Exploring the regulation mechanism of signaling molecules on algal-bacterial granular sludge through different N-acyl-homoserine lactones
Xibei Tan , Rongrong Wang , Naif Abdullah Al-Dhabi , Bin Wang , Rongfan Chen , Qian Zhang , Dao Zhou , Wangwang Tang , Hongyu Wang
Due to the high efficiency of nitrogen and phosphorus removal, low carbon dioxide emission rates, and the absence of the need for additional nutrients, microalgae wastewater treatment technology has attracted increasing attention [1, 2]. The photosynthesis of microalgae and the respiration of bacteria establish a self-sustaining algal-bacterial metabolic loop, eliminating the need for external nutrients and reducing the demand for external aeration and CO2 emission [3, 4]. However, the suspended characteristics of microalgae sludge can impact water quality and result in long hydraulic retention times. Moreover, maintaining a single or pure microalgae microbial system during the biological treatment process is challenging [2, 5]. To address these issues, there is a growing interest in integrating sludge granulation technology with microalgae processes. Compared to the algae-bacteria suspended sludge process, the algae-bacteria granular sludge (ABGS) process, allows effective separation of treated wastewater from biomass and superior effluent quality due to larger size and excellent settling performance of granular sludge [6, 7]. Therefore, the ABGS process recently become a research hotspot and is considered as a "composite factory" for water-energy-resource-carbon neutrality. Research has revealed that the adoption of the ABGS process can achieve a reduction of 100% in aeration energy consumption and 63% in carbon emissions [8]. Moreover, ABGS proliferates by utilizing nitrogen, phosphorus, and organic substances in wastewater. The produced microalgae biomass can be utilized for the production of biofuels and other renewable energy sources, bringing economic benefits to wastewater treatment projects [5]. In this light, ABGS appears to offer a low-energy, carbon-neutral, environmentally sustainable development strategies for future wastewater treatment [9].
Nevertheless, ABGS also has certain drawbacks, such as long granulation time, unstable long-term operation, fluctuating denitrification effect [10]. Very few efforts have been made to improve the drawbacks of ABGS. Further optimization of the ABGS system could make the entire process more environmentally friendly and efficient. In traditional activated sludge processes, signaling molecules play a crucial role in intercellular communication and have been extensively studied and identified by numerous scholars [11, 12]. Signaling molecules are often utilized to enhance the pollutant removal efficiency of activated sludge, promote system stability, and regulate numerous biological functions beyond the capability of individual microorganisms [13]. N-acyl-homoserine lactone (AHL)-mediated quorum sensing (QS) is considered as the primary signaling molecule influencing the structure and performance of microbial communities [14]. Feng et al. [15] detected four types of AHLs from two types of sequencing batch reactor (SBR) reactors, namely activated sludge and biofilm reactors. These AHLs include N-butanoyl-l-homoserine lactone (C4-HSL), N-hexanoyl-l-homoserine lactone (C6-HSL), N-octanoyl-l-homoserine lactone (C8-HSL), and N-(3-hydroxyoctanoyl)-dl-homoserine lactone (3-oxo-C8-HSL). These AHLs exhibit significant correlations with nitrite accumulation and ammonia nitrogen removal. Based on this, the present study aimed to investigate the impact mechanisms of different signaling molecules on ABGS systems by constructing various SBR systems. However, the role of signaling molecules in the aggregation and growth of microalgae and bacteria forming ABGS is still unclear, and there is a lack of systematic and in-depth research on the regulatory mechanisms of different types of signaling molecules. Therefore, this study aims to explore the effects of different signaling molecules on ABGS and investigate the mechanisms by which signaling molecules influence the formation of bacterial-algal granular sludge. Simultaneously, signaling molecules are utilized to further optimize the ABGS system.
According to the relevant research of others in the team [16] and the previous literature research, this study constructed ABGS systems mediated by different AHLs (N-hexanoyl-l-homoserine lactone (C6-HSL) and N-(3-oxodecanoyl)-l-homoserine lactone (3-oxo-C12-HSL)) to analyze and compare changes in sludge morphology, extracellular polymeric substances (EPS) secretion, pollutant removal efficiency, microbial community structure, and other aspects. The main objectives of this research include the following three parts: (1) exploring the mechanisms of enhancing the formation and pollutant removal performance of ABGS under the influence of different types of signaling molecules; (2) revealing the succession patterns of microbial community structure in ABGS systems mediated by different types of signaling molecules; (3) studying the influence mechanisms between microalgae and bacteria in ABGS systems under the influence of different types of signaling molecules.
Previous studies had indicated that C6-HSL and 3-oxo-C12-HSL were the predominant signaling molecules in the initiation and long-term operation processes of the ABGS system [17]. Bottle shaking experiments had shown that the addition of exogenous C6-HSL and 3-oxo-C12-HSL at a concentration of 5 nmol/L both could enhance the denitrification and phosphorus removal efficiency of the algae-bacteria symbiotic system. Three identical columnar SBR reactors were established for this experiment, labeled as R1, R2, and R3, respectively. R1 served as the control group, while R2 and R3 were experimental groups with exogenous additions of 5 nmol/L C6-HSL and 3-oxo-C12-HSL, respectively. The reactors were made of organic glass with an inner diameter of 100 mm, a height of 300 mm, an effective volume of 2.2 L, and a volume exchange ratio of 50% [16]. LED light strips were attached to the external surface of the reactors to serve as a light source, with an intensity of 40 µmol m−2 s−1 and a light/dark cycle of 12 h/12 h [18]. Tin foil was wrapped around the reactors to ensure stable illumination inside.
The reactors operated in an anaerobic/aerobic/anoxic (A/O/A) mode with a cycle set at 6 h and a hydraulic retention time of 12 h. The duration of each stage within the operational cycle was controlled by a microcomputer timer switch as follows: influent for 2 min, anaerobic stage for 120 min, aerobic stage for 90 min, anoxic stage with settling time totaling 138 min, and effluent for 10 min. This operational mode could effectively achieve synchronized nitrification-denitrification phosphorus removal functions in the system [18]. Throughout the process, the stirring speed was maintained at 250 rpm during the anaerobic, aerobic, and anoxic stages, while the aeration rate during the aerobic stage was set at 200 mL/min. Dissolved oxygen in the aerobic stage was continuously monitored and maintained above 2 mg/L. By adjusting the timing of stirring cessation and gradually reducing settling time, this experiment aimed to provide increasing selective pressure to achieve rapid granulation of floc sludge. The operational schemes for different stages of the reactors were summarized in the supplementary materials (Table S1 in Supporting information). The reactor configuration was shown in supplementary materials (Fig. S1 in Supporting information).
