Low-concentration methanol effect on the microorganisms, nitrogen removal, and recovery of the completely autotrophic nitrogen removal over nitrite

As the functional bacteria of the CANON process, the AOB and AnAOB are the autotrophic microorganisms. Previous researches have investigated that several kinds of parameters, such as dissolved oxygen (DO) concentration, temperature and pH, could affect the activities and growths of AOB and AnAOB (Kim et al. 2020; Pedrouso et al. 2021; Hausherr et al. 2022), resulting in the fluctuation of the CANON process performance. In addition, organic matter is also a critical parameter of the CANON process (Wang et al. 2020). The presence of organic matter can inhibit the activity and growth of AnAOB. Thus, the CANON process is suitable for treating high-ammonium-concentration wastewaters with a low chemical oxygen demand/ammonium ratio owing to the lack of an organic carbon source. However, in reality, most ammonium nitrogen-containing wastewaters contain small numbers of naturally living organic materials or substances (Zhang et al. 2020a, 2020b). Therefore, it is necessary to study the effects of organic matter on nitrogen removal performance of the CANON process. Furthermore, different types of organic matters have diverse effects on the CANON process. Acetate and glucose are the commonly encountered organic matters. Low concentrations of these organic matters were found to not inhibit AnAOB and sustain a stable total nitrogen removal rate from the anammox granule biomass (Pereira et al. 2021). The extracellular polymeric substance contents of AnAOB tended to increase gradually. Protein and polysaccharide enrichment could be the ion-transport channel between the bulk liquid and AnAOB, thereby maintaining the ‘lung-like breathing’ behaviour and increasing the mass transfer efficiency (Chai et al. 2019; Li et al. 2021). At the optimum concentrations of these organic matters, AnAOB and denitrification bacteria can coexist in one reactor and enhance the nitrogen-removal ability of the CANON process (Huang et al. 2022; Sanjaya et al. 2022). However, in the presence of excessive organic matter, heterotrophic denitrifiers overgrow and compete with AnAOB for NO₂⁻-N, resulting in the elimination of AnAOB (Liu et al. 2021), and therefore, elimination of the CANON process. These results indicate that the types and concentrations of organic matter influence AnAOB and nitrogen removal in the CANON process, and therefore, further research on this topic is necessary.

Methanol is also a typical characteristic organic matter of industrial wastewaters. In nitrification and denitrification processes, methanol is usually used as an excellent organic carbon resource to provide nutrients for denitrifying bacteria, leading to improving the biological nitrogen removal performance of this process. Xu et al. (2018) confirmed that methanol could promote the AOB activity resulting in the improvement of nitrosation reaction. In addition, the phyla Acidobacteria and Planctomycetes were found to decrease in proportion in the presence of methanol. It is implied that methanol could have an important influence on partial nitrification reaction in the CANON process, which has few reports until now. Furthermore, some papers reported that sludge containing methanol could be suitable for AnAOB enrichment. The Candidatus Brocadia was the dominant population in the enriched biomass (Tang et al. 2010). However, others showed that methanol could almost completely inhibit AnAOB activity, and have an irreversible inhibitory effect on the anammox process, which can be caused by enzyme inactivation or cell death, wherein suppression of the threshold differed from studies (Kim et al. 2020; Isaka et al. 2021; Chen et al. 2022). The results signify that methanol has a marked impact on the CANON process performance, which needs further research, though it has not been reported. Furthermore, nitrifying oxidizing bacteria (NOB) inhibition is critical for stable CANON performance. Some previous research reported that NOB enrichment was destructive to the CANON process, as NOB competed with the AnAOB for NO₂⁻-N acting as an electron acceptor and inhibited the total nitrogen removal ability of the CANON system (Zhang et al. 2020a, 2020b; Yang et al. 2023). Organic carbon matter is one of the factors affecting NOB and different types of organic carbon matters have different effects on NOB (Chen et al. 2023). In particular, methanol can significantly affect the activity of NOB. Xu et al. (2018) reported that the methanol addition could suppress the phylum Nitrospirae (belonged to the NOB). Thus, the methanol effect on NOB needs further study to increase the stability of the CANON process for treating practical wastewaters.

