Cultivation of anammox bacteria from a tropical lake in Indonesia using a novel filter bioreactor to enhance nitrogen removal efficiency Open Access

In the environment, anaerobic ammonium oxidation (anammox) bacteria convert ammonium into nitrogen gas under anaerobic conditions using nitrites as donor electrons (Van Kemenade et al. 2024). Anammox process is more efficient than the conventional nitrification–denitrification process, which can achieve a 60% reduction in oxygen demand, eliminate the need for organic carbon addition, and reduce land requirements. Additionally, the lower production of N₂O greenhouse gases (i.e., N₂O) and waste active sludge makes this technology more environmentally friendly (Ali & Okabe 2015). This process is widely utilized for nitrogen removal from wastewater, with over 114 large-scale anammox reactors currently in operation worldwide, and the number is steadily growing (Lackner et al. 2014). Furthermore, it fosters the exploration and enrichment of anammox bacteria across diverse ecosystems. Presently, 23 species spanning seven genera have been identified, including Ca. Anammoxoglobus, Ca. Anammoximicrobium, Ca. Brasilis, Ca. Brocadia, Ca. Jettenia, Ca. Kuenenia, and the marine anammox group within Ca. Scalindua (Narita et al. 2017; Miao et al. 2019). The distribution of known anammox species encompasses regions, such as Europe, China, Japan, Russia, and Brazil. However, they are predominantly located in sub-tropical zones (except for Brazil), rather than tropical areas.

Anammox bacteria represent a subset of slow-growing microorganisms, as indicated by their doubling time of 10–12 days (Van Der Star et al. 2007); although faster rates have been reported ranging from 2.1 to 3.9 days (Zhang et al. 2017). This characteristic poses challenges for their practical application, rendering conventional microbiological cultivation techniques ineffective (Fujii et al. 2002). Moreover, their low environmental abundance requires laboratory enrichment from environmental samples for identification purposes, yet achieving pure cultures remains elusive. Various enrichment strategies have been explored using different reactor configurations such as sequencing batch reactors (SBRs), fluidized bed reactors (FBRs), gas-lift reactors (GLRs), and membrane bioreactors (MBRs) (Nsenga Kumwimba et al. 2020). MBRs emerge as particularly promising due to their ability to enrich slow-growing bacteria, including anammox species. However, the widespread adoption of MBRs is hindered by their high procurement costs, limited market availability, and operational complexities associated with membrane fouling (Wu et al. 2023). Previous efforts by Zulkarnaini et al. (2018) demonstrated the use of a string wound filter for cultivating anammox biofilms with Candidatus Brocadia sinica granules at 35 °C and employed filter feeding strategies to grow anammox biofilm before start-up one-stage nitritation/anammox process (Zulkarnaini et al. 2024). Building upon this work, they introduced a novel reactor, termed the filter bioreactor (FtBR), in which the same string wound filter was installed as a supporting medium on cartridge housing filters. This innovative setup facilitated the cultivation of anammox bacteria using environmental sludge as an inoculum, thus extending the applicability of anammox biofilm cultivation techniques (Gumelar et al. 2024; Zulkarnaini et al. 2024).

In addition to the enrichment strategy, procuring anammox inoculants is important for developing a bioreactor-based anammox process. Anammox bacteria are omnipresent across diverse ecosystems and geographical regions due to their crucial role in aquatic and marine nitrogen removal (Ismail et al. 2022; Gumelar et al. 2024). Exploring and discovering anammox bacteria within local environments offers significant advantages over importing strains from external sources. However, the existing body of information and research pertaining to this subject remains relatively scarce, especially for the anammox inoculants originating from tropical countries (Nsenga Kumwimba et al. 2020). Given Indonesia's status as a tropical country, it represents a promising venue for exploring anammox bacteria within its indigenous environmental contexts.