In this experiment, the initial sludge inoculum was obtained from the aeration tank of the Longwangzui Sewage Treatment Plant in Wuhan city. Initially, the sludge appeared as yellow-brown flocs. After retrieving the sludge, it was acclimated with simulated domestic wastewater for 7 days (d) before being inoculated into the reactors to enhance the sludge's adaptability to domestic wastewater. The initial mixed liquor suspended solids (MLSS) of the sludge were 4141.41 mg/L, mixed liquor volatile suspended solids (MLVSS) were 2070.71 mg/L, and the ratio of MLVSS to MLSS was 0.50.
Throughout the entire experiment, the influent for the reactors consisted of artificially synthesized simulated domestic wastewater. In the construction and long-term operation of the ABGS system, the influent concentrations of chemical oxygen demand (COD), NH4+-N, and TP were controlled at 220, 20, and 3 mg/L, respectively, while the temperature was maintained at 24 ± 5 ℃. Additionally, appropriate amounts of calcium, magnesium ions, and a solution containing essential trace elements required for microbial growth were added to the simulated domestic wastewater. The specific dosages of the additives and the composition of the trace element solution in the influent were listed in supplementary materials (Table S2 in Supporting information). The signaling molecules were prepared as solutions and stored at 4 ℃ for later use. The signaling molecule solutions were then added to the influent. Each of the reactors, R1, R2, and R3, received influent independently. Reactor R2 received influent with an addition of 5 nmol/L C6-HSL, while reactor R3 received influent with an addition of 5 nmol/L 3-oxo-C12-HSL.
COD, TP, NH4+-N, NO2−-N, and NO3−-N were measured according to international standard methods (AHPA) [19]. MLSS and MLVSS were determined using the weight method. In this study, an improved thermal extraction method was used to extract EPS from ABGS, which included loosely bound EPS (LB-EPS) and tightly bound EPS (TB-EPS) [20]. Protein (PN) concentration was determined using the Coomassie Brilliant Blue colorimetric method, while polysaccharide (PS) concentration was determined using the sulfuric acid-anthrone colorimetric method. Three-dimensional fluorescence (HITACHI, F-7000, Japan) and Fourier-transform infrared spectroscopy (FTIR, FR-6500) were employed for the determination and analysis of EPS. Portable pH meters were used to measure pH. Sludge morphology was observed using a scanning electron microscope (SEM, QUANTA, Netherlands). The particle size distribution of the sludge was determined using a laser particle size analyzer (Mastersizer 2000, UK). Chlorophyll content was measured using the acetone extraction method. Total lipid content and fatty acid composition in ABGS were determined using organic solvent extraction and gas chromatography (Agilent, 6890 N, Palo Alto, CA).
Adequate amounts of sludge were stored at −20 ℃ during different stages of reactor operation for subsequent analysis. Sludge samples were sent to Shanghai Meiji Biomedical Sequencing Co., Ltd. for Majorbio Cloud Platform (www.majorbio.com) high-throughput sequencing. The prokaryotic microbial region targeted was the 16S rRNA V3-V4 variable region, with primers 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′). The eukaryotic microbial region targeted was the 18S rRNA V4 region, with primers 528F (5′-GCGGTAATTCCAG CTCCAA-3′) and 706R (5′-AATCCRAGAATTTCACCTCT-3′). Majorbio Cloud platform (PE300, CA, USA) was used for DNA extraction and sequencing, followed by bioinformatics analysis to study the community structure of both prokaryotic and eukaryotic microorganisms.
From the perspective of the complete granulation process, all three reactors went through stages of floc-like sludge agglomeration, formation of loosely structured granular sludge, and stabilization of granular structure. The formation times of different granular sludge were different [21]. Looking at the overall operation of the reactors, on the 35th day, as seen in Figs. 1a and b, it was evident that reactors R1, R2, and R3 all showed partially well-formed granules, with the sludge gradually converting from dark yellow and flocculent to yellow-green and granular. The average particle sizes on the 40th day were 250.10, 694.73, and 333.36 µm for R1, R2, and R3, respectively (all exceeding 200 µm), indicating the initial granulation of sludge. Based on the experimental results, the operational phases were divided into the startup period (1–40 d) and the long-term stable operation period (41–120 d) for subsequent discussion.
In terms of the start-up period of the reactors, R1 exhibited the slowest granulation process, R2 had the fastest granulation process but with structural instability, and R3 had a relatively fast granulation process with stable particle size growth, forming mature granular sludge with a regular shape. In terms of particle size distribution, R2 and R3 had smaller distribution intervals than R1, and overall, their distribution uniformity was better than that of R1 during the granulation process. In the initial granulation process, the particle sizes of R2 and R3 were larger than that of R1. R2 showed the earliest granulation trend with an average particle size significantly higher than that of R1 and R3. This indicated that the addition of the exogenous signal molecules C6-HSL and 3-oxo-C12-HSL could accelerate the granulation process of algae-bacterial sludge. C6-HSL, in particular, showed a more pronounced promoting effect in the initial stages, quickly promoting the formation of granules in algal-bacterial sludge. On the 120th day, that was, after the long-term stable operation of the reactor, as shown in Fig. 1b, all three reactors exhibited predominantly deep green granules with a compact structure and regular shape, with average particle sizes reaching R1: 475.95 µm, R2: 687.89 µm, and R3: 925.31 µm. From the perspective of the overall reaction stage, it could be observed that the granulation process of R1 showed a trend of increasing particle size followed by stabilization, R2 showed a trend of increasing particle size followed by a decrease, and R3 showed a continuous increase in particle size. This could prove that the granular structure formed by R3 was denser and more stable than that of R1 and R2, also indicating that the addition of exogenous 3-oxo-C12-HSL had a more stable and effective enhancing effect on the granulation process.
For the ABGS surface in the long-term stable operation stage of reactors R1, R2, and R3, sludge elemental spectrum (EDX) analysis was conducted. From Fig. 1c, it could be observed that the elements C, N, and O were the most abundant in R1, R2, and R3, with respective abundances of 98.91%, 83.38%, and 94.86%, which showed that the sludge in each reactor had a high level of microbial activity. The abundances of Mg, P, K, and Ca were in the order of R2 > R3 > R1. The presence of Ca2+ and Mg2+ accelerated the granulation process, and their mechanisms included neutralizing the negative charge on the bacterial cell surface to accelerate aggregation, using precipitates as granule cores for granulation (with Ca2+ as the main component), forming ion bonds on the granule surface to enhance granulation [22], and Ca2+ could also combine with EPS to form cross-linked structures to improve granule structural stability [23]. It could be observed that R2 particles had the highest abundance of Ca2+ and Mg2+, which corresponded to the fastest particle formation rate in R2. This to some extent indicated that C6-HSL promoted an increase in calcium ion abundance, thereby exerting a positive effect on sludge granulation.
