Unclassified Anammox bacterium responds to robust nitrogen removal in a sequencing batch reactor fed with landfill leachate

Highlights

• An unclassified Anammox bacterium responds to N removal in an SBR fed with leachate.

• The unclassified Anammox bacterium is distinctly related to known Anammox bacteria.

• PN/A was achieved from activated sludge in an SBR without biomass carriers.

• SBR performed robust N removal and achieved a peak NRR of 1.91 kg N/m³/d.

• SBR likely performed arsenate reduction and degradation of drugs and antibiotics.

Abstract

Treatment of landfill leachate was conducted in a lab-scale sequencing batch reactor (SBR). The SBR was started through inoculating activated sludge with controlling dissolved oxygen of 0.5–1.0 mg/L. Anammox reaction took place within around three months. The SBR established robust nitrogen removal with incremental NLRs of 0.25–2.17 kg N/m³/d. At the final phase, it achieved elevated nitrogen removals of 1.68–1.91 kg N/m³/d. 16S rRNA gene amplicon sequencing analysis revealed Nitrosomonas, unclassified Anammox bacterium, and diverse denitrifying populations coexisted and accounted for 4.02%, 20.05% and 34.69%, respectively. Phylogenic analysis and average nucleotide identity comparison jointly suggested the unclassified Anammox bacterium potentially pertained to a novel Anammox lineage. The functional profiles’ prediction suggested sulfate reduction, arsenate reduction and eliminations of antibiotics and drugs likely occurred in the SBR. The finding from this study suggests contribution of unclassified Anammox bacteria in influencing nitrogen budget in natural and engineering systems is currently being underestimated.

Introduction

Landfilling of municipal solid waste (MSW) is the dominant strategy in today’s context of waste management worldwide (Sri Shalini & Joseph, 2012). Leachate percolated from solid wastes has become a great threat to the surroundings as it contains complex constituents including refractory compounds (i.e., phenols and humic substances), toxic pollutants (i.e., heavy metal ions and arsenate), ammonia, and high salt content (Kjeldsen et al., 2002). Efforts have been invested to address the ammonia issue in particular cause its overload provokes severe water pollution issues such as eutrophication, methemoglobinemia, poisoning of aquatic animals, and offensive smells.

Among the treatment strategies, partial nitritation and Anammox (PN/A) has been affirmed as an energy-saving and eco-friendly approach in comparison with the conventional nitrification and denitrification (N/DN). Hitherto, the PN/A has been widely applied from lab- to full-scale to treat active and mature landfill leachate (Azari et al., 2017, Chen et al., 2016, Phan et al., 2017, Wang et al., 2010). Emphases have been placed on the achievements of high-rate nitrogen removal and process stability. For instance, an internal circulation reactor has been applied to treat landfill leachate and a maximum nitrogen removal rate (NRR) of 9.52 kg N/m³/d was obtained (Phan et al., 2017). Nitrogen removal efficiency (NRE) of over 90% has been normally achieved in Anammox-based processes treating landfill leachate (Li et al., 2020, Zhang et al., 2017). Thus, in short, it is realizable to obtain stable and high-rate nitrogen removal with leachate while Anammox process was applied. As for the microbial analysis, peer studies either identified the functional microbes (e.g., AOB and Anammox bacteria) using fluorescence in situ hybridization (FISH) (Azari et al., 2017, Wang et al., 2010) and quantitative real-time polymerase chain reaction (qRT-PCR) (Wu et al., 2018) or determined the microbial community analysis with a single biomass sample (Chen et al., 2016, Phan et al., 2017). However, tracking the dynamics of the microbial community along with long-term process operation have not been well interpreted yet. Understanding the dynamics of microbial community especially for functional microflora in response to variates during the long-term operation, e.g., incremental nitrogen load, is beneficial to process manipulation and optimization.