Typically, anammox bacteria grow in nitrogen-polluted environments or those with elevated nitrogen concentrations, where they often play a crucial role in mitigating eutrophication in lakes, as demonstrated in Japan (Yoshinaga et al. 2011) and China (Zhu et al. 2013). In Indonesia, Lake Koto Baru has become a focal point for studying eutrophication, a consequence of nutrient accumulation from surrounding agricultural areas. The lake surface is predominantly covered by Salvinia with a lesser presence of Eichhornia crassipes, while the shallow lake bed is characterized by the accumulation of decaying plant matter over several years (Putra et al. 2020). Given these ecological conditions, Lake Koto Baru presents an ideal candidate location for investigating and characterizing anammox bacteria within tropical environments. This study aims to cultivate and identify anammox bacteria within this setting using a FtBR approach. Sediment samples were collected from Lake Koto Baru, Tanah Datar, Indonesia, and inoculated into the FtBR reactors. Two operational runs were conducted for 200 days: one at a range of tropical ambient temperatures (22–28 °C) in Reactor 1, and the other at a constant temperature of 35 °C in Reactor 2. Microbial abundance was assessed via Illumina MiSeq sequencing.

Lake Koto Baru, situated in Tanah Datar, Indonesia, spans an area of 126,963 m². Approximately 85.3% of its surface has transitioned into marshland, encroached upon by vegetation. Originally boasting a depth of approximately 20 m, the lake has experienced a shallowing phenomenon, with depths ranging from 0.5 to 4 m, attributed to sedimentation and the accumulation of organic matter from plant debris. Surrounding agricultural activities have further exacerbated nutrient and soil deposition, including nitrogen compounds, in the lake ecosystem.

Dissolved oxygen (DO) and pH were measured onsite. Collected samples were immediately transported to the laboratory, where total solids (TS) and volatile suspended solids (VS) concentrations were measured. The filtered samples’ ammonium, nitrate, and nitrite concentrations were also determined.

FtBR was made of a housing filter (10 inches, Nanotec, China), and a string wound filter (0.5 μm pore size; PURETREX, USA) was mounted in the center of the reactor as a biofilm supporting media. The reactor was covered with aluminum foil to prevent the growth of photosynthetic bacteria. The sediment mud mixture filled around 70% of the reactors. The substrate is fed into the reactor through the hollow inner part of the filter using a peristaltic pump. The flow rate was controlled for 24-h hydraulic retention time (HRT). The substrate container was equipped with a Tedlar bag containing nitrogen gas to maintain anoxic conditions.

Two FtBRs were operated at different temperatures, i.e., tropical ambient temperature (22–28 °C) in Reactor 1 and 35 °C in Reactor 2. Table 1 shows the operational conditions of FtBRs. At the beginning of the reactor's operation (Period 1), 70 mg-N/L (NH₄)₂SO₄ and NaNO₂ were added to the mineral medium (per liter of distillate water) containing 500 mg KHCO₃; 27.2 mg KH₂PO₄; 300 mg MgSO₄·7H₂O; 180 mg CaCl₂·7H₂O; and 1 mL trace elements I and II (Zulkarnaini et al. 2021). At the end of Period 1, all suspended solids derived from inoculum were withdrawn from the reactor since biofilm was attached to the filter. Ammonium and nitrite concentrations were gradually increased from 70 to 150 mg-N/L in Periods 2–4.

Table 1

Comparison of operational conditions between Reactor 1 (22–28 °C) and Reactor 2 (35 °C)

Period .	Time (d) .	Concentration (mg-N/L) .		HRT (h) .	Temperature (°C) .		Note .
		.	.		Reactor 1 .	Reactor 2 .
1	0–62	70	70	24	22–28	35
2	63–91	70	70	24	22–28	35	Seeding sludge removed at day 63
3	92–105	100	100	24	22–28	35
4	106–200	150	150	24	22–28	35

Period .	Time (d) .	Concentration (mg-N/L) .		HRT (h) .	Temperature (°C) .		Note .
		.	.		Reactor 1 .	Reactor 2 .
1	0–62	70	70	24	22–28	35
2	63–91	70	70	24	22–28	35	Seeding sludge removed at day 63
3	92–105	100	100	24	22–28	35
4	106–200	150	150	24	22–28	35

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Temperature and pH were monitored every day. Influent and effluent were sampled twice weekly, and the concentrations of ammonium, nitrite, and nitrate were analyzed. Observations of the anammox process were analyzed based on calculations using the ammonium conversion efficiency (ACE, %), nitrogen removal efficiency (NRE, %), and nitrogen removal rate (NRR, kg-N/m³·d).