In order to ensure that the sludge in the early stage of granulation with shortened settling time and under conditions of enhanced hydraulic selection pressure, sufficient biomass was maintained to sustain the stable operation of the system. The initial inoculum sludge concentrations for R1, R2, and R3 were all controlled at around 4000 mg/L, with a sludge MLVSS/MLSS ratio of approximately 0.50. As shown in Fig. 1d, with the continuous decrease in settling time, the MLVSS/MLSS in reactors R1, R2, and R3 still showed a rising trend, indicating that the sludge performance in the reactors improved with the operation of the system. In order to ensure that the sludge in the early stage of granulation with shortened settling time and under conditions of enhanced hydraulic selection pressure, sufficient biomass was maintained to sustain the stable operation of the system.
The early growth trend of MLVSS/MLSS in reactor R2 was significantly greater than that of R1 and R3, but in the later stages, the MLVSS/MLSS in reactor R3 surpassed that of R1 and R2. These data also demonstrated that the addition of C6-HSL and 3-oxo-C12-HSL could promote the rapid growth of microorganisms in the initial stage of ABGS system startup, increase the proportion of organic matter in the sludge, and play a "protective" role in reducing sludge loss in the initial stage of startup, thereby improving the success rate of rapid startup of the system. C6-HSL exhibited significant advantages in rapidly increasing MLVSS/MLSS in the early stage, while 3-oxo-C12-HSL could effectively and stably enhance the microbial growth rate during long-term operation.
Due to the initial settling performance of the sludge being relatively good (SVI30 < 80 mL/g), the variation range of SVI30 during the sludge granulation process was small, making SVI5 more suitable for reflecting changes in sludge settling performance. As shown in Fig. 1e, under the pressure of sedimentation selection, the flocculent sludge with poor settling performance and lighter mass in each reactor was eliminated from the system with the effluent. The SVI5 values of R1, R2, and R3 rapidly decreased from the initial sludge values of 169.02 mL/g to 80.54, 66.87, and 78.70 mL/g, respectively, within a short period (10 d). At this point, R2 exhibited the smallest SVI5 and the most rapid improvement in settling performance, consistent with the earliest observation of fine particles under the microscope in R2. As the settling time gradually decreased from 10 min to 2 min, the system accelerated the selection of large particles. From day 61 to day 120, due to the adjustment of the settling time to 5 min, the system's selection effect on large particles weakened, leading to slight increases in SVI5 for R1 and R2 to 71.83 mL/g and 76.44 mL/g, respectively, while R3 decreased to 46.39 mL/g, indicating that R3 had formed a more compact and stable particle structure during the mature and stable operation stage, which played a certain role in preventing sludge loss, consistent with the above conclusions.
The total chlorophyll content (Chl a + Chl b) was closely related to the microalgae content in ABGS [24] and was an important indicator in the algae-bacteria system, reflecting the biomass of microalgae in ABGS and its coupling degree with particulate sludge. Changes in Chl a/Chl b could reflect changes in the algal community structure to some extent. The changes in chlorophyll content during operation were shown in Fig. 1f. The overall trend of chlorophyll content changed in each reactor was an initial increase followed by stabilization in fluctuations, consistent with the results of Zhang [25] in SBR reactors. In the initial granulation stage (1–40 days), it was observed that the Chl a + Chl b in R2 was significantly higher than in R1 and R3, reaching the highest level of 0.74 mg g−1 ss−1 on day 20, indicating that the addition of exogenous C6-HSL effectively promoted the rapid growth of microalgae in the initial stage of system startup, strengthened QS between algae and bacteria, and accelerated the construction of the algae-bacteria symbiotic system.
The initial growth of algae improved the system's photosynthetic oxygen production capacity, which was of great significance for building the internal spatial structure of ABGS, forming a good symbiotic relationship between algae and bacteria, and effectively accelerating the process of sludge granulation. It could be inferred that the key reason why R2 granulated faster than R1 and R3 in the initial granulation process was the stimulating effect of C6-HSL on algal growth. In the long-term stable operation stage (41–120 days), the overall trend of Chl a + Chl b and Chl a/Chl b in each reactor was an initial increase followed by a decrease, reaching the highest level on day 80. An appropriate ratio of algal to bacterial growth was conducive to maintaining a good particle structure and performance of ABGS. At this time, excessively high levels of algal growth in ABGS might be an important reason for the relatively loose particle structure. On day 120, there was not much difference in the total chlorophyll content and distribution among the reactors, indicating that the final distribution of microalgae in the mature ABGS was relatively similar, and stable algae-bacteria symbiotic relationships had been formed in each reactor. The addition of exogenous C6-HSL during the startup stage could stimulate algal growth, thereby achieving rapid granulation, but excessively large algal-to-bacterial growth ratios could lead to the formation of loose and unstable granular sludge. Conversely, adding exogenous 3-oxo-C12-HSL could stabilize and increase the growth rate of microorganisms in the algae-bacteria symbiotic system, forming stable granular sludge. According to Fig. 2, the average effluent concentrations of COD for R1, R2, and R3 were 2.17, 2.40, and 3.73 mg/L, respectively, all of which were below 5 mg/L. The average removal rates of COD were 99.08%, 99.00%, and 98.48%, respectively, stabilizing at above 98%. It could be seen that R1, R2, and R3 had good organic matter removal effects with no significant differences between them (P < 0.05). During the establishment and stable operation of each ABGS system, the COD removal rates could be maintained at a high level, indicating that ABGS had good and stable heterotrophic microbial activity in SBR reactors.
Each reactor initially showed a fluctuating trend in COD effluent concentration, which tended to stabilize and achieve a COD removal rate of over 99% within 10 d. It was found that with the good COD removal capability of the ABGS system, the addition of signal molecules did not have a significant additional effect on COD removal. However, around the 20th day, there was a small range of fluctuations in the COD effluent concentrations of R1 and R2 (COD effluent concentrations on the 21st day: R1 8.31 mg/L, R2 4.09 mg/L, R3 1.87 mg/L), which might be due to a significant loss of sludge concentration under sedimentation selection. However, at this time, R3 did not show a significant COD fluctuation, indicating that exogenous 3-oxo-C12-HSL could "protect" the initial sludge concentration of granulation and improve the stability of the reactor effluent water quality.
From Fig. 2, the average effluent concentrations of NH4+-N for R1, R2, and R3 were 0.61, 0.30, and 0.70 mg/L, respectively, all of which were below 1 mg/L. The average removal rates of NH4+-N for the entire reactor were 97.17%, 98.64%, and 96.78%, respectively, all of which were above 96%. The removal of NH4+-N in the ABGS system mainly relied on the assimilation and nitrification of microorganisms [26]. Each reactor maintained a high level of dissolved oxygen during aeration, and microalgae could produce a certain amount of oxygen, ensuring the sufficient progress of ammonia oxidation. Therefore, R1, R2, and R3 all exhibited stable and efficient removal performance for NH4+-N.
