3.1 Nitrogen removal performance
Figure 1 depicts the nitrogen components. The control reactor, R0, was neither exposed to any signaling molecules nor methanol. The mean influent ammonia nitrogen was quantified at a concentration of 173.4 ± 6.9 mg L− 1. As time progressed, both the levels of ammonia nitrogen and nitrite in the effluent gradually escalated, peaking at 54.3 ± 8.9 and 65.0 ± 2.6 mg L− 1, respectively. This led to a sustained reduction in TRE down to 27.6 ± 6.1%. The absence of mechanical aeration, coupled with limited oxygen transfer, might have restricted the microbial activity in contrast to the parent reactor.
Figure 2 presents the nitrogen removal efficiencies of the six reactors, each fed with different signaling molecules. Reactor R1, to which only methanol was added, resulted in an average effluent ammonia nitrogen and nitrite nitrogen concentration of 53.0 ± 6.4 and 47.4 ± 4.2 mg L− 1, respectively, after 30 days. Furthermore, the average TRE and ARE reached 37.8 ± 4.1% and 67.9 ± 3.4% respectively, in the final days. It is plausible that this outcome is due to methanol acting as a substrate, leading to the induction of denitrifying bacteria. Section 3.3 provides further evidence supporting this hypothesis. Consequently, the introduction of methanol appears to induce denitrifying bacteria and enhance nitrogen removal.
The addition of 2 µM C6-HSL to R2 resulted in a gradual rise in the concentration of ammonia and nitrite nitrogen, reaching 65.5 ± 4.9 and 64.7 ± 3.3 mg L− 1, respectively, after 30 days. This escalation could be ascribed to the inhibitory effect of C6-HSL, which consequently delayed nitrite consumption and led to a decrease in the TRE. Consequently, both ARE and TRE were observed to decrement to 63.7 ± 3.7% and 26.8 ± 3.9%, respectively.
Observations of R3, R4, R5, and R6 revealed parallel outcomes, where both effluent ammonia nitrogen and nitrite concentrations escalated whilst Total Removal Efficiency (TRE) gradually reduced to 26.0 ± 3.4%, 28.1 ± 5.2%, 27.6 ± 4.5%, and 27.7 ± 5.5%, respectively. In conjunction, the ARE witnessed a decrease to 62.8 ± 4.7%, 62.6 ± 4.8±%, 63.7 ± 7.1%, and 62.9 ± 5.8%, respectively. This data suggests that the integration of signal molecules curtailed the activity of both AOB and AnAOB. Despite potentially augmenting denitrifying bacteria, these changes impaired the nitrogen removal process.
Research conducted by De Clippeleir et al. suggests that the activity of AnAOB in autotrophic nitrification-denitrification biofilms can be augmented by the addition of C12-HSL, although it exhibits limited impact on AOB(6). Another study posits that the activity of AnAOB is positively influenced by the presence of C8-HSL, while C6-HSL elevates both the activity of AnAOB and its growth rate. However, C12-HSL has been shown to encourage the proliferation of heterotrophic bacteria, which, in turn, depresses the activity of AnAOB (31). The inconsistencies observed in various studies surrounding the effect of exogenously added C12-HSL on AnAOB may be attributed to variations in operating conditions and the structure of the bacterial flora(21, 30). The nitrogen removal capabilities of microorganisms in a wastewater treatment reactor can be shaped by the kind and amount of exogenous AHLs (13). Nonetheless, in this study, inhibition was seen regardless of the signal molecules introduced, potentially attributable to the high concentrations of the added signal molecules.
3.2 Sludge properties
Figure 3(a) showcases the outcomes of the EPS determination. In the Control Reactor R0, the PRO and PS contents were recorded as 26.6 and 6.4 mg g− 1 SS, respectively. Strikingly, Reactor R1 exhibits notably augmented quantities of both PRO and PS, at 45.2 and 10.2 mg g− 1 SS, respectively. This observed increment is attributable to the amplifying effect of methanol on denitrifying bacteria, resulting in a marked increase in EPS production.
The levels of PRO demonstrated an increase after the addition of 2 µM signal molecules. However, in comparison to R1, the PRO levels exhibited a decrease to 30.7, 37.9, 39.0, 37.3, 29.7 mg g− 1 SS under the application of 2 µM C6-HSL, C8-HSL, C10-HSL, C12-HSL, and 3-oxo-C8-HSL. This decrease is likely the result of the inhibitory effects these signal molecules have on denitrifying bacteria, ultimately leading to a reduction in PRO levels. High concentrations of these signal molecules have been demonstrated in studies to lead to excessive PRO secretion and impaired nitrogen removal(39). This decrease in nitrogen removal performance was also observed in the present study. The application of these signal molecules also reduced PS levels, which may be due to denitrifying bacteria growth. PS is utilized as a substrate during endogenous denitrification, hence, a pronounced reduction in its levels was observed along with a decrease in nitrate concentration in the effluent. C8-HSL, C10-HSL, C12-HSL, and 3-oxo-C8-HSL have been established to aid in microorganism growth (12, 29). These microorganisms may metabolize PS during growth, thereby decreasing its concentration. Consistent with the conclusion drawn in this study, a slight reduction in biofilm weight, from 835 to 827 mg, was observed when the concentration of the signal molecule was 1 µM (11). The PS content in EPS was reduced from 6.4 to 10.2, 4.1, 5.9, 5.4, 6.4, 5.3 mg g− 1 SS by signal molecules C6-HSL, C8-HSL, C10-HSL, C12-HSL, and 3-oxo-C8-HSL. Thus, the introduction of 2 µM signal molecules could potentially reduce PS secretion on EPS.