This experiment studies the low-concentration methanol effect on the AnAOB growth, NOB elimination, and nitrogen removal of the CANON performance in a submerged aerated biological filter (SABF) system at a low DO level. The nitrogen removal ability and stability of this CANON process could indicate the AnAOB and NOB activities with low-concentration methanol. A 16S rDNA sequencing technology analyzed the CANON process's microorganisms and these relative abundances at phylum and genus levels, respectively, to demonstrate the low-concentration methanol effect on the AnAOB and NOB survivals. This research supplies a reference for practical engineering design and application by improving the understanding of the effects of low-concentration methanol on CANON process performance and is vital for optimizing CANON process performance in a SABF system.

The combined carriers were fastened in the SABF, which consisted of plastic fibres, annulus, and a centre copper wire. Because of their high specific surface areas (1500 m²/m³), the combined carriers could improve the attachment of the microorganisms to achieve a favourable adsorption capability. Moreover, AnAOB and AOB could be absorbed on the inside and outside of the biofilms, respectively, owing to the different concentrations of DO on the inside and outside of the biofilms.

Water distribution of the CANON system: In the system, the peristaltic pump transported the simulated wastewater to the bottom of the CANON system, after which the effluent was outflowed from the upper outlet of the system. The flow rate of the system was 2.5 mL·min⁻¹. To achieve a reactor's temperature of 30.1–31.5 °C, the submerged pump transported the heated water into the inlet of the water bath layer at the base of the reactors, after which the effluent was outflowed from the upper outlet of the water bath layer into the radiator.

Beyond that, a temperature controller controlled the average temperature at 30.8 °C. This reactor was warmed by water insulation. An air pressure pump supplied the Dissolved Oxygen (DO), the concentration of which was controlled at 0.1–0.5 mg·L⁻¹ in the start-up period and 0.1–0.3 mg·L⁻¹ in the performance period. The hydraulic residence time (HRT) was 20 h. A temperature controller controlled the temperature at 30.1–31.5 °C. A constant flow pump pumped the influent from the bottom of the reactor.

The ingredients of the artificial wastewater are shown in Tables 1 and 2. The influent water quality parameters of this experiment are shown in Table 3.

Effect of low-concentration methanol on nitrogen removal and microorganisms. This experiment added the methanol by 0, 20.0, 27.0 and 38 mg·L⁻¹, adjusting the average influent COD concentration to 0, 29, 39, and 58 mg·L⁻¹, respectively.

The samples of sludge were analysed by a 16S rRNA gene sequencing technology (Guangzhou RiboBio Co., Ltd). The process of detection is described in supplementary material A.

The water samples of the CANON biofilm system were filtered through a qualitative filter paper. The detections of the COD, NH₄⁺-N, NO₂⁻-N, and NO₃⁻-N concentrations were based on the instructions of the Standard Methods (APHA) (APHA 2005).

The integrity of the nucleic acids in the sludge was measured using a TapeStation 2200 instrument (Agilent Technologies Co. Ltd, America), and the concentration and mass of the nucleic acids were measured using a Qubit 2.0 fluorimeter (Life Technologies Co. Ltd, America) and an ND-1000 Nanodrop spectrophotometer (Thermo Fisher Scientific Co. Ltd, America). The Q30 ratio and total data were detected using a Miseq sequencer (Illumina Ltd, America).

When the low-concentration methanol increased from 0 to 38.0 mg·L⁻¹ in this CANON process, the AOB was suppressed with its average activity reducing from 2.42 to 2.09 mg·L⁻¹·h⁻¹ (Figure 3(a)), causing the average NH₄⁺-N_eff concentration collection from 3.5 to 30.0 mg·L⁻¹ (Figure 3(c)) and the average ammonium nitrogen removal rate reduction from 96.6 to 70.7% (Figure 3(b)). Low-concentration methanol also inhibited the AnAOB activity, with the average value from 3.48 mg·L⁻¹·h⁻¹ to 1.89 mg·L⁻¹·h⁻¹ (Figure 3(a)), resulting in the total nitrogen removal rate reduction from 80.6 to 43.6%, as Figure 3(b) shows (the average FA_inf concentration and average ratio of FA_inf concentration/NH₄⁺-N_inf concentration were 2.57 mg·L⁻¹ and 2.53, respectively, which were not enough to affect the CANON reaction system). The reason for this phenomenon is that low-concentration methanol as a toxicant could cause the microbial toxication or enzyme deactivation of anammox bacteria, which was named formaldehyde inhibition (Huang et al. 2022). As a critical anammox enzyme, a hydroxylamine oxidoreductase might change methanol into intracellular formaldehyde, which cross-linked the peptide chains to damaged enzyme and protein activity. These results were similar to that of previous studies (Xu et al. 2018; Isaka et al. 2021). In addition, the average COD consumption in this system increased to 57.2 mg·L⁻¹ (Figure 3(d)), which might be due to heterotrophic microorganism accumulation (Cao et al. 2020) and competed with the anammox bacteria for space and electron acceptor (NO₂⁻-N) (Du et al. 2020; Li et al. 2020). Thus, low-concentration methanol eliminated the anammox bacteria and reduced the CANON process's total nitrogen removal ability in a SABF system.