In the initial stage of reactor operation, the NH4+-N effluent fluctuation of R1 was relatively large compared to R2 and R3. After stable operation, the NH4+-N effluent fluctuation of R2 and R3 was greater than that of R1. It could be concluded that exogenous C6-HSL and 3-oxo-C12-HSL could improve the stability of NH4+-N removal in the startup stage of the ABGS system. However, as the initial granulation was completed, the larger particle size granular structure formed by R2 and R3 around the 20th day was looser, and the internal spatial structure was unstable, resulting in an increased fluctuation in NH4+-N effluent [27].
The average effluent concentrations of the effluent total inorganic nitrogen (TIN) for R1, R2, and R3 were 2.96, 3.71, and 1.53 mg/L from Fig. 2, respectively. The average removal rates were 87.49%, 84.12%, and 93.50%, respectively, all of which were above 84%. Unlike the similarity in COD and NH4+-N removal effects of R1, R2, and R3, R3 performed better in TIN removal than R1 and R2. This indicated that exogenous 3-oxo-C12-HSL significantly improved the TIN removal rate of the ABGS system. Most of the TIN remaining in the water was NO2−-N and NO3−-N. The high TIN removal rate of R3 might be attributed to the promotion of the activity of denitrifying bacteria by exogenous 3-oxo-C12-HSL, which was confirmed by the high abundance of denitrifying functional bacteria in R3 in the subsequent high-throughput sequencing results, showing a more significant enhancement of denitrification.
It could be inferred from the above conclusions that the differences in denitrification performance of R1, R2, and R3 mainly lay in the accumulation of NO2−-N and NO3−-N. In order to comprehensively analyze the denitrification situation in each reactor, the effluent concentrations of NO2−-N and NO3−-N were detected during the reactor operation. From Fig. 2, it could be seen that the accumulation of NO2−-N in the three reactors was maintained at a low level and does not differ much. The difference was that the fluctuation of NO2−-N in R3 was smaller, and the accumulation of NO3−-N in the effluent of R3 was significantly lower than that of R1 and R2. This proved that the nitrite oxidation bacteria (NOB) in R1, R2, and R3 were all highly active, and the excellent denitrification performance of R3 was because it had a stronger denitrification ability. Comparing the accumulation of NO2−-N and NO3−-N before and after granulation in the system, it was found that as the sludge particle size gradually increased, the accumulation of NO2−-N in R1 and R2 increased, while that in R3 decreased, indicating that the ABGS system regulated by exogenous 3-oxo-C12-HSL was less affected by the increase in sludge particle size and was more conducive to the growth of NOB.
The average removal rates of phosphorus for R1, R2, and R3 (Fig. S2 in Supporting information) were 94.86%, 96.98%, and 98.18%, respectively. The performance of R3 in phosphorus removal was consistent with its denitrification effect, both of which were higher than R1 and R2. Therefore, it was easy to see that the ABGS system regulated by exogenous 3-oxo-C12-HSL had the best denitrification and phosphorus removal performance, and exogenous C6-HSL also had a certain promoting effect on denitrification and phosphorus removal in the system. However, overall, the promoting effect of exogenous 3-oxo-C12-HSL was better than that of exogenous C6-HSL. The regulating strategy of adding exogenous 3-oxo-C12-HSL had a significant effect on improving the effluent water quality of the ABGS system, providing theoretical support for the application of ABGS technology in the upgrading and reconstruction of traditional sewage treatment plants.
To provide a more in-depth explanation of the denitrification and phosphorus removal mechanism of the ABGS system regulated by exogenous C6-HSL and 3-oxo-C12-HSL, the concentrations of C, N, and P in each reactor were monitored during a typical cycle (6 h), as depicted in Fig. 3 illustrating the water quality changes. Due to the volume exchange ratio designed for the reactors being 50%, the actual initial pollutant concentrations within one operational cycle were reduced under dilution effects. Simultaneously, they were influenced by the remaining pollutants from the previous operational cycle.
In R1, the actual initial concentration of NO2−-N was significantly higher than R2 and R3 (R1: 0.94 mg/L, R2: 0.10 mg/L, R3: 0.22 mg/L), indicating a higher accumulation of NO2−-N in R1 from the previous cycle. During the anaerobic phase, COD concentrations in R1, R2, and R3 rapidly decreased within 30 min, where most organic compounds were absorbed and degraded by polyphosphate-accumulating organisms (PAOs), glycogen-accumulating organisms (GAOs), and mixotrophic algae. Under anaerobic conditions, PAOs hydrolyze polyphosphates to release phosphorus and rapidly absorb volatile fatty acids (VFAs) produced by the hydrolysis of organic carbon sources in the influent, synthesizing intracellular carbon sources (PHAs) [27].
At 30 min, R1, R2, and R3 all reached their highest phosphorus release levels, with TP concentrations of 19.32, 25.93, and 27.09 mg/L, respectively, indicating high growth activity of polyphosphate microorganisms in the stable ABGS system. The oxygen produced by algae in photosynthesis might inhibit phosphorus released during the anaerobic phase, and the larger particle size in the ABGS interior was more favorable for the formation of anaerobic zones. Therefore, the higher phosphorus released levels in R2 and R3 compared to R1 during this period might be related to the larger particle size in R2 and R3, promoted by exogenous signaling molecules.
The removal of NH4+-N during the anaerobic phase might rely on simultaneous nitrification-denitrification and assimilation processes. Algal photosynthesis created a micro-oxygen environment in the anaerobic phase, advancing some of the NH4+-N removal from the aerobic phase to the anaerobic phase [28]. At 30 min, NO2−-N and NO3−-N concentrations rapidly decreased in R1 and R2, while increasing in R3. This might be attributed to the strong photosynthetic oxygen production by algae in R3, oxidizing some NH4+-N to nitrate. Additionally, the addition of exogenous 3-oxo-C12-HSL might enhance the metabolic activity of ammonia-oxidizing bacteria (AOB). At the end of the anaerobic phase, COD was almost completely absorbed and degraded in R1 and R2, while R3 retained a COD concentration of 36.47 mg/L, possibly due to a closer algal-bacterial symbiosis in R3 providing bacteria with additional carbon sources [27].