Figure 3b presents the results of SMP. In R0, the concentrations of PRO and PS in SMP were 54.2 mg L− 1 and 10.5 mg L− 1, respectively. In R1, PRO concentration decreased to 44.7 mg L− 1 as PS concentration climbed to 22.5 mg L− 1. This increase in PS is likely the outcome of methanol inducing heterotrophic microorganisms' growth. As these organisms proliferated, they used SMP as a nutrient, causing its decline. The addition of C6-HSL escalated the concentrations of PRO and PS to 77.7 and 19.9 mg L− 1, respectively. This increase might be attributed to the role of C6-HSL in boosting microorganisms' growth. As a result, there is an amplified secretion of SMP. However, in R3, R4, R5, and R6, the concentrations of PRO declined to 49.8, 43.0, 48.1, and 44.7 mg L− 1 respectively, while PS concentrations escalated to 22.7, 10.3, 21.0, and 18.1 mg L− 1 respectively. This implies that compounds C8-HSL, C10-HSL, C12-HSL, and 3-oxo-C8-HSL exerted more minor influences on SMP secretion compared to C6-HSL.
Hydroxylamine oxidoreductase (HAO) is a widely scrutinized enzyme with a significant role in Anammox metabolism. It catalyzes hydrazine oxidation (1), and its relationship to not only Aerobic Oxidizing Bacteria (AOB), but also Anaerobic Ammonia-Oxidizing Bacteria (AnAOB) and denitrifying bacteria has been determined. As a potent catalyst, HAO can facilitate both the oxidation of hydroxylamine to nitrite and the reduction of nitrite to hydroxylamine. Therefore, the efficiency of nitrogen removal in autotrophic nitrogen removal systems directly corresponds to HAO activity (8, 24). Figure 3(c) indicates that the HAO enzyme activity in R0 was 0.4449 EU g− 1 SS, increasing to 0.7983 EU g− 1 SS in R1. This notable increase is predominantly attributable to methanol-enhanced denitrifying bacteria. Furthermore, the HAO enzyme activity in R3 and R5 rose to 1.137 EU g− 1 SS and 1.111 EU g− 1 SS, respectively. However, there was variation in R2, R4, and R6. Analysis of denitrification performance revealed that adding 2 µM of signal molecules amplified denitrifying bacteria activity, triggering an increase in HAO enzyme activity. Additionally, C8-HSL and C12-HSL can bolster heterotrophic bacteria growth, further contributing to the HAO enzyme activity boost.
Heme-c, a critical coenzyme in the Anammox process, has a concentration directly associated with AnAOB activity. Research has established the presence of Heme-c in cells as a significant contributing factor to the characteristic red color of AnAOB (18). As illustrated in Fig. 3 (d), the reactor's Heme-c concentration was examined and studied. The minimal Heme-c content in R0 reflects the relatively scarce abundance of AnAOB bacteria in the reactor, a conclusion corroborated in section 3.3. This low AnAOB abundance might result from inhibition due to environmental variation. The Heme-c concentration in R1 dipped to 0.00834 µmol g− 1 SS, attributed to an overgrowth of heterotrophic bacteria triggered by methanol. Conversely, in R2, R5, and R6, Heme-c rose to 0.01753, 0.01605, and 0.02121 µmol g− 1 SS, respectively. This outcome suggests that C6-HSL, C12-HSL, and 3-oxo-C8-HSL might positively impact AnAOB growth, though further investigation is required to substantiate this claim.
3.3 Microbial community structures
The taxonomic findings at the genus level, as obtained by leveraging the Silva database, are demonstrated in Fig. 4. The relative frequencies of AOB and denitrifying bacteria are summarized in Table 2. Nitrosomonas and Arenimonas emerged as the primary nitrogen-eliminating microorganisms in the seed sludge, possessing relative abundances of 4.78% and 16.61% respectively. In Reactor 0, the relative frequency of Nitrosomonas elevated from 4.78–7.93%, concurrent with a corresponding Areal Removal Efficiency (ARE) of 70% in the reactor. The relative frequency of Arenimonas reduced from 16.61–8.45%, with a corresponding TRE of 27.6 ± 6.1%. Following the transfer of sludge into experimental reactors from the seeding reactor, the increase in Nitrosomonas and decrease in Arenimonas could possibly be attributed to environmental changes. Such changes seem to inhibit AnAOB, leading to an inability to consume nitrite timely, which promoted the growth of denitrifying bacteria by utilizing nitrite. In Reactor 1, Hyphomicrobium, the denitrifying bacterium, emerged as the dominant microorganism, with the relative abundance escalating from 3.60–20.55%. With a TRE of 37.8 ± 4.1% in Reactor 1, it can be hypothesized that nitrogen removal in the reactor was primarily driven by the denitrification pathway. The escalation of denitrifying bacteria could have been facilitated by the addition of methanol, used as the organic carbon source for denitrification. As a result, the abundance of denitrifying bacteria surged as SMP were utilized as substrate, leading to a decline in SMP. In Reactor 2, the relative frequency of denitrifying bacteria experienced a drop, perhaps because C6-Homoserine Lactone (C6-HSL) exerted a certain inhibitory influence on the denitrification bacteria.
