strain and its application to saline wastewater treatment in constructed wetlands

Highlights

• Zobellella sp. A63 was isolated as a novel denitrifying bacterium.

• Addition of A63 increased nitrogen removal from saline wastewater in CW.

• A63 induced partial nitrification/aerobic denitrification in the top layer of CW.

• A63 increased the abundance of nirK in the top layer of CW.

Abstract

A salt-tolerant aerobic denitrifying bacterium, Zobellella denitrificans strain A63, was isolated, and its effects on the efficiency of denitrification of saline wastewater and the denitrifying microbial community structure in the matrix were studied in vertical-flow constructed wetlands (VFCWs). In a VFCW system with strain A63, the removal efficiencies of NH₄⁺-N, NO₃⁻-N, and total nitrogen reached 79.2%, 95.7%, and 89.9%, respectively. Quantitative PCR analysis indicated that the amoA gene from ammonia-oxidizing archaea (AOA) was highly abundant, whereas amoA from ammonia-oxidizing bacteria and nxrA from nitrite-oxidizing bacteria were lowly abundant because of the influent salinity, irrespective of whether strain A63 was added. However, the addition of strain A63 significantly increased the abundance of nirK in the top layer of the VFCW. Therefore, AOA-driven partial nitrification and aerobic denitrification by strain A63 occurred in VFCWs. Our findings suggest that adding salt-tolerant denitrifying strains to constructed wetlands can enhance denitrification for saline wastewater treatment.

Introduction

With the development of mariculture and the seafood processing industry in coastal areas, the discharge of large amounts of wastewater has led to increased eutrophication of the seawater and frequent red tides (Paliaga et al., 2017, Tan et al., 2017). This wastewater is characterized by a high organic load, high nitrogen content, and high salinity. Because of the high salinity, the efficiency of biological nitrogen removal is only 47.5–62.8% (Leung et al., 2016).

Previous studies have shown that salinity alters the osmotic pressure in microbes and thereby reduces their enzymatic activities, including metabolic activity associated with nitrogen removal (Hong et al., 2013, Wang et al., 2015a, Wang et al., 2015b). Ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) are particularly sensitive to salt concentration, and inhibition of their activities under high salinity leads to a significant decrease in ammonia nitrogen oxidation, and consequently, in nitrogen removal efficiency (Wang et al., 2015a, Wang et al., 2015b). To increase the efficiency of nitrogen removal from salty wastewater, various salt-tolerant (halophilic) nitrogen-removing microorganisms, including ammonia-oxidizing archaea (AOA) (Li et al., 2011), Zobellella sp., which are aerobic denitrifying bacteria (DB) (Fu et al., 2018), and the heterotrophic nitrifying/aerobic denitrifying bacterium Vibrio diabolicus SF16 (Duan et al., 2015), have been isolated and studied. Among these microorganisms, aerobic DB have the benefits of high salt tolerance and high growth and denitrification rates (Wang et al., 2015a, Wang et al., 2015b). Compared with anaerobic DB, aerobic DB have two clear advantages. First, aerobic DB can perform denitrification under aerobic conditions and thus, the nitrification and denitrification processes can be carried out in a single reactor, reducing space requirements and cutting costs for engineering and construction (Huang et al., 2015). Second, the acids and alkali generated during nitrification and denitrification are mutually neutralized, so that the pH of the system basically need not be adjusted, which greatly reduces the operation cost (Ji et al., 2015). Various aerobic DB, including Thauera, Zobellella, Pseudomonas, Aeromonas, Enterobacter, Acinetobacter, and Bacillus, which exhibit salt tolerance and efficient total nitrogen (TN) removal rates of up to 90.39%), were enriched in the early stage in mangrove constructed wetlands (CWs) (Fu et al., 2019). Zobellella abundance in these CWs was as high as 36.49%, making it the most important aerobic denitrifier in the system (Fu et al., 2019). However, it is unknown whether Zobellella can also effectively remove nitrogen from other wetlands.

Microbe-enhanced nitrogen removal is an in-situ remediation technique that involves adding specific types of bacteria or flora to contaminated soil or water to promote the degradation and conversion of nitrogen-containing contaminants and thus enhance the quality of these environments (Perelo, 2010). Microbe-enhanced nitrogen removal techniques for CWs are currently in an exploratory stage. Addition of the denitrifying bacterium Paenibacillus sp. XP1 to a pilot-scale reed CW increased the NO₃⁻-N and TN removal rates in secondary effluent of rural domestic wastewater (Pei et al., 2016). Other studies have demonstrated enhanced nitrogen removal in the presence of a complex flora (Zhao et al., 2016), low concentrations of a sediment/microbial community suspension (Zaytsev et al., 2011), or electricity-producing microorganisms (Xu et al., 2018, Liu et al., 2019). However, the addition of salt-tolerant microbes has not been explored, and thus, it is unknown whether such strains will have an impact on the microbial population.

In this study, a Zobellella sp. strain was isolated, purified, and added to a newly designed CW system to study whether it could improve the efficiency of nitrogen removal from wetland treated with salty wastewater. Further, the effect of addition of this bacterium on the wetland microbial community structure was analyzed to provide a preliminary experimental basis for future application of this strain in nitrogen pollution remediation of coastal wetlands.