In the aerobic phase, R3 utilized the remaining COD from the anaerobic phase. The phosphates released during the anaerobic phase were synthesized into Poly-P within polyphosphate bacteria under aeration conditions, possibly involving polyphosphate functional algae. The PHAs synthesized and stored during the anaerobic phase provide carbon sources for polyphosphates, and the removal of phosphorus from wastewater mainly involved discharging residual activated sludge containing high levels of Poly-P [29]. TIN removal in all systems was primarily achieved in both anaerobic and aerobic phases. Actual concentrations of NH4+-N in R1, R2, and R3 decreased from initial values of 9.65, 11.86, and 10.46 mg/L to 0.24, 0.32, and 0.09 mg/L, respectively, at the end of the aerobic phase. NO2−-N and NO3−-N concentrations remained below 1 and 3 mg/L, indicating TIN removal in the ABGS relies on bacterial simultaneous nitrification-denitrification (SND) and microbial assimilation [30].
Further calculations revealed that 75.76%, 72.60%, and 99.50% of NH4+-N removal in R1, R2, and R3, respectively, was attributed to SND and assimilation. R3 exhibited the highest proportion of SND and assimilation, and nitrate concentrations fluctuated the least throughout the entire cycle, suggesting that the ABGS structure regulated by exogenous 3-oxo-C12-HSL was more stable and favorable for SND and assimilation. All reactors were capable of removing most of the C, N, and P from the influent in both anaerobic and aerobic phases, with further reduction of COD and TP concentrations in the anoxic phase. It was observed that nitrate concentrations fluctuate at different levels during the anoxic phase, with R1 consistently having higher NO3−-N concentrations than R2 and R3. Moreover, R3 effluent showed almost no accumulation of nitrate. This might be due to the complete depletion of internal carbon sources stored by cells in R1 during the anaerobic phase, while the tighter algal-bacterial synergy in R3 under the influence of exogenous signaling molecules provided additional supplementary carbon sources. This aligned with the higher residual COD in R3 at the end of the anaerobic phase, further confirming the improved comprehensive pollutant removal performance of the ABGS under the regulation of exogenous 3-oxo-C12-HSL. EPS played a crucial role in the formation of granular sludge by facilitating aggregation and protecting cell structures under hydraulic shear forces [31]. EPS included loosely bound EPS (LB-EPS) and tightly bound EPS (TB-EPS). As observed in Fig. 4a, the trends in LB-EPS variation among the three reactors were similar. The LB-EPS content in the sludge generally increased significantly during the initial granulation process in all reactors, while gradually decreasing and stabilizing during the mature phase. The addition of exogenous AHLs could expedite the induction of QS phenomena and EPS secretion by reducing the time required to reach the threshold concentration of signaling molecules, thereby accelerating bacterial adhesion [32]. The secretion of LB-EPS contributed significantly to the rapid granulation of flocculent sludge in the initial stages, but excessive content could reduce cell surface adhesion ability and granule structural stability [33]. On the 10th day, the LB-EPS content in R1, R2, and R3 was 4.27, 11.43, and 2.84 mg g−1 vss−1, respectively. The higher LB-EPS content in R2 during the initial granulation stage might contribute to its fastest granulation process, but it was a crucial factor in the insufficient stability of granule structure during the mature phase. The above results indicated that C6-HSL could increase LB-EPS in the early stages of granular sludge formation to promote sludge granulation. From day 20 to day 60, the LB-EPS content in each reactor remained between 5 and 10 mg g−1 vss−1, which facilitated rapid granule growth. From day 61 to day 120, while LB-EPS gradually decreased, the granule structure tended to stabilize. The LB-EPS in R3 was significantly lower than in R1 and R2 during this stage, and the granule structure in R3 was the most compact and regular, indicating that lower LB-EPS in mature ABGS might be more conducive to granule structural stability. From Fig. 4b, it could be observed that the TB-EPS content steadily increased with the granulation process, demonstrating its more significant role in forming and maintaining granule structure in ABGS compared to LB-EPS. This finding aligned with Wang's [34] discovery that during the sludge granulation process, the effect of LB-EPS on flocculent sludge aggregation shifted from attraction to repulsion, while TB-EPS could alter the sludge surface zeta potential and hydrophobicity, facilitating adhesion between sludge cells and playing a critical role in driving granule formation. Moreover, the PS/PN ratio in TB-EPS also increased continuously [35]. The addition of exogenous AHLs such as C6-HSL and 3-oxo-C12-HSL induces QS, leading to PS secretion and subsequently increasing PS/PN. This indicated that the PS secretion pathway was more sensitive to AHL-based QS, and PS also played an essential role in ABGS formation [36]. PS in EPS acted as a binder to promote the aggregation of dispersed cells and could also serve as a bridge between smaller particles to form larger granules.
To further investigate the profound changes in the composition of EPS during the start-up and stable operation phases in ABGS, three-dimensional fluorescence spectroscopy (3D-EEM) analysis was conducted on the terminal TB-EPS samples at the start-up end (day 40) and stable operation end (day 120). The analysis results were depicted in Fig. 5. Observation of the main peaks in the figure suggested that the major components of TB-EPS in all samples consist of three categories of substances: tyrosine-like proteins, soluble microbial by-products, and humic acid-like organic matter [37], corresponding to peaks A, B, and C in Fig. 5c, respectively. This correlation was related to the types of carbon sources employed in simulating domestic wastewater. However, it's noteworthy that the analysis of the terminal R3 sample at the start-up phase also distinctly revealed the presence of fulvic acid-like organic matter (corresponding to peak D), indicating a richer organic composition of TB-EPS in R3.
Figs. 5a–c illustrated that during the initial granulation stage, the fluorescence intensities of peaks A, B, and C in R3 were significantly higher than those in R1 and R2, indicating higher levels of tyrosine-like proteins, soluble microbial by-products, and humic acid-like organic matter. This suggested that the addition of 3-oxo-C12-HSL significantly promoted the secretion of various organic compounds in TB-EPS. Compared to R1, R2 exhibited higher fluorescence intensity in peak A and lower intensity in peak C, indicating that the addition of C6-HSL could adjust the organic composition of TB-EPS by increasing tyrosine-like proteins and reducing humic acid-like organic matter. The increase in tyrosine-like proteins in R2 and R3 compared to R1 might be mainly attributed to the perception of higher levels of AHLs by microalgae, enhancing the secretion of aromatic proteins, regulating cell hydrophobicity, and promoting bio-flocculation [38].
Figs. 5d–f demonstrated that the differences in organic composition of TB-EPS among the systems reduced during the mature stable operation stage, indicating similar organic contents in mature ABGS. AHLs had little impact on the organic content of ABGS after maturation. Comparing the intensities of peaks at the end of the start-up and stable operation phases, it was found that soluble microbial by-products increased significantly in R1, R2, and R3 during long-term operation, while the content of tyrosine-like proteins decreased. It was speculated that tyrosine-like proteins might play a more important role during the initial granulation. Meanwhile, humic acid-like organic matter increased in R1 and R2 during long-term operation, and although it decreased in R3, it remained at a higher level compared to other reactors, indicating that soluble microbial by-products and humic acid-like organic matter were the main growth components of TB-EPS in the mature stable operation stage of ABGS.
