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Understanding of aerobic sludge granulation enhanced by sludge retention time in the aspect of quorum sensing
Accepted Manuscript Understanding of aerobic sludge granulation enhanced by sludge retention time in the aspect of quorum sensing Zhiming Zhang, Zhuodong Yu, Zihao Wang, Ke Ma, Xiangyang Xu, Pedro J.J. Alvarezc, Liang Zhu PII: DOI: Reference:
S0960-8524(18)31448-2 https://doi.org/10.1016/j.biortech.2018.10.027 BITE 20591
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Bioresource Technology
Received Date: Revised Date: Accepted Date:
5 September 2018 9 October 2018 11 October 2018
Please cite this article as: Zhang, Z., Yu, Z., Wang, Z., Ma, K., Xu, X., Alvarezc, P.J.J., Zhu, L., Understanding of aerobic sludge granulation enhanced by sludge retention time in the aspect of quorum sensing, Bioresource Technology (2018), doi: https://doi.org/10.1016/j.biortech.2018.10.027
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Understanding of aerobic sludge granulation enhanced by sludge retention time in the aspect of quorum sensing Zhiming Zhanga, Zhuodong Yua, Zihao Wanga, Ke Maa, Xiangyang Xua,b,c, Pedro J.J. Alvarezcd, Liang Zhua,b,c* a. Institute of Environmental Pollution Control and Treatment, Zhejiang University, Hangzhou 310058, China b. Zhejiang Province Key Laboratory for Water Pollution Control and Environmental Safety, Hangzhou 310058, China c. Zhejiang Provincial Engineering Laboratory of Water Pollution Control, 388 Yuhangtang Road, Hangzhou 310058, China d. Department of Civil and Environmental Engineering, Rice University, 6100 Main St., Houston, TX 77005, USA *Corresponding
author: Liang Zhu
Address: Department of Environmental Engineering, Zhejiang University, No. 866 Yuhangtang Road, Hangzhou 310058, PR China. Tel (fax): +86 571 88982343 E-mail address: [email protected]. Abstract Aerobic granular sludge (AGS) reactors with different sludge retention times (SRTs) were established for enhanced functional microorganism enrichment and granular formation. Results showed that higher total nitrogen (TN) removal efficiency and compact granules were achieved in the 6-day-SRT reactor. Also, Xanthomonadaceae, Rhodobacteraceae and Hyphomonadaceae with AHL-producing and EPS-secreting functions also enriched under 6-day SRT. For investigating the enhanced mechanism of sludge granulation, typical quorum sensing signals of acylated-homoserine-lactones
(AHLs) and extracellular polymeric substances (EPS) were analyzed. Tryptophanand-protein-like substances were major EPS components in granules formed at 6-day SRT. Meanwhile, most detected AHLs, i.e. C8-HSL and 3OHC8-HSL, were correlated positively with contents of tryptophan-and-protein-like substances. Accroding to AHLs add-back test, AHLs especially those with 8-carbon sidechains, played important roles in aerobic sludge granulation via secreting special extracellular proteins by functional microbes enrichment. Keywords: Aerobic granular sludge; sludge retention time (SRT); acylated homoserine lactones (AHLs); extracellular polymeric substance (EPS); quorum sensing (QS) 1. Introduction Aerobic granular sludge has been investigated extensively in the past two decades because of its excellent settling ability, enhanced pollutants removal efficiency and high biomass retention (de Kreuk et al., 2007; Show et al., 2012; Liu et al., 2015; Derlon et al., 2016; Li et al., 2017), and it has been widely applied to industrial and municipal wastewater treatment (Su and Yu, 2005; Pronk et al., 2015; Corsino et al., 2016; Derlon et al., 2016). The stability of aerobic granular sludge is essential for long-term aerobic granule processes. However, granular disintegration occurs in both lab-scale and pilot-scale reactors, which impedes the optimization and application of this technology. As is well known, the seeding sludge, type and loading of the substrate, reactor operating mode, and sludge retention time (SRT) are the main factors affecting the granular disintegration, related to variations in the microbial community and extracellular polymeric substances (EPS) of sludge (de Kreuk et al., 2007; Show et al., 2012). Therein, many papers showed that the activated sludge of laboratory-scale sequencing batch bioreactors (SBR) has higher diversity at shorter