Table 2
The relative abundance of the nitrogen removal-related bacteria (%).
Reactor | AOB | Denitrifying bacteria | Total Denitrifying bacteria |
Nitrosomonas | Hyphomicrobium | Arenimonas | Denitratisoma | Thermomonas |
Seed | 4.87 | 3.83 | 16.61 | 2.06 | 0.15 | 22.65 |
R0 | 7.93 | 3.60 | 8.45 | 2.00 | 0.26 | 14.31 |
R1 | 5.83 | 20.55 | 7.36 | 1.91 | 0.27 | 30.09 |
R2 | 6.11 | 16.88 | 5.77 | 1.49 | 0.25 | 24.39 |
R3 | 5.51 | 28.55 | 6.50 | 1.81 | 0.19 | 37.00 |
R4 | 4.80 | 26.92 | 7.23 | 1.40 | 0.21 | 35.76 |
R5 | 7.57 | 19.45 | 5.76 | 1.16 | 0.19 | 26.56 |
R6 | 5.36 | 29.42 | 6.00 | 1.27 | 0.17 | 36.86 |
Upon the addition of C8-HSL, C10-HSL, and 3-oxo-C8-HSL, the relative abundance of denitrifying bacteria rose to 37.00%, 35.76% and 36.86%, respectively. The enhancement in nitrification and denitrification efficiency of the activated sludge system due to these signal molecules potentially caused this trend, leading to a rise in the denitrifying bacteria's relative abundance (35). Moreover, a promotion of heterotrophic bacteria growth in the community is linked to C8-HSL, C10-HSL, and 3-oxo-C8-HSL (13, 19). Despite the similar situation with C6-HSL, feeding R5 with C12-HSL resulted in a decrease in the relative abundance of denitrifying bacteria.
From the findings, it can be inferred that the introduction of methanol promoted the growth of denitrifying bacteria. However, the presence of signal molecules such as C6-HSL and C12-HSL reduced the relative abundance of these denitrifying bacteria while simultaneously increasing the relative abundance of AOB. Conversely, molecules such as C8-HSL, C10-HSL, and 3-oxo-C8-HSL had the opposite effect, leading to an increase in the abundance of denitrifying bacteria and a decrease in AOB.
3.4 Mechanism and the prospect
Existing literature demonstrates that nitrogen removal can be promoted by low concentrations of signal molecules (31, 35), while high concentrations may impede this process (30). The current study suggests that the amount of signal molecules added may have exceeded an optimal level, causing inhibition of microbial activity, suppression of associated gene expression, and thus a decline in nitrogen removal performance.
Research consistently underscores the pivotal role of AHLs in enabling bacterial aggregation through EPS concentration regulation. More specifically, a positive correlation has been identified between the EPS content and AHLs such as C8-HSL and C10-HSL, which are typically present in granular sludge (14, 23). This promotion of sludge granulation by AHLs can predominantly be attributed to their capacity to control EPS content, with the standout being C4-HSL that significantly enhances PRO formation (7). Comparative observations indicate that granular sludge fortified with AHLs like C6-HSL and 3-oxo-C6-HSL possesses a higher EPS content relative to sludge devoid of AHLs (19, 25). Given that EPS constitutes an organic substrate that stimulates denitrifying bacterial proliferation, this discovery has substantial implications for the efficiency of microbial processes in wastewater systems.
The incorporation of methanol into the system could adversely affect nitrogen removal and promote the growth of heterotrophic denitrifying bacteria. Consequently, it is advisable to refrain from using organic solvents such as methanol when introducing signal molecules. Instead, the union of signal molecules with carriers should be considered. For example, magnetic enzyme carriers have been effectively generated through immobilizing acylase on magnetic carriers, which have exhibited high recyclability and resilience, even after repetitive trials (36). Another alternative method involves immobilizing acylase on magnetic circular mesoporous silica via adsorption and cross-linking, a method that has proven to successfully foster and delay biofilm formation over extended periods (17). This synergy of signal molecules with carriers can extend their duration of action while renouncing the use of organic solvents.
While the AHLs did not enhance nitrogen removal in this study, the findings remain significant for future research. It was discovered that higher concentrations might inflict inhibition, thus future studies should investigate the impact of reduced AHLs concentration. Additionally, it is imperative to devise new strategies for integrating AHLs into the nitrogen removal system, necessitating thorough exploration, with a particular focus on mitigating methanol toxicity. Finally, the value of in situ experimental systems was highlighted in this study since observed environmental changes led to the inhibition of AnAOB.