Fourier transform infrared spectroscopy (FTIR) analysis was performed on the terminal TB-EPS (120 d) during stable operation to further compare the functional groups and structural differences of mature ABGS of EPS in various reactors. Detailed results could be found in the attachment (Fig. S3 in Supporting information). The addition of exogenous signaling molecules did not significantly alter the molecular structure and chemical composition of TB-EPS. However, the intensity at the same absorption peak varied among the samples, indicating that the addition of signaling molecules changed the content of various organic components in EPS to some extent. Among them, the absorption peak at 3430 cm−1 [39] representing polysaccharide (-OH) was the most prominent and had the highest intensity throughout the detection range, indicating a significant proportion of PS in TB-EPS components of all reactors, consistent with the higher PS/PN values in the EPS of R1, R2, and R3. The increased intensity of the absorption peak at 3430 cm−1 in R3 compared to R1 and R2 also indicates the significant role of PS in the long-term stable operation of the ABGS system. The higher intensities of the absorption peaks at 1640 and 1265 cm−1 [40], representing amide C=O and protein secondary structure C=O, respectively, in R2 and R3 compared to R1, suggested that exogenous C6-HSL and 3-oxo-C12-HSL could promote the secretion of hydrophobic functional groups, thereby accelerating cell aggregation.
Microalgae, as a third-generation biomass energy source, had enormous potential in wastewater treatment. With the popularity of microalgae systems, an increasing number of researchers were also focusing on the resource utilization of algal residue in ABGS, including its application as biofuels, algal membranes, and more [41]. Algae were typically rich in lipids, which could be extracted and converted into biofuels such as biodiesel or biogas [42]. In order to study the content and composition of lipids for the purpose of microalgae resource utilization, measurements of lipid content and composition were taken to monitor whether different AHLs would affect lipid changes in ABGS. The lipid content of mature ABGS in the three reactors and the composition of fatty acid methyl esters (FAMEs) after methylation treatment were shown in supplementary materials (Fig. S4 in Supporting information). The lipid contents of mature ABGS in R1, R2, and R3 were 10.4%, 13.0%, and 15.1%, respectively, indicating that the addition of exogenous AHLs, such as C6-HSL and 3-oxo-C12-HSL, could promote lipid synthesis in ABGS, with 3-oxo-C12-HSL showing a more significant promoting effect. However, as shown in Fig. 1f, R3 did not have an advantage in terms of microalgae growth at the end of long-term stable operation, further indicating that the promoting effect of exogenous 3-oxo-C12-HSL on lipid production was achieved by enhancing metabolic activities related to cell growth and lipid accumulation.
Further analysis of FAMEs composition revealed that the main component of sludge lipids in R1, R2, and R3 was palmitic acid (C16:0), accounting for 43.02%, 64.43%, and 62.06%, respectively. Both exogenous C6-HSL and 3-oxo-C12-HSL could increase the proportion of the dominant component (palmitic acid) in lipids. Palmitic acid (C16:0) was the preferred component of biodiesel. In addition, only palmitoleic acid (C16:1) was detected in R2, while oleic acid (C18:1) was not detected in R2, and myristic acid (C14:0) was not detected in R3, indicating that the addition of exogenous AHLs could alter some lipid components in ABGS. The stability of biodiesel usually depended on the composition of fatty acids, with a higher content of saturated fatty acids being more favorable for stability [43]. The proportions of saturated fatty acids accumulated in R1, R2, and R3 lipids were 79.56%, 88.55%, and 86.52%, respectively, indicating that the advantages of lipids as biodiesel feedstock in R2 and R3 were higher than in R1, and the addition of exogenous AHLs could further improve the utilization of lipids in the algal-bacterial system.
The mature ABGS samples extracted from reactors in long-term stable operation stages (R1, R2, R3) and the seeding sludge (S0) samples used for comparative reference were subjected to high-throughput sequencing analysis. The sample coverage (Coverage index) of each sample during sequencing was above 0.99, which fully demonstrates that this sequencing could comprehensively and accurately reflect the distribution of microorganisms in the samples, while ensuring the reliability of subsequent analyses. The diversity indices of various prokaryotic organisms were observed as R1 > R2 > S0 > R3, with significantly higher OUT numbers, Ace index, and Chao index in R1 without added signal molecules compared to S0, indicating that microalgae grown under light induction could influence the community composition of prokaryotes in the system and increase the species richness of prokaryotic communities (Table S3 in Supporting information). The diversity indices of eukaryotic communities revealed an overall trend of S0 > R1 > R3 > R2, suggesting that the addition of exogenous C6-HSL and 3-oxo-C12-HSL might enhance microbial selection, promote the growth of dominant species in the algae-fungi community, accelerate the elimination of inferior species, and thus improve the adaptability of granular sludge to environmental changes (Table S4 in Supporting information). Moreover, it could be demonstrated that 3-oxo-C12-HSL had the most significant selective effect on bacteria, while C6-HSL had a more significant selective effect on eukaryotic algae. The Shannon index and Simpson index indicated that the species diversity in the ABGS community formed in each reactor (R1, R2, R3) was lower than that in the sludge (S0), indicating a higher enrichment level of dominant species during the establishment of the ABGS system, which was conducive to the establishment of stable synergistic relationships between algae and bacteria [44]. When comparing prokaryotic species diversity, it was found that the Shannon index was lowest and the Simpson index was highest in R2, indicating that exogenous C6-HSL could significantly accelerate species turnover, consistent with the significant advantage demonstrated by R2 in terms of granulation speed. The dilution curves of various indices were relatively flat, as shown in detail in the attachment (Fig. S5 in Supporting information), demonstrating that the data volume of this sequencing was sufficient and the reliability of the obtained indices was high.
According to Fig. 6a Venn diagram, it could be observed that the number of common operational taxonomic units (OUTs) in samples S0, R1, R2, and R3 was 243, while the number of common OTUs in samples R1, R2, and R3 was 471. This indicated significant changes in the prokaryotic microbial community species during the establishment process of each reactor ABGS system, while the similarity between mature granular sludge in different reaction phases was high. The unique OTU numbers in each sample were 351, 160, 83, and 89, respectively and the numbers of shared OTUs between R1 and R2, R1 and R3, R2 and R3 were 657, 590, and 537, respectively. The overlap between prokaryotic microbial community species in sample R3 and other samples was the lowest, indicating that exogenous 3-oxo-C12-HSL had a more pronounced effect on the structure of prokaryotic microbial communities in ABGS compared to C6-HSL.