SRTs (de Kreuk et al., 2007; Liebana et al., 2016). As described by Ahmed et al. (2007), the bound-EPS per unit of biomass increased with decreased SRT as the microorganisms shifted. Additionally, EPS is the matrix for granular sludge, and it is secreted by bacteria in the system to promote microorganism enrichment and provide a suitable ecological niche in stable aerobic granules, but the mechanism of EPS secretion in aerobic granular sludge is still vague (Flemming and Wingender, 2010; Sheng et al., 2010; Xuan et al., 2010; Ju and Zhang, 2014; Sasikumar et al., 2017). Quorum sensing (QS) is a cell-to-cell communication process that mostly occurs among microorganisms and is biomass density dependent (Fuqua et al., 1996; Papenfort and Bassler, 2016; Donato et al., 2017). QS was studied extensively as an important factor for EPS secretion and microbial aggregation, and can control many bacterial behaviors such as biofilm formation, bioluminescence, and public good production (Fuqua and Greenberg, 2002; Dandekar et al., 2012). Lots of studied showed that bacteria with function of nitrogen and phosphate removal and EPS secretion, such as Rhodobacter spp., Pseudomonas spp. and Xanthomonadaceae, are related to AHL-mediated QS, and these bacteria are enriched at biological wastewater treatment (Zhang et al., 2012; Tan et al., 2014; Duan et al., 2018; Figdore et al., 2018). It has been extensively reported that acyl-homoserine lactone (AHL)-mediated QS is related to aerobic granular sludge (Fuqua and Greenberg, 2002; Dandekar et al., 2012; Li et al., 2014; Donato et al., 2017), According to Liu et al. (2016), the starvation period during SBR cycle could enhance QS in aerobic granular sludge system, leading to EPS secretion and granular stability. Li et al. (2014) found that inactivation of AHLs led to reduction of EPS component and deteriorate granular structure. Tan et al. (2014) also highlighted that low concentrations of AHLs in aerobic granular sludge was a common feature to coordinate community behavior. It
seemed that QS was the key factor for aerobic sludge granulation. Nevertheless, the effect of AHLs-based QS mechanism on EPS-component secretion and functional microbes selective enrichment in aerobic granular sludge is still not clear. According to the effect of SRT on the selection of functional microbes, aerobic sludge granulation reactors with different sludge retention times (SRTs) were established in this study. The objectives are as follows: 1) the SRT is controlled to enhance the microbial richness and QS system in aerobic granular sludge; 2) AHLs are quantified to investigate the relationship between QS and the properties of granular sludge; and 3) AHLs are added back to investigate the effect of the enhancement of QS on EPS-component secretion and the stability of aerobic granular sludge. 2. Materials and methods 2.1 Reactor and operation Three identical sequencing batch reactors (50 cm in height and 10 cm in diameter with a corking volume of 4 L in each reactor) were operated in parallel with different SRTs: uncontrolled (R1), 6 days (R2), and 12 days (R3). Aeration was set at an airflow rate of 8.5 L/min. The SBR cycle was 4 h with a volume exchange ratio of 50% for all the reactors. The operation modes, including feeding, aeration, settling, and decanting, are shown in Appendix A. The SRT was maintained by discharging excess sludge from the middle of the reactor at the end of aeration in R2 and R3. The reactors were inoculated with 4.0 ± 0.1 g/L seeding sludge from a Sewage Treatment plant in Hangzhou. Synthetic wastewater used as feeding water, comprised the following (mg/L): sodium acetate, 1008.3; NH4Cl, 185.35; KH2PO4, 27.17; K2HPO4•5H2O, 34.72; yeast, 16.15; peptone, 24.2; CaCl2, 80; MgSO4, 30; and a trace element solution composed of the following components: H3BO3, 0.05; CuSO4•5H2O,