DO was measured using a DO meter (DO-5510, Lutron, Taiwan). Temperature and pH were measured using a thermo-hygrometer (Corona gl-89, China) and pH meter (STARTER300, Ohaus, USA), respectively. Chemical oxygen demand (COD), TS, and VS concentrations were analyzed according to APHA (2017). Total phosphorus (TP) and total organic carbon (TOC) concentrations were analyzed using a perchloric acid method and a TOC analyzer (TOC-L, Shimadzu, Japan). The concentrations of ammonia and nitrite were analyzed by the Nessler and spectrophotometric method, while nitrate was measured by an ultraviolet spectrophotometric method with a UV–Vis spectrophotometer (Shimadzu 1800, Kyoto, Japan), according to APHA (2017).

At the end of Period 3, the biofilm was sampled and the microbial community was analyzed. The total DNA from biofilm samples was extracted using a PowerSoil DNA Isolation Kit (QIAGEN, Germany) following the manufacturer's protocol. The V3–V4 region of 16S rRNA gene was amplified from the extracted DNA using universal forward primer 515F (5′-ACTCCTACGGGAGGCAGCAG-3′) and reverse primer 806R 5′-GGACTACHVGGGTWTCTAAT-3′) for bacterial community (Caporaso et al. 2012) and 340F (5′-CCTACGGGNGGCWGCAG-3′) and 806Rb (5′-GGACTACHVGGGTWTCTAAT-3′) primers for archaeal community (Gantner et al. 2011; Apprill et al. 2015). The PCR products were sequenced using the Illumina MiSeq sequencing (Illumina, USA). The UPARSE (Edgar 2013) and the QIIME2 platform (Caporaso et al. 2010) were used to process and analyze the sequences as described by Mawarda et al. (2022). Phylogenetic tree was constructed using ARB software.

Table 2 shows the characteristics of the collected mud samples. TS and VS concentrations were 40.2–42 and 25.7–27.6 g/L, respectively. There were no significant differences in mud sample characteristics across the sampling points. The VS/TS ratios ranged from 64 to 65%, indicating a high organic content in the mud. Whereas the dissolved oxygen carbon (DOC) concentrations were approximately 40 mg/L, with ammonium and nitrate being present in the supernatant, suggesting that the lake was eutrophic. The TP concentration, ranging from 1.56 to 1.82 mg-P/L, exceeded the water quality standard criteria in the Indonesian Government Regulation. These characteristics are comparable to those of lakes in Indonesia, where eutrophication is a common phenomenon (Soeprobowati et al. 2021; Izzati et al. 2022; Ruthena et al. 2022; Komala et al. 2024).

Table 2

Characteristics of the mud samples collected from Lake Koto Baru

Sampling points .		I .	II .	III .	IV .
Depth	m	0.6	0.5	1.0	0.5
pH		6.6	7.1	7.4	7.3
Temperature	°C	25.2	25.6	25.5	25.2
DO	mg/L	4.2	3.1	3.4	7.4
TS	g/L	40.2	42.2	41.0	40.6
VS	g/L	25.7	27.6	26.2	26.1
	mg-N/L	5.6	5.2	5.2	4.7
	mg-N/L	0.3	0.3	0.3	0.3
	mg-N/L	2.7	2.1	2.2	2.1
DOC	mg-C/L	37.3	41.0	39.5	38.6
TP	mg-P/L	1.82	1.62	1.58	1.56
COD sediment	mg/L	2,688a