As the granulation process progresses, analysis of the prokaryotic microbial community structure at the phylum level in mature ABGS from Fig. 6b indicated a significant increase in the relative abundance of Proteobacteria (S0: 27.77%, R1: 35.61%, R2: 56.58%, R3: 56.51%) and Cyanobacteria (S0: 0.00%, R1: 12.91%, R2: 17.83%, R3: 7.91%) in R1, R2, and R3 compared to S0, demonstrating enhanced pollutant removal potential of the ABGS system compared to traditional activated sludge. Under the action of exogenous signal molecules, the abundance of main colonies in the ABGS system would be further increased, and the decontamination performance would be better [45].
Analysis of the prokaryotic microbial structure at the genus level in Fig. 6c heatmap indicated significant changes in dominant bacterial genera composition in R1, R2, and R3 compared to the seed sludge, with R1 and R3 showing more similarity in dominant bacterial genera composition, while R2 exhibited significant differences. Candidatus_Competibacter secreted EPS with strong adhesion under shear force stimulation, effectively protecting the external morphology of bacteria and promoting the aggregation of flocculent sludge [46]. In mature granular sludge samples, the absolute dominant bacterial genera in R1 and R3 were Candidatus_Competibacter, with relative abundances of 21.04% and 17.07%, respectively, while the abundance of filamentous bacteria Thiothrix (31.75%) in R2 was significantly higher than that of Candidatus_Competibacter (14.93%), consistent with the relatively poor settling performance of R2 at the end of the maturation period. Recent studies had shown that norank_f__Saprospiraceae was suitable for survival under dynamic aerobic-anoxic conditions, could grow attached to the surface of filamentous bacteria, and could utilize polysaccharides in EPS as a carbon source, demonstrating the potential to degrade polysaccharides [47]. The relative abundance of norank_f__Saprospiraceae in R3 was significantly higher than in R1 and R2 (S0: 5.19%, R1: 2.29%, R2: 2.74%, R3: 9.55%), which might be the main reason why R3 could maintain a lower level of LB-EPS concentration during long-term stable operation. This might have contributed to the stable and efficient pollutant removal performance of R3. In addition, the effective utilization of endogenous polysaccharides by norank_f__Saprospiraceae could reduce the demand for exogenous carbon in the ABGS, thereby enhancing the adaptation of the ABGS system to low-carbon conditions, consistent with the previous finding that R3 still maintains a certain level of COD concentration at the end of the anaerobic stage.
To comprehensively investigate the biological basis of ABGS in pollutant removal and structural performance maintenance in each reactor, various major functional bacterial genera were summarized in Table S5 (Supporting information). The sum of the relative abundances of major denitrifying bacteria (DNB) in S0, R1, R2, and R3 was 5.13%, 14.31%, 10.35%, and 15.58%, respectively, significantly higher than that of the seed sludge S0. Zhou et al. [28] showed that the presence of algae could increase the efficiency of organic matter denitrification utilization and the relative abundance of denitrifying bacteria in activated sludge systems, consistent with the results of this experiment. The abundance of various denitrifying functional bacterial genera (Hyphomicrobium, unclassified_f__Comamonadaceae, etc.) in R3 was significantly higher than in R1 and R2, and the addition of exogenous 3-oxo-C12-HSL had a significant promoting effect on DNB. Combined with the very low sum of AOB abundance (< 0.5%) and the presence of nitrification in the full cycle analysis, it could be inferred that AOB in the ABGS maintains high biological activity [48]. Although the sum of AOB abundance in R3 was the lowest, it did not cause a decrease in NH4+-N removal rate. This was partly because the assimilation effect of microalgae supplements the denitrification capacity and partly because exogenous 3-oxo-C12-HSL could increase the ammonia oxidation rate of AOB [49]. The enrichment level of NOB in R2 and R3 was significantly lower than in R1, reducing the inhibition of denitrification by NOB reduced the carbon source demand for denitrification and benefited the improvement of bacterial denitrification efficiency, and the inhibitory effect of exogenous 3-oxo-C12-HSL on NOB was more pronounced. DNPAOs such as Dechloromonas could use NO2−-N or NO3−-N as electron acceptors to achieve simultaneous denitrification and phosphorus removal. Although the abundance of PAOs and DNPAOs in R3 was low, they still exhibited good phosphorus removal effects, indicating that the polyphosphate accumulation and assimilation effects of microalgae might play a more critical role in the phosphorus removal process in R3. During the long-term operation of the SBR, the addition of exogenous 3-oxo-C12-HSL could promote the growth of QS-related bacterial communities (AOB, DNB, NOB), thereby regulating the nitrogen transformation in the SBR.
In the ABGS, the sum of the relative abundances of the major filamentous bacteria in S0, R1, R2, and R3 were 0.75%, 13.75%, 37.88%, and 13.86% respectively. The higher filamentous bacteria content in mature ABGS than floc sludge was mainly due to the filamentous bacteria playing a role in the structure of the granular sludge. The excessively high filamentous bacteria content in R2 was mainly due to the abundant proliferation of Thiothrix under the action of exogenous C6-HSL, which posed a risk of filamentous bacteria expansion during long-term stable operation. Studies had shown that the addition of exogenous C6-HSL could enhance the QS between algae to significantly promote the growth of Thiothrix. However, excessive proliferation of filamentous bacteria would inhibit the enrichment of bacteria that form flocs, leading to the loss of microbial population structure diversity in the flocs of sludge [50], which was consistent with the lowest bacterial community diversity (Shannon index lowest, Simpson index highest) in R2. At the same time, it could be found that the addition of exogenous 3-oxo-C12-HSL did not significantly change the growth level of filamentous bacteria, and the dense fur observed on the surface of R3 granules was mainly composed of filamentous algae. This distribution of microorganisms was more conducive to the stability of the ABGS structure. Filamentous algae could play a backbone role in microbial aggregates and preserve more EPS by providing more attachment points and carrying points [51]. Dominant bacteria belonging to the phylum Cyanobacteria in each reactor were mostly filamentous, while the algae of eukaryotic microalgae were mostly spherical or spindle-shaped, which could be inferred that the filamentous algae formed on the surface of ABGS in this experiment were mainly composed of cyanobacteria. That was to say, the addition of exogenous C6-HSL would lead to an increase in cyanobacteria, thereby reducing the stability of granules.