0.05; ZuSO4•7H2O, 0.05; AlCl3, 0.09; CoCl2, 0.05; MnSO4•H2O, 0.05; (NH4)2Mo7O24, 0.05; NiCl2•6 H2O, 0.09; and FeSO4•7H2O, 0.05. The ambient temperature in the laboratory was 25±2°C. 2.2 Experiments of AHL add-back test Batch experiments were conducted to further investigate the effect of AHLs on EPS secretion based on the results from the reactors. A total of 100 mL of activated sludge collected from the Sewage Treatment plant in Hangzhou was washed three times with phosphate buffer solution (PBS) and added into 250 mL flasks with an MLSS of 4000 ±500 mg/L. The main AHLs, i.e., C4-HSL, C8-HSL, 3OHC8-HSL and 3OHC12-HSL, were added into the flasks at a final concentration of 10 or 1000 μg/L. The higher concentration of AHLs was added to reduce the complex effect from environmental factors during long-term operation and observing the significant changes of sludge in short time (Tan et al., 2014; Ding et al., 2015; Li et al., 2015).Furthermore, the shorter reaction times in this study could ensure that the AHLs added and the EPS component secreted were not metabolized by the microbial community as substrate. Methanol was added into the flask as a negative control. All flasks were incubated at 150 rpm for 2 h at 25°C. The sludge was taken from the flasks at the end of batch experiment for EPS analysis. 2.3 Analytical methods 2.3.1 Water quality Total nitrogen (TN) concentrations were analyzed by sampling the effluent three times every week after it was filtered through a 0.45 μm cellulose filter. Their concentrations were determined following the standard methods (APHA, 2005). 2.3.2 Analysis of sludge physical properties The sludge physical properties included the mixed liquor suspended solids (MLSS),
sludge volume index (SVI30), microstructure and morphology. MLSS and SVI30 were measured according to the standard methods (APHA, 2005) three times every week. The mean size and size density distribution of the aerobic granular sludge were measured by analyzers for particle size and shape (QICPIC, Sympatec, Germany) according to the international standards (ISO, 2001). The sludge microstructure was measured using microscopy (Leica DM300, Germany) and scanning electron microscopy (SEM). The sludge sample from reactors was fixed with 2.5% glutaraldehyde in phosphate buffer (pH 7.0) for more than 4 hours, and then was washed three times in phosphate buffer, post-fixed with 1% OsO4 in phosphate buffer (pH 7.0) for 1 hour and washed three times in phosphate buffer. The sludge sample was dehydrated by a graded series of ethanol (50%, 70%, 80%, 90%, 95% and 100%) for approximately 15 to 20 minutes at each step. Finally, the sample was dehydrated and coated with gold-palladium, then observed in the SEM (Hitachi Model SU8010FE, Japan). The granular strength was described as the integrity coefficient and measured according to Ghangrekar et al. (2005). Briefly, granules taken from reactors were added into flasks and diluted using distilled water to a final volume of 100 mL. The flasks were agitated using an orbital shaker at 200 rpm for 5 min, and then, the mixed solution was put into a 150 mL-measuring cylinder to separate ruptured granules. The dry weights of the settled granules and the residual granules in the supernatant were measured. The ratio of the solid in the supernatant to the total weight of the granular sludge was used for the granular strength measurement, which is expressed in percentages as an integrity coefficient. 2.3.3 Analysis of EPS content and components EPS was extracted from the sludge sample using a heating method (Bourven et al., 2011). The polysaccharide (PS) content in the EPS was then quantified using the
