Abundance and Role of Comammox Nitrospira in Biological Nitrogen Removal Systems in the Tropics

Abstract:

Nitrifying communities in activated sludge play a crucial role in biological nitrogen removal processes of municipal wastewater treatment plants. While extensive research has been conducted in temperate regions, the nitrifiers in engineered systems in tropical regions remain poorly understood. This project elucidated nitrifying communities in two full-scale municipal wastewater treatment plants in Malaysia operated under low-dissolved oxygen (0.2-0.7 mg-DO/L) or high-dissolved oxygen (2.0-5.5 mg-DO/L) conditions at 30°C. Core nitrifiers in the tropical municipal wastewater treatment plants were Nitrosomonas (ammonia-oxidizing bacterium, AOB) and Nitrospira bacteria (nitrite-oxidizing bacteria or complete ammonia-oxidizing comammox organisms) as determined by 16S rRNA gene amplicon sequencing analysis, which was further supported by fluorescence in-situ hybridization. Quantitative polymerase chain reaction (qPCR) assays targeting amoA genes revealed a wide distribution of comammox Nitrospira and betaproteobacterial AOB in the municipal wastewater treatment plants. In contrast to temperate regions, where ammonia-oxidizing archaea (AOA) are rarely detected in wastewater treatment systems, AOA were temporally abundant in the analyzed tropical municipal wastewater treatment plant operated under high-DO conditions. These findings enhance the understanding of nitrifying communities in tropical municipal wastewater treatment plants.

Group photo in the Centre for Microbiology and Enviromental Systems Science, University of Vienna (from left to right: Dr. Loi Jia Xing, UM; Prof. Ir. Dr. Adeline Chua, UM; Prof. Holger Daims, UW)

© CeMESS

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Implementation Period:

04/2024 – 06/2024

Project:

Project Objectives and Impact

Biological nitrogen removal (BNR) processes comprising nitrification-denitrification in wastewater treatment plants (WWTPs) are crucial to prevent eutrophication in water environments. Nitrification is the rate-limiting step in the BNR process, involving a diverse functional group of microorganisms. For >120 years, microbiologists firmly assumed that nitrification requires the activity of two different microbial groups, one that oxidizes ammonia to nitrite, represented by ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB), and another that oxidizes nitrite to nitrate by nitrite-oxidizing bacteria (NOB). Only recently, ‘comammox bacteria’ were discovered that completely oxidize ammonia to nitrate in the same microbial cell. This discovery was made independently by a research group at the University of Vienna and by a Dutch group [1, 2], and it has overturned our picture of nitrification in the nitrogen cycle. The comammox bacteria belong to the genus Nitrospira and, thus far, Nitrospira inopinata is the only successfully isolated comammox culture (it was isolated and is maintained in the team of Profs Holger Daims and Michael Wagner at the University of Vienna). Comammox bacteria have been ubiquitously detected in WWTPs in temperate regions [1, 2], and little is known about the abundance and roles of comammox bacteria in tropical WWTPs [3, 4]. Temperature is a critical environmental factor affecting microbial growth, and wastewater treatment plants in tropical regions are expected to harbor distinct nitrifying communities from those found in temperate regions.  A few previous studies conducted in tropical regions found the population of nitrifiers including AOB, AOA, and Nitrospira bacteria in tropical wastewater treatment plants [5, 6], while comprehensive insight of nitrifying communities in tropical region is still lacking.

Besides temperature, another critical factor affecting nitrifying communities is dissolved oxygen (DO). Aeration for supplying DO in the conventional nitrification accounts for about half of the energy costs in a typical WWTP. High energy requirements for aeration are not a sustainable wastewater treatment practice for developing countries. The research team of Prof Adeline Chua recently proposed a low‐DO oxic‐anoxic system as a simple and low‐cost retrofit to the conventional BNR in the developing countries [7]. AOB, AOA, NOB and comammox-bacteria have different affinities for DO [8]. By enriching those nitrifying communities that can grow at low DO levels, the low‐DO BNR process is able to reduce the amount of oxygen needed in the aeration step. The low‐DO BNR attained 99% NH₄⁺‐N removal at a DO level around 0.5 ppm, contributing to 23% saving in aeration energy when compared to a high‐DO (2 ppm) system. This process offers an exciting innovation to achieve energy‐saving wastewater treatment. To drive this innovation into operation, several fundamental aspects including the microbiology involved need to be addressed. Based on preliminary analyses of 16S rRNA gene sequences, Prof. Chua and co‐workers found potentially novel comammox Nitrospira in the low‐DO BNR system. However, this finding has yet to be confirmed.

Through the mobility program, the teams have collaborated to investigate the abundance and composition of nitrifying communities in tropical municipal wastewater treatment plants through a 7-month sampling campaign. Activated sludge samples were collected from two full-scale municipal wastewater treatment plants (WWTP-A and WWTP-B) in Malaysia operated under high-DO (2.0-5.5 mg-O₂/L) and low-DO (0.2-0.7 mg-O₂/L) conditions. A comparison of the nitrifying communities between high-DO and low-DO process enables to examine different nitrifying communities. The nitrifying communities were thoroughly investigated using a combination of 16S rRNA gene amplicon sequencing, quantitative PCR (qPCR), and fluorescence in situ hybridization (FISH) analysis targeting bacterial/archaeal 16S rRNA genes and AOB, AOA, and comammox amoA genes encoding ammonia monooxygenase subunit A.