Sampling points .		I .	II .	III .	IV .
Depth	m	0.6	0.5	1.0	0.5
pH		6.6	7.1	7.4	7.3
Temperature	°C	25.2	25.6	25.5	25.2
DO	mg/L	4.2	3.1	3.4	7.4
TS	g/L	40.2	42.2	41.0	40.6
VS	g/L	25.7	27.6	26.2	26.1
	mg-N/L	5.6	5.2	5.2	4.7
	mg-N/L	0.3	0.3	0.3	0.3
	mg-N/L	2.7	2.1	2.2	2.1
DOC	mg-C/L	37.3	41.0	39.5	38.6
TP	mg-P/L	1.82	1.62	1.58	1.56
COD sediment	mg/L	2,688a

^aComposite samples as inoculum.

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The catabolism-produced ammonium was more rapid in Reactor 2 than in Reactor 1 due to higher temperature operation (Figure 3). In contrast, the nitrite concentration in effluent decreased rapidly in both reactors. This result is consistent with the previous reports, which reported a rapid reduction in nitrite concentration during the start-up period when using a mixture of aerobic activated sludge and nitrifying sludge (Wang et al. 2013) or sludge from an anaerobic digester of wastewater treatment plants (Chen et al. 2012). The rapid decline in nitrite concentration was likely associated with the activity of denitrifying bacteria, which potentially utilized organic compounds in the seeding sludge (Wang et al. 2013). The anammox activity appeared after 1 month of cultivation in the above experiment.

In contrast, the sluggish period continued over 2 months in this study. Since the lake was eutrophic, the sediment was estimated to contain a high concentration of biodegradable organic matter (COD 2,688 mg/L). In addition, the heterotrophic denitrification activity was likely high.

The maximum nitrogen removal performance during the first period in Reactor 1 based on ACE, NRE, and NRR values was 25.6%, 53.3%, and 0.085 kg-N/m³·d, respectively. The results in Reactor 2 were higher than in Reactor 1, where ACE, NRE, and NRR were 54.4%, 74.9%, and 0.107 kg-N/m³·d, respectively. Meanwhile, on day 63 (Period 2), the sludge was discharged from both reactors to reduce the effect of organic carbon, while the attached biofilm was preserved on the filter. On day 92 (Period 3), the ammonium and nitrite concentrations in the substrate were increased to 100 mg-N/L. Both effluent concentrations of ammonium and nitrite decreased significantly. Almost all nitrogen was removed from the reactor. In this third period, ACE, NRE, and NRR in Reactor 1 were 97.1%, 91.9%, and 0.19 kg-N/m³·d, respectively, whereas in Reactor 2, they were 94.7%, 92.0%, and 0.16 kg-N/m³·d, respectively.

The fourth period started on the 106th day when the concentration of ammonium and nitrite increased to 150 mg-N/L. At the beginning of this stage, nitrogen removal performance declined because of the shock loading; then, the ammonium and nitrite concentrations gradually decreased in Reactor 1. During this period, the maximum ACE, NRE, and NRR values in Reactor 1 were 98.2%, 93.0%, and 0.303 kg-N/m³·d, respectively. In contrast, ammonium and nitrite concentrations were unstable in the effluent from Reactor 2. Meanwhile, the maximum values of ACE, NRE, and NRR were 88.6%, 68.9%, and 0.214 kg-N/m³·d, respectively. This indicates that the reactor performances in Reactor 1 were better than those in Reactor 2 in this period (Figure 4). The results showed that temperature influenced the performance of nitrogen removal where sudden changes in temperature (temperature shock) may affect the activity and viability of indigenous microorganisms on Reactor 2. The water temperature in Lake Koto Baru at the sampling date (31 June 2020) was 25.8 °C, whereas the experimental temperature in Reactor 1 was 22–28 °C (23.9 °C on average). Enrichment temperature near the origin environment showed better performances.