Furthermore, NOB suppression is vital to the CANON process performance. As Figure 3(a) shows, when the low-concentration methanol was added, the average NOB activity increased from 0 to 0.10 mg·L⁻¹·h⁻¹, and the average NO₃⁻-N_eff concentration increased from 20.1 to 58.6 mg·L⁻¹, as Figure 3(c) shows. It was because the NOB inhibited the AnAOB, obsolete at lower DO concentrations without mass transfer limitation in biofilms when organic matter existed (Yang et al. 2023). Thus, the low-concentration methanol facilitated the NOB survival, inhibiting ammonium nitrogen oxidation and total nitrogen removal ability of the CANON process, resulting in the deterioration of this SABF system. Beyond that, as Figure 3(f) shows, the ratio of △NO₃⁻-N/△NH₄⁺-N maintained at 0.07–0.11, signifying no denitrification occurred. This result was different from other studies reporting anammox and denitrification coexisting in a one-stage system with organic matters (Peng et al. 2020; Chen et al. 2023). The reason for this phenomenon was likely that the low-concentration methanol could suppress the denitrifying bacteria enrichment (Zhang et al. 2022). Furthermore, the low-concentration methanol was challenging to diffuse into the thick biofilm for denitrifying growing bacteria and therefore inhibited the nitrogen removal of this system.

When the CANON process in a SABF system was stably performed without methanol, the biofilm's dominant microorganisms were phyla Acidobacteria, Chloroflexi, Planctomycetes, Proteobacteria, and Verrucomicrobia, accounting for >77.6% of all microorganisms detected. Among these microbes, the phyla Planctomycetes and Proteobacteria were the critical actors in this CANON system's adequate biological nitrogen removal. The phylum Proteobacteria attached to AOB, NOB and denitrifiers, and the relative abundances were in the upper biofilm by 21.4% and in the bottom biofilm by 18.0%. The phylum Planctomycetes belonged to the AnAOB (Chen et al. 2022) with relative abundances in the upper biofilm by 31.0% and in the bottom biofilm by 38.1%. Moreover, the phylum Chloroflexi enrichment often appeared in the CANON process, and its relative abundances were 10.4% in the upper biofilm and 9.1% in the bottom biofilm. The reason was that the autotrophic phyla Planctomycetes and Proteobacteria grew well, and their metabolic products provided enough nutrients for phylum Chloroflexi accumulation (Qian et al. 2022). Furthermore, as the conventional microorganisms, the phyla Acidobacteria and Verrucomicrobia often exist in wastewater treatment plants. This stable microbial community resulted in a regular CANON operation in a SABF system.

When the low-concentration methanol increased from 0 to 38.0 mg·L⁻¹, the CANON's microbial community in a SABF system was varied. The enzyme of phylum Planctomycetes converted methanol into formaldehyde, resulting in the phylum Planctomycetes suppression with relative abundances from 31.0 to 24.4% in the upper biofilm and from 38.1 to 28.5% in the bottom biofilm. The result agreed with some reports on low-concentration methanol inhibiting the phylum Planctomycetes as an AnAOB, leading to the anammox inhibition (Qian et al. 2022). Moreover, the phylum Proteobacteria inhibition occurred due to the low-concentration methanol. Its relative abundances decreased in the upper biofilm from 21.4 to 18.5% and in the bottom biofilm from 18.0 to 16.3%, resulting in ammonium nitrogen removal rate reduction in the CANON process. Furthermore, the phyla Planctomycetes and Proteobacteria inhibition resulted in fewer metabolic products, sufficient for the phylum Chloroflexi survival. Thus, the phylum Chloroflexi was suppressed with relative abundances in the upper biofilm by 3.6% and in the bottom biofilm by 7.2%. However, associated with NOB, the phylum Nitrospirae accumulated relative abundances of 9.3% in the upper biofilm and 1.7% in the bottom biofilm. The reason was that the surplus DO due to the AOB inhibition possibly promoted the NOB growth, which competed with the AnAOB for survival space and the AOB for the electron acceptors (O₂). Thus, the low-concentration methanol adversely influenced the stable CANON process’ functional microorganisms in a SABF system.