Our findings advanced a comprehensive understanding of the role of different nitrifying communities in BNR processes in tropical WWTPs. This program allowed Prof Holger’s team to work directly with tropical low-DO activated sludge samples, offering new insights into comammox microbiology in WWTPs under different climatic and operational conditions. The mobility program facilitated knowledge transfer in molecular biology analysis methods, including functional gene analysis and chemical imaging, enabling Prof Adeline’s team to integrate cutting-edge molecular approaches into their research. The collaboration has provided an opportunity for Prof Adeline and her graduate student to engage with the broader scientific community at the University of Vienna. The academic outputs are one oral presentation in an international conference and the expected publication of one peer-reviewed journal article in a high-impact international scientific journal. The output will broaden the outreach of the findings within the global scientific community. The exchange of knowledge, skills, and resources has been mutually beneficial, ensuring that the impact of this program extends beyond its immediate outcomes to influence future research directions in microbial ecology of tropical wastewater treatment systems.

Results and Contributions

The canonical ammonia-oxidizing microbes detected were Nitrosomonas bacteria, and archaea related to the genera Nitrososphaera and Candidatus Nitrosotenuis. AOB other than Nitrosomonas (e.g., Nitrosospira and Nitrosococcus) [9], were not detected. The AOA were much more abundant in WWTP-B operated under high-DO conditions (up to 3.8% of the total community). However, their abundance showed a temporal variation and decreased to <0.2% after Feb. 2024. The site-specific distribution of AOA indicates that AOA have a defined ecological niche in WWTPs. AOA preferentially thrive in low-DO environments [10-12], and high abundance of AOA in municipal wastewater treatment plant under high-DO condition has not been described. High abundance of AOA under high-DO condition might reflect the bioavailability of copper. AOA possess a multi-copper-based electron transport system [13], and the decrease of copper availability inhibited the growth of AOA [14]. Operational conditions of the WWTP-B_lowDO (>30°C, 177 mg-COD/L, and 0.2–0.7 mg-O₂/L) are physiological conditions favoring sulfate reducers, and sulfate reduction causes the formation of hydrogen sulfide. Hydrogen sulfide easily reacts with soluble copper and decreases the bioavailability of copper [15]. However, further investigation including measurements of copper concentrations to elucidate the ecological niche(s) that facilitates the growth of AOA in tropical WWTPs will be required. Such investigation could be a next phase of collaboration between University of Vienna and University of Malaya.

Notably, the Nitrosomonas AOB and the AOA were often undetectable or less abundant in the WWTPs operated at low-DO conditions; i.e., the relative abundance of Nitrosomonas ASVs was <0.1% to 0.2% and no AOA ASV was detected in WWTP-A_lowDO. This suggests the involvement of other ammonia-oxidizing microbes in the WWTP-A_lowDO (e.g., comammox bacteria).

The 16S rRNA gene amplicon sequencing analysis detected Nitrospira (potentially, canonical NOB and comammox) in both low-DO (0.2-0.7 mg-O₂/L) and high-DO (2.0-5.5 mg-O₂/L) conditions, suggesting their capability to adapt to a wide range of DO environments. The presence of comammox Nitrospira was confirmed by the qPCR assay targeting comammox amoA genes, revealing the presence of comammox Nitrospira in tropical WWTPs. The combined results of 16S rRNA gene amplicon sequencing, qPCR, and FISH analysis revealed that the abundance of (comammox and NOB) Nitrospira cells in activated sludge was comparable with that of Nitrosomonas cells.

The findings of this project are the first comprehensive description of AOB, AOA, NOB and comammox populations in tropical municipal WWTPs. The findings contribute to advance the understanding of tropical nitrifiers in order to optimize sewage treatment processes in tropical climates. For instance, an overgrowth of AOA was observed in the WWTP-A_{high DO}, which can enhance the environmental impact because the NH₄⁺ oxidation by AOA discharge less amounts of nitrous oxide (N₂O) gas known as strong greenhouse gas [16]. Specific ecological niche(s), however, needs to be identified in the future studies to control the population of those functional microbes in the tropical WWTPs. Also, the contribution of AOA, AOB, NOB, and comammox bacteria to overall nitrification rates in tropical sewage treatment plants remains unclear due to a lack of kinetic data. Therefore, future studies should include year-round sampling campaigns, batch incubation experiments using specific inhibitors for AOB (allylthiourea (ATU)), AOA (2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide (PTIO)) and comammox bacteria (allylthiourea (ATU)) [17, 18], and advanced techniques, such as ¹⁵N stable isotope tracer analysis, to quantify the functional roles of various nitrifiers under dynamic environmental conditions in full-scale systems.

Reference:

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