phenol-sulfuric acid method with glucose as the standard (Dubois et al., 1956). The protein (PN) content in the EPS was further determined by a modified Lowry colorimetric method with bovine albumin serum as the standard (Lowry et al., 1951). The excitation-emission matrix of the EPS extracted above was analyzed using a fluorospectrophotometer (Shimadzu F-4500) according to Luo et al. (2014) and as follows: Scanning emission spectra was obtained from 250 to 550 nm in 5-nm increments, and the excitation wavelength varied from 200 to 400 nm in 5-nm increments. Excitation and emission slits were kept at 5 nm and 10 nm, respectively, while the scanning speed was set at 1200 nm/min. A parallel factor (PARAFAC) analysis was employed to process the EEM data to obtain the variation in the EPS components. Raman scattering and Rayleigh scattering were eliminated according to Sheng et al. (2013). MatLab R2016a (MathWorks Inc., USA) software was employed for handling the EEM data. 2.3.4 AHL extraction and analysis Standard substances, N-butyryl-DL-homoserine lactone (C4-HSL), N-hexanoyl-DLhomoserine lactone (C6-HSL), N-(3-oxohexanoyl)-DL-homoserine lactone (3OC6HSL), N-octanoyl-DL-homoserine lactone (C8-HSL), N-(3-hydroxydodecanoyl)-DLhomoserine lactone (3OHC8-HSL), N-decanoyl-DL-homoserine lactone (C10-HSL), N-(3-oxodecanoyl)-L-homoserine lactone (3OC10-HSL), N-dodecanoyl-DLhomoserine lactone (C12-HSL), N-(3-oxododecanoyl)-L-homoserine lactone (3OC12-HSL), and N-(3-hydroxydodecanoyl)-DL-homoserine lactone (3OHC12HSL), were purchased from Sigma-Aldrich. The effluents from the reactors were used for AHL extraction after filtration with 0.45 μm cellulose filters according to Shaw et al. (1997) with slight modifications. Briefly, 100 mL of filtered effluents was extracted three times with one volume of
dichloromethane. All dichloromethane extracts were concentrated by rotary evaporators (Buchi R210) at 30 °C and resuspended in 100 μL of methanol:water (1:1 v/v). The resuspended solution was analyzed by HPLC-MS/MS (Agilent 1290/6460). The chromatographic column used for HPLC was XR-ODS C18, and the samples were chromatographed at a flow rate of 0.3 mL/min. The mobile phase consisted of a linear gradient (40%-90%) of solvent A (2 mM ammonium acetate with 0.1% formic acid) and solvent B (acetonitrile). Effluents were ionized by electrospray positive ionization (ESI+) and scanned by multiple reaction monitoring (MRM). For AHLs quantification, matrix-matched standard curves, ranging from 20 to 2000 ng/L, were constructed. The samples were tested three times for the data credibility. 2.3.5 Microbial community analysis The genomic DNA of the biomass in granule samples was extracted following the protocol of the Power Soil DNA extraction kit (MO BIO Laboratories Inc.). The total DNA extracted from the samples was used as a template, and the V3-V4 region of the bacterial 16S rRNA was amplified with the primers (338F 5'ACTCCTACGGGAGGCAGCAG-3'; 806R 5'-GGACTACHVGGGTWTCTAAT-3'). All reactions were carried out in 25 µL (total volume) mixtures containing approximately 25 ng of genomic DNA extract, 12.5 µL of PCR Premix, 2.5 µL of each primer, and PCR-grade water to adjust the volume. PCR reactions were performed in a Master Cycler Gradient thermocycler (Eppendorf, Hamburg, Germany) set to the following conditions: initial denaturation at 98 °C for 30 seconds; 35 cycles of denaturation at 98 °C for 10 seconds, annealing at 53 °C for 30 seconds, and extension at 72 °C for 45 seconds; and final extension at 72 °C for 10 minutes. The PCR products of the samples were sequenced with the Illumina MiSeq platform (PE300, CA, USA).
