Manganese oxides in Phragmites rhizosphere accelerates ammonia oxidation in constructed wetlands

Highlights

• Enriched microbes in Phragmites rhizosphere are largely involved in Mn oxidation.

• Mn oxide is a significant factor shaping rhizosphere microbiome composition.

• 92% of Mn-oxidizing microbes potentially participate in nitrogen cycling.

• Mn-oxidizing genes are positively correlated with comammox Nitrospira.

• Ammonia oxidation is enhanced in Mn-amending constructed wetlands.

Abstract

Phragmites reeds are widely used in constructed wetlands (CWs) for treating wastewater. The enrichment of microorganisms and Fe/Mn plaque in Phragmites rhizospheres largely contributes to pollutant removal. However, their interactions and potential synergistic roles in water purification are poorly understood. To address the issue, we first compared the microbial community traits in the Phragmites rhizosphere and adjacent bulk soil in six long-term operated CWs. Results showed that enriched microbes and functional genes in the Phragmites rhizosphere were largely involved in Mn oxidation, resulting in a two to three times enrichment of Mn oxides in the rhizosphere. In turn, the enriched Mn oxides played significant roles in driving microbial community composition and function. To further understand the biological manganese oxidation in the rhizosphere, we identified Mn-oxidizing bacteria using genome-centric analysis and found that 92% of identified Mn-oxidizing bacteria potentially participated in nitrogen cycling. We then conducted relationships between Mn-oxidizing genes and different nitrogen cycling genes and found Mn-oxidizing gene abundance was significantly correlated with ammonia oxidation gene amoA (R = 0.65). Remarkably, complete ammonia oxidation (comammox) Nitrospira, accounting for 39.11% of ammonia oxidizers, also positively correlated with Mn-oxidizing microbes. Based on the above observations, we inferred that the use of Mn oxides as a substrate in CWs may enhance ammonia oxidation. To apply this to actual engineering, we explored treatment performance in a pilot-scale Mn-amending CW. As expected, ammonia removal capacity improved by 23.34%, on average, in the Mn-amending CW. In addition, the abundance of amoA genes increased significantly in the Mn-amending CW, indicating improved biological processes rather than chemical reactions.

Introduction

Constructed wetlands (CWs) are engineered systems that have developed rapidly in recent decades. As a natural alternative to technical wastewater treatment, CWs exhibit strong pollution remediation via the combined roles of plants, microorganisms, and soil (Stottmeister et al., 2003). Increasing evidence has demonstrated that plant rhizosphere microbiomes play a vital role in pollutant transformation (Jiang et al., 2020; Saravanan et al., 2019; Thomas et al., 2019). Reeds (Phragmites) are one of the most commonly applied plants in CWs. Taxonomically, Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes are abundant at the phylum level in the Phragmites rhizosphere (Borruso et al., 2014). The Phragmites rhizosphere microbiome can biodegrade technical nonylphenol (Toyama et al., 2011b), pyrene, and benzo[α]pyrene (Toyama et al., 2011a). Nevertheless, a comprehensive understanding of Phragmites rhizosphere microbiome traits is still needed, e.g., which environmental factors shape the composition and function of the rhizosphere microbiome.

Besides microbiome, Iron (Fe) and Manganese (Mn) plaque is a common component of the plant rhizosphere environment (Liu et al., 2006), which contributes to the transformation and stabilization of pollutants, such as arsenic (As) (Wang et al., 2019) and phosphate (Warrinnier et al., 2020). Mn oxides derived from biological processes (i.e., biogenic Mn oxides) are one of the main components of Fe/Mn plaque (Learman et al., 2011). Moreover, Mn-oxidizing microbes are common in plants’ rhizosphere and mostly aerobic (Maisch et al., 2019; Weiss et al., 2003). Due to their high oxidizing ability, Mn oxides can further transform and degrade pollutants absorbed by Fe/Mn plaque. In natural environments, Fe/Mn plaque accumulation can account for up to 10% of Phragmites’ root dry weight and extends as much as 15–17 µm into the rhizosphere (Hansel et al., 2001), thus providing an ecological foundation for its application in wastewater treatment. Therefore, we inferred that Mn oxides should be enriched in the rhizosphere environment. However, to what extent Mn oxides are enriched in the Phragmites rhizosphere environment and how they influence the performance of CWs requires further exploration.

The average concentration of Mn in wetlands is ∼100 mg/kg (ppm) (Vymazal and Švehla, 2013). Therefore, Mn-amending methods, i.e., Mn ore (Yang et al., 2019), Mn-coated biochar (Guo et al., 2020), and birnessite-coated sand substrates (Xie et al., 2018), have been applied in CWs to promote pollutant removal. Mn-amending CWs can promote nitrogen transformation, phosphate removal, and emerging organic micropollutant removal at mg/L or ng/L levels (Li et al., 2019). Of note, nitrogen removal efficiency is closely related to the microbial community influenced by Mn oxides, and the application of Mn oxides can improve total nitrogen removal efficiency (Zhao et al., 2020). Furthermore, some Mn-oxidizing bacteria possess the ability to denitrify, which can improve the removal efficiency of nitrate in underground water where Mn(II) and nitrate are found at high concentrations (Bai et al., 2020; Su et al., 2020). However, previous advances about improved nitrogen removal in CWs led by amending Mn oxides constrained into lab-scale reactors. The pilot-scale applications and related gene-level mechanisms have yet to be explored. The nitrogen transformation and fixation in CWs are complexed (Jahangir et al., 2020) and varied with environmental factors (for example, temperature) (Wang et al., 2021). Therefore, two key questions were proposed “which process is most significantly influenced by Mn oxides in CWs” and “what are the underlying mechanisms of the influence”.

Overall, information is still lacking on Mn oxides in plant rhizospheres (e.g., Phragmites) and their interactions with attached microorganisms, hindering the application of Mn-amending CWs in wastewater treatment. Here, we chose six CWs that have been in operation for at least five years as study sites and applied metagenomic sequencing to profile the Phragmites rhizosphere microbiome and Mn-oxidizing bacteria. We also investigated the relationships among Mn oxides, Mn-oxidizing genes, and the rhizosphere microbiome. Based on the finding from metagenomic analysis, we performed a one-year pilot-scale experiment on Mn-amending CWs to investigate the causal relationship between Mn amendment and augmented biological processes.