Nonetheless, this indicates that the biomass in the FtBR exhibited the characteristics of anammox bacterial biomass, consistent with the reactor's anaerobic nitrogen removal performance. At the end of the experiment, the red carmine biofilm became thicker and covered the filter. This observation was similar to the study by Zhang et al. (2015), who reported that the enrichment of anammox bacteria was successful if the sludge color changed from brown to reddish and bright red. According to Ma et al. (2011), the red color is also typical of anammox bacteria enrichment. The biofilm attached to the filter and housing of FtBR. On the other hand, there was no difference in the biofilm's color of inoculum in Reactor 2, in which NRR was lower than in Reactor 1. The microbial community analysis below explains the above phenomenon.

The low abundance of the Anaerolineaceae family was observed, where these microorganisms live mutually with anammox bacteria by utilizing organic material from dead cells or organic material from seeding sludge (Kindaichi et al. 2012). In contrast, Reactor 2 had five genera with an abundance higher than 5%, which are Mycobacterium (14.24%), PHOS-HE51 (11.00%), Candidatus Anammoxoglobus (7.64%), Ignavibacterium (7.08%), and Candidatus Brocadia_1 (6.84%). Another anammox bacteria identified in FtBR operated at 35 °C (Reactor 2) was Candidatus Brocadia_3 (1.77%). It was reported that Ca. Anammoxoglobus outcompeted in the presence of an organic substance such as propionate (Kartal et al. 2007) or sulfate (Liu et al. 2008) and operated in a controlled temperature of 33–35 °C. These results indicate that anammox bacteria were successfully enriched in tropical ambient temperature and 35 °C operation (both reactors), though the dominant anammox consortium was different.

In this study, a high-rate anammox reaction was successfully established within 90 days, resulting in the enrichment of anammox bacteria by over 25% under tropical ambient temperatures, using the newly developed reactor and procedure. These findings suggest the potential applicability of the reactor for developing anammox-based wastewater treatment under tropical ambient conditions. Additionally, the dominant anammox bacterial species identified showed close phylogenetic relationships with Ca. Brocadia fulgida.

Different bioreactor treatments in this study involved variations in operational temperature: Reactor 1 operated at tropical ambient temperature (22–28 °C) and Reactor 2 at 35 °C. These differences resulted in the variation of relative abundance and the diversity of the dominant species of the growing anammox. This indicates that temperature is indeed a crucial factor in cultivating anammox bacteria. The optimal temperature for the activity of many anammox species ranges between 30 and 40 °C (Tomaszewski et al. 2017). Interestingly, this study revealed that the optimal temperature, indicated by the highest relative abundance of the anammox bacteria, falls within the tropical ambient temperatures, ranging from 22 to 28 °C.

Furthermore, the highest variation of anammox bacteria species diversity was observed at the operational temperature of 35 °C. Careful temperature control during the anammox process is essential, as temperatures exceeding 45 °C could permanently reduce anammox bacterial activity (Fernández et al. 2012). Yet, Vandekerckhove et al. (2020) reported the successful adaptation of thermophilic anammox bacteria by gradually increasing the temperature of a mesophilic inoculum to 50 °C. Additionally, it is well-established that the tropical temperature range is conducive to the anammox process for nitrogen removal, with suitable temperatures ranging from 25 to 38 °C (Trigo et al. 2006), 20–30 °C (Isaka et al. 2007), and approximately 20 °C (Ma et al. 2013). This substantiates that the anammox process can be operated at various temperatures; however, selecting the appropriate species of anammox bacteria is critical due to variations in anammox activity among different species.

The species Ca. Brocadia sinica had the smallest abundance within the biomass of Reactor 1. This is because Ca. Brocadia sinica cannot thrive well at tropical ambient temperatures, since the bacterial optimum growth is at 45 °C (Oshiki et al. 2011). However, at 35 °C, Ca. Brocadia sinica exhibits improved growth rates, as shown by an increase in abundance to 1.77%. Meanwhile, the identification of the species Ca. Anammoxoglobus propionicus can be considered rare among anammox researchers (Hsu et al. 2014). This species was initially discovered through laboratory-scale reactor studies involving the addition of the chemical compound propionate to the substrate (Kartal et al. 2007), although research conducted by Hsu et al. (2014) successfully obtained the species Ca. Anammoxoglobus propionicus without the addition of propionate. Interestingly, Ca. Anammoxoglobus propionicus was not found in Reactor 1, indicating that this species cannot survive and adapt to tropical ambient temperatures in tropical climates.