Another critical issue is the overlook and underestimation of the contribution of unclassified Anammox-like microorganism in the landfill leachate treatment processes. Anammox contributed to approximate 10% of the nitrogen removal in a bioreactor treating mature landfill leachate based on isotopic ¹⁵N tracing study, however, Candidatus Kuenenia was detected at below 0.01% and no other known Anammox bacterium was detected (Xie et al., 2013). It is notable that 59.2% of the total Planctomyces reads were unclassified to lower taxonomic levels, which were hypothesized to contain some novel anammox bacteria. Such phenomenon has been also observed in other Anammox bioreactors. Kuenenia shifted to an unclassified Brocadiaceae whilst salinity of 1.8% was introduced, and the latter further predominated the Anammox population and mainly responded to the NRR of up to 9.72 kg N/m³/d (Lu et al., 2019). An unclassified bacterium affiliated with the family of Brocadiaceae was the corresponding Anammox bacterium in a simultaneous partial nitritation, anammox, and denitrification (SNAD) process treating ammonia-rich organic wastewater (Zhou et al., 2018). Similarly, microorganism affiliated with Brocadiaceae was the corresponding Anammox bacterium in a lab-scale up-flow bioreactor, and no other recognized Anammox genera proliferated during over 200 days’ operation (Wang et al., 2020). It has been hypothesized that, aside from Candidatus Anammoximicrobium (i.e., 0.08%), there should be novel Anammox bacterium contributing to the nitrogen removal in the simultaneous sludge fermentation, denitrification and anammox process (Wang et al., 2016). In brief, these peer studies indicated the prevalence of unclassified Anammox bacterium in practical applications. However, its contribution to overall nitrogen removal is being overlooked, and the survival niche of these unclassified Anammox lineages has not been interpreted yet. The co-occurrence network analysis is one advantageous means to disclose interaction networks of dominant microflora (Ma et al., 2016). The co-occurrence patterns between hub species and neighbors reveal niche spaces shared by symbiotic members (Deng et al., 2016). Besides, the exploitation of potential functionalities via FARPROTAX and Tax4Fun offers insights into the potential metabolic networks of microbial community (Aßhauer et al., 2015, Louca et al., 2016). These analytical approaches could be united to decrypt the microbial survival niche, which have been not applied in above-mentioned studies yet.

Globally, the availability of mature Anammox seed is considered as the primary factor hindering the wide application of Anammox process. Aside from the European countries and the United States, Anammox seed is usually not available in many places of other countries (Lackner et al., 2014). Hitherto, most PN/As have been initiated through introducing Anammox seed and nitrifying sludge as summarized by Huang et al. (2016a). Alternatively, it has been validated that operating PN under limited oxygen conditions could realize PN/A with the membrane bioreactor (MBR) (Huang et al., 2016a, Zhang et al., 2013). Most recently, PN/A was achieved from activated sludge with an MBR in around two months through controlling the DO at 0.89–1.38 mg/L (Huang et al., 2020). The utilization of MBR was considered as the principle factor leading to these achievements owing to that the MBR is capable of complete biomass retention. The biomass retention is the key factor determining the success or failure of the startup of PN/A whist activated sludge was utilized as the sole inoculant. Either MBR or introduction of biomass carriers was considered previously as the bare essential to prosper PN/A (Lackner et al., 2014). The sequencing batch reactor (SBR) was shown to be a powerful tool for cultivating slowly growing Anammox bacteria due to its efficient biomass retention and a homogeneous distribution of substrates (Strous et al., 1998). Conceivably, it could be a proper candidate reactor configuration for initiating PN/A from activated sludge without introducing biomass carriers. Such study has not been carried out yet.

In this study, treatment of landfill leachate has been carried out in a laboratory-scale sequencing batch reactor (SBR) with inoculating activated sludge solely. Anammox reaction took place in around three months. The reactor performance including nitrogen removal and COD removal was routinely monitored. 16S rRNA gene amplicon sequencing was performed to scrutinize the microbial community dynamics. Interestingly, unclassified Anammox bacterium was enriched and responded to the nitrogen removal. The overarching objectives of this study were 1) to evaluate the reactor performance and process stability with incremental nitrogen loads; 2) to examine time-varying dynamics and potential functionalities of microbial community; 3) attempting to understand the survival niche of the detected unclassified Anammox bacterium.