As for the algal community, based on the phylum-level analysis of the community structure of eukaryotes in Fig. 6d, it could be seen that the most abundant algae at the phylum level in each ABGS sample were Chlorophyta (S0: 0.76%, R1: 24.04%, R2: 28.03%, R3: 20.36%). In addition, two types of fungi, Cryptomycota (S0: 1.43%, R1: 24.37%, R2: 1.27%, R3: 7.17%) and unclassified_kFungi (S0: 0.49%, R1: 7.00%, R2: 11.98%, R3: 0.47%), were also enriched at the phylum level. Cryptomycota was a newly discovered type of fungi that was widely found in water environments. It usually parasitized algae or other fungi and absorbed nutrients through phagocytosis, thereby directly affecting the growth of the host [52]. From Fig. 6e, it could be seen that the dominant algal orders at the order level in each sample were Chlorophyceae (S0: 0.16%, R1: 22.68%, R2: 27.18%, R3: 18.88%) and trebouxiophyceae (S0: 0.57%, R1: 1.36%, R2: 0.83%, R3: 1.48%), which contained many algal genera that were spherical or spindle-shaped and mainly exist in the inner layer of the granules, helping to enhance the structure and settling ability of the granules. At the same time, some protozoa (single-cell) such as ciliates Oligohymenophorea and some micro-invertebrates (multicellular) such as rotifers Eurotatoria and nematodes Chromadorea were also detected, indicating the diversity of microorganisms in the ABGS system and the good quality of the effluent water. Rotifers could feed on algae, and a longer food chain in the community often represented a higher degree of stability in the ecosystem [53]. In conclusion, the introduction of AHLs alters the microbial community structure to some extent but does not affect the dominant algal species in ABGS systems.
In this study, the different effects of reactors R1, R2, and R3 on the granulation process were comprehensively analyzed by contrasting the blank reactor with reactors containing different AHLs. The changes in sludge morphology, physicochemical properties, and EPS secretion during the granulation process were examined.
The addition of exogenous signaling molecules does not alter the main factors and essential stages of the granulation process. Instead, it accelerates the granulation process or optimizes granule performance by regulating specific factors during different stages of granule formation. The addition of exogenous C6-HSL and 3-oxo-C12-HSL affects the ABGS system in three main ways:
(1) Enhanced nitrogen metabolism efficiency; the addition of exogenous signaling molecules improved the stability of the ABGS system during the startup period and had a positive effect on the nitrogen removal rate.
(2) Affected EPS secretion and the formation of granular sludge; the addition of both signaling molecules increased TB-EPS and PN, facilitating the formation of granular sludge.
(3) Altered the microbial community structure, such as an increase in DNB with the addition of exogenous 3-oxo-C12-HSL, promoting denitrification and increasing denitrification efficiency. The addition of exogenous C6-HSL leaded to an increase in cyanobacteria and so on.
The mechanisms of action of C6-HSL and 3-oxo-C12-HSL differ significantly. The former primarily accelerates the granulation process in the early stages by promoting LB-EPS secretion, filamentous bacterial growth, and the establishment of algae-bacteria symbiotic relationships. The latter, through the promotion of TB-PS and aromatic protein secretion, accelerates the granulation process to some extent, with more significant benefits for the structural stability and denitrification and phosphorus removal effects of mature ABGS. In summary, both C6-HSL and 3-oxo-C12-HSL could expedite the construction of the ABGS system, but the reinforcing effect of 3-oxo-C12-HSL was more advantageous during long-term stable operation.
This study addresses research gaps in the roles of different signaling molecules in the ABGS system, the signaling molecule-mediated mechanisms of rapid granulation and steady-state operation of the ABGS system, and the mechanisms underlying the enhancement of ABGS pollutant removal performance using signaling molecules. Notably, the research also provides several potential applications for the future development of ABGS systems.
(a) Enhancing pollutant removal in ABGS systems with poor startup performance or unstable mature granules by adding signaling molecules such as 3-oxo-C12-HSL to strengthen pollutant removal and improve the structural stability of mature ABGS.
(b) Increasing the potential for resource recovery; the biomass in the ABGS, enriched with nutrients during the photo granulation process, holds promise for applications such as biofuel, fertilizers and pharmaceuticals.
Despite the various advantages of adding different signaling molecules to ABGS systems, their practical application is currently limited to the laboratory stage. Further research in pilot or full-scale facilities is necessary to provide real-world application data, extend operation processes and cost analysis, and verify the long-term effects of implementing this strategy. To push this promising technology towards engineering applications, the development of ABGS systems should progress from a single function (nutrient removal) to multifunctionality (wastewater treatment - energy production - resource recovery) to maximize environmental sustainability and economic feasibility.
The study achieved rapid start-up of an ABGS system using floc-like sludge as seeding sludge by constructing different SBR reactors. The regulatory mechanisms of two different signaling molecules on ABGS systems are also distinct. With the addition of exogenous C6-HSL, the growth of microalgae in the early operation stages was the fastest and the trend of granulation was observed first among three reactors, while the stability of granular structure suffered to some extent. Exogenous 3-oxo-C12-HSL contributed to faster granulation process and more regular shape with optimal particle size distribution compared to control group. Introducing the 3-oxo-C12-HSL signaling molecule than C6-HSL can further optimize ABGS systems during rapid construction and enhancement of ABGS performance and stability.
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Xibei Tan: Writing – original draft, Data curation, Conceptualization. Rongrong Wang: Writing – original draft, Data curation, Conceptualization. Naif Abdullah Al-Dhabi: Methodology, Investigation. Bin Wang: Writing – review & editing, Resources, Funding acquisition, Formal analysis. Rongfan Chen: Writing – review & editing, Supervision, Methodology, Formal analysis. Qian Zhang: Writing – review & editing, Visualization, Validation, Conceptualization. Dao Zhou: Writing – review & editing, Resources, Project administration, Methodology. Wangwang Tang: Supervision, Investigation. Hongyu Wang: Writing – review & editing, Validation, Supervision, Investigation.
This work was financially supported by the Open Project of Sanya Science and Education Innovation Park of Wuhan University of Technology (No. 2022KF0005). The authors would like to extend their appreciation to Researchers Supporting Project (No. RSP-2024- R20), King Saud University, Riyadh, Saudi Arabia.
Supplementary material associated with this article can be found, in the online version, at doi:
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Figure 1 (a) Forty times microscope photographs. (b) The particle size distribution. (c) Energy spectrum analysis of mature ABGS sludge (120 days). (d) The changes of MLSS and MLVSS. (e) The change of SVI5. (f) The change of chlorophyll.
Figure 2 Concentration changes of TIN, COD, NH4+-N, NO2−-N, NO3−-N and removal efficiency of TIN, COD in R1, R2 and R3.
Figure 3 The concentration of C, N and P in each reactor changed in a typical cycle (6 h).
Figure 4 (a) The change of LB-EPS content in sludge during operation. (b) The change of TB-EPS content in sludge during operation.
Figure 5 The three-dimensional fluorescence images of TB-EPS at the end of the start-up period (a-c) and the end of the stable operation period (d-f).
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