The species Ca. Brocadia fulgida emerges as the most dominant species in terms of abundance percentage in both reactors, accounting for 20.04% in Reactor 1 and 6.85% in Reactor 2. In Reactor 1 specifically, Ca. Brocadia fulgida was the most dominant species. This indicates that the tropical ambient temperature maintained in this study, within the range of 22–28 °C, falls within the optimum temperature range for the activity of this species. Yet, despite its non-dominant status in Reactor 2, Ca. Brocadia fulgida continues to grow together with the dominant species Ca. Anammoxoglobus propionicus, with a difference in abundance of <2%. The relative abundance difference of the Ca. Brocadia fulgida between Reactor 1 and Reactor 2 indicates that the temperature of 35 °C lies outside the optimum condition for this bacterium, possibly leading to its decreased activity. However, given that the temperature variance falls within approximately ±5 °C of the optimum range, this temperature remains conducive to the growth, activity, and competitive coexistence of Ca. Brocadia fulgida alongside other species of anammox bacteria.

Ca. Brocadia fulgida was first identified in a study on cultivating anammox bacteria with organic acetic acid (Kartal et al. 2008). One of the beneficial features of Ca. Brocadia fulgida is its ability to oxidize acetate, consequently establishing dominance over other anammox bacterial species within the reactor in the presence of acetate (Jenni et al. 2014). Additionally, Ca. Brocadia fulgida can effectively coexist with Nitrosomonas in forming biofilm thickness (Liu et al. 2017). Therefore, the opportunity to carry out a one-stage anammox process becomes more feasible with a combination of Nitrosomonas AOB (ammonia-oxidizing bacteria) and Ca. Brocadia fulgida as the anammox bacteria. Furthermore, the most beneficial advantage of the anammox process using Ca. Brocadia fulgida is a substantial reduction in nitrate production by 40–68% compared with other bacterial species (Winkler et al. 2012).

Candidatus Brocadia and Candidatus Kuenenia are frequently found in wastewater treatment facilities (Kuenen 2008). Despite ongoing efforts, the precise ecological niche of anammox bacteria remains rudimentary, with elevated nitrogen concentrations serving as a principal indicator of their environmental presence. Ammonium serves as a primary substrate for anammox bacteria and other microorganisms; thus, AOB in the environment supplies nitrite for nitrogen removal. At the time of sampling, the water of Lake Koto Baru, which had experienced eutrophication, contains ammonium, nitrite, and nitrate. Remineralization processes, such as anaerobic degradation of organic matter from decomposing vegetation at the lake bottom, constitute a potential ammonium source for anammox, AOB, and ammonia-oxidizing archaea (AOA), functioning as crucial participants in the lake's nitrogen cycle. Additionally, ammonium inputs from residual fertilizers in the surrounding agricultural areas, dissolved and accumulated in the lake, further contribute to the nitrogen load. AOB-mediated ammonium oxidation at a depth of 1–1.5 m in the lake surface produces nitrites, which can subsequently be converted into nitrogen gas by anammox bacteria.

The discovery of anammox bacteria in tropical regions underscores their pivotal role in nitrogen removal processes. Anammox bacteria exhibit adaptability to diverse environmental conditions, with their presence being contingent upon the availability of ammonium and nitrite substrates. These findings serve as a valuable reference for investigating anammox bacteria across various ecosystems in tropical regions. Indigenous anammox bacteria from the local environment are expected to outperform exogenous strains in nitrogen removal, owing to the former's established adaptation to local environmental conditions, thereby ensuring enhanced operational efficiency. Agustina et al. (2017) reported high anammox-nitrogen removal, a maximum NRR of 1.05 kg-N/m³·d, at tropical temperatures using a non-woven and UASB reactor with the KSU-1 strain of anammox bacteria from Osaka University. Similar results were reported by Zulkarnaini et al. (2020) with various carriers, including plastic and palm fiber (organic), using Ca. Brocadia sinica from Hiroshima University for low nitrogen concentration. However, the color of anammox biomass changed from red carmine to brown because it was operated below the optimum temperature. This could be an obstacle to future application of the anammox process due to the high cost of temperature control. Consequently, future implementations of the anammox process for nitrogen removal in wastewater treatment or environmental contexts can be globally applicable by utilizing enriched anammox bacteria sourced from the local area. This approach might offer cost efficiencies during the initiation phase of anammox reactors, as it obviates the need for transporting biomass from distant regions.