As shown in Figure 4(b), the genus Candidatus Brocadia, belonging to the phylum Planctomycetes, was the nitrogen removal contributor to the CANON process. Its relative abundances were in the upper biofilm by 25.5% and in the bottom biofilm by 28.6% without methanol addition. When the low-concentration methanol increased from 0 to 38.0 mg·L⁻¹, the genus Candidatus Brocadia was restrained with relative abundances in the upper biofilm by 12.5% and in the bottom biofilm by 16.2%. This result was similar to other research that reported that methanol feeding significantly limits genus Candidatus Brocadia activity and accumulation (Isaka et al. 2021).

The genus Nitrospira, which belonged to the phylum Nitrospirae (NOB), was enriched because of the low-concentration methanol addition, with relative abundances of 9.2% in the upper biofilm and 1.7% in the bottom biofilm. The genus Nitrospira enrichment harmed the CANON process. One reason was that the genus Nitrospira accumulation led to the AnAOB elimination at lower DO concentrations with no mass transfer limitation in the biofilm in the presence of methanol. Another reason was that the NOB enrichment could compete with the genus Candidatus Brocadia for nitrite nitrogen as the electron acceptor, inhibiting the system's total nitrogen removal ability (Wang et al. 2021). Therefore, the low-concentration methanol promoted the NOB enrichment and AnAOB elimination, breaking down the stability of the CANON process (Wu et al. 2021).

Furthermore, some researches showed that the methanol addition generally promoted the growth of denitrifying microorganisms, such as genera Azospira, Dechloromonas, Denitratisoma, Flavobacterium, Longilinea, Ornatilinea, Pseudomonas Thermomarinilinea, Thauera, unclassified Chlorobiales, Zoogloea, etc. Partial nitrification, anammox, and denitrification were also reported to exist in a single reactor depending on the organic matter concentration (Zhang et al. 2020a, 2020b). However, as Table 4 shows, these denitrifying microorganisms were virtually absent in this system. The reason was that the low-concentration methanol did not provide sufficient organic carbon sources for denitrifying microorganisms’ growth. Therefore, low-concentration methanol negatively affected the dominant CANON process’ microorganisms in a SABF system.

Furthermore, in the stable CANON process performance, the NOB is a potential spoilage organism to compete for O₂ with AOB and NO₂⁻-N with AnAOB, thus needs to be restrained. Generally, the experiment used ΔNO₃⁻-N/ΔNH₄⁺-N as a standard for NOB overgrowth of the CANON system stability. As Figure 5(f) shows, the average practical ΔNO₃⁻-N/ΔNH₄⁺-N standard for NOB overgrowth was 0.10, nearly 0.13 as a theoretical value (Zhao et al. 2021). The result implied that relatively few NOB existed in this system.

From the above, through the adverse effects of low-concentration methanol, the CANON process operation in a SABF system recovered rapidly, and the system maintained a stable total nitrogen removal capacity. The results verified that the CANON process in a SABF system had an excellent self-healing ability, which could upgrade the future CANON process's development.

This study demonstrated that the low-concentration methanol harmed the CANON process in a SABF system. The AOB and AnAOB of the CANON process were suppressed with the low-concentration methanol addition, respectively. In particular, the genus Candidatus Brocadia (belonging to phylum Planctomycetes) was obviously restrained. However, low-concentration methanol promoted the enrichment of NOB. The genus Nitrospira (belonging to phylum Nitrospirae) rapidly grew with the low-concentration methanol addition. These results led to the average ammonium nitrogen removal rate reduction from 95.0 to 70.7% and the average total nitrogen removal rate decreased from 81.3 to 43.6%. It is demonstrated that the low-concentration methanol harmed the CANON process. Fortunately, when the methanol was stopped, the CANON system had an excellent self-healing ability with the average ammonium and total nitrogen removal rate returning to 95.5 and 80.9%, respectively.

This work was supported by the National Natural Science Foundation of China (No.51808157), the Science and Technology Program of Guangzhou, China (No. 202002030374), the Provincial Natural Science Foundation of Guangdong, China (No. 2018A0303130014), the Science and Technology Planning Project of Guangzhou (No.202102021119), and the Education Commission of Guangdong Province (No.2020ZDZX2077,No.2021KTSCX198).

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

The authors declare there is no conflict.