This study, for the first time, attempted to cultivate native anammox bacteria from a tropical lake in Indonesia, through a novel FtBR. This research has the potential to advance future efforts in cultivating anammox bacteria in Indonesia and other tropical countries. The identification of four anammox bacteria (Candidatus Brocadia caroliensis, Candidatus Anammoxoglobus propionicus, Candidatus Brocadia sinica, and Candidatus Brocadia fulgida) provides a crucial perspective on the potential discovery of further genera and species within this bacterial group, capable of growing in tropical ambient temperatures, prevalent in tropical climates. This research sheds light on the presence of anammox bacteria within tropical ecosystems and lays the groundwork for the development of comprehensive, large-scale anammox reactors. However, we acknowledge that there are several limitations in our study. First, multiple reactors are needed to confirm the reproducibility and repeatability of the results, providing a more robust conclusion for statistical analyses. Second, future research must aim for multiple sampling points to monitor the dynamic of anammox bacteria diversity and growth. This way we would be able to track the abundance of these bacteria across different operational stages.

Nevertheless, this research serves as the first step for more advanced application of anammox bacteria in the treatment of domestic and industrial wastewater. The effluents discharged from municipal wastewater treatment plants consistently fall short of meeting Indonesian effluent quality standards, resulting in widespread eutrophication issues in the country's aquatic environments. Eutrophication, predominantly leads to oxygen depletion, fish mortality, unpleasant odors, and toxicity concerns, exemplified by the eutrophication incident in Lake Koto Baru. While initiatives have been undertaken to leverage the anammox process to address these challenges (Wijaya & Soedjono 2018), the limited research on anammox in Indonesia poses a barrier to its implementation, despite the process being discovered over 29 years ago and having been successfully applied in full-scale reactors worldwide.

Therefore, there are significant opportunities to integrate anammox processes into wastewater treatment systems, aiming to mitigate eutrophication concerns in the tropical environment by utilizing indigenous anammox bacteria. Simultaneously, the development of large-scale anammox reactors could serve to supplement or replace existing processes, such as the wetland system employed in the fertilizer industry. This application holds promise in establishing a national standard for effluent quality, thereby addressing the eutrophication issue in Indonesia.

The successful cultivation of the anammox culture from tropical environmental sediment highlights the remarkable adaptability and resilience of these bacteria in such conditions. Temperature plays a crucial role in their activity, with optimal performance observed within the tropical ambient range of 22–28 °C, outperforming operations at 35 °C. The pioneering discovery of anammox bacteria from Indonesia, facilitated by a novel FtBR, paves the way for future anammox research in tropical regions. The identification of two distinct anammox species, Ca. Brocadia fulgida and Ca. Brocadia caroliensis, which were dominant in this temperature range, offers valuable insights for the potential development of large-scale anammox reactors under similar conditions. Despite challenges in meeting effluent standards, leveraging indigenous anammox bacteria presents a promising alternative approach for wastewater treatment and combating eutrophication in tropical water bodies. This finding not only advances the understanding of anammox processes in tropical climates but also underscores the potential for sustainable and efficient wastewater management in these regions.

This research was supported by the Integrated Instrument Laboratory Polytechnic of ATI Padang and the Laboratory of Water Environment and Technology, Kanazawa University, Japan.

During the preparation of this work, the authors used ChatGPT in order to improve the language and readability. After using this tool/service, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.