2.4 Statistical analysis Statistical analysis between all the variables was conducted by the t-test using SPSS (SPSS 17.0). p < 0.05 was considered to be statistically different, and p < 0.01 was considered to be significantly different. 3. Results 3.1 Reactor performance under different SRTs The operation of all the reactors was divided into three phases: phase I (days 1-15) was the initial phase when sludge flocs were converted to a granular shape, phase II (days 15-32) was the mature phase when more sludge flocs accumulated and granular sludge was formed gradually, and phase III (days 32-55) was the maintenance phase when mature granules were operated steadily. The changes in MLSS at different SRTs are shown in Fig. 1a. It is apparent that during the first 20 days of operation, the MLSS in the three reactors increase gradually from 3.5±0.5 g/L to 8.0±0.3 g/L, 4.0±0.2 g/L and 6.0±0.4 g/L in R1, R2 and R3, respectively. Although the MLSS increase from approximately 4 g/L to 6 g/L as the SRT increases from 6 days to 12 days, granules with excellent ability to settle were formed in R2 with an SRT of 6 days. The SVI5 in R2 decreased sharply to 60 mL/g, while the SVI5 in R3 (SRT=12 days) was maintained between 250 and 300 mL/g during phase I and phase II. The shorter SRT led to excellent granules. After 20 days of operation, granules appeared and the size distribution curves of the three reactors were similar. The mean granular sizes were 145.7, 184.2 and 160.1 μm in R1, R2 and R3, respectively (Fig. 1c). The granules gradually matured after 30 days of operation, and the size distribution curves of the three reactors during maintenance phase were distinctive, especially granules in R2 with a mean granular size of 561.3 μm, which is larger than those in R1 and R3 (Fig. 1d). The integrity
coefficients of aerobic granular sludge in R1 and R2 were 0.11 and 0.08, respectively, which were significantly lower than that in R3 (p<0.05) at 45 days of operation (Fig. 1e). SEM images of aerobic granular sludge in R1, R2 and R3 after 50 days of operation are shown in Appendix A. Granules with compact structures were observed in R1 and R2, while loose granules were observed in R3. Results showed that granular sludge with a controlled SRT could maintain large and compact structures, especially in the sludge with an SRT of 6 days. --------------------------Figure 1-------------------------As shown in Appendix A, the TN removal efficiency of the three reactors was approximately 58% when seeded with activated sludge, and gradually increased to 72%, 85% and 80% in R1, R2 and R3 respectively during the maintenance phase. The results showed that the TN removal efficiency increased with aerobic sludge granulation, and the stable granular sludge reactor with an SRT of 6 days had the granules with a larger size, more compact structure and higher TN removal efficiency (Fig. 1a-e). 3.2 Variation of the sludge EPS components The content of the sludge EPS in three reactors is shown in Fig. S3. It was observed that the EPS components (PS and PN) increased by 44.59 and 42.85 mg/g VSS in R1 (from 172.85 to 217.44 mg/g VSS) and R2 (from 193.18 to 236.03 mg/g VSS), respectively. These increases were higher than that in R3 of 20.59 mg/g VSS (from 200.86 to 221.45 mg/g VSS) during the first 20 days. On the other hand, the PS content of granules in R1 and R2 was 34.28 mg/g VSS and 38.45 mg/g VSS, respectively, at 20 days of operation. By contrast, the PS content of granules in R3 was 24.53 mg/g VSS, which was lower than that in R1 and R2. The sludge with the suitable SRT of 6 days can produce more EPS for adherent bacteria and granule
formation during the initial phase. The EPS contents and PN/PS ratios were similar (2.51, 2.56 and 2.32) for each of the three reactors after 20 days of operation, and it was necessary to further analyze the EPS components of granular sludge. 3D-EEM was used to confirm the main components of the EPS during slduge granulation, which were further analyzed by PARAFAC (Fig. 2). The results indicated that there were three major components in the 3D-EEM spectra, i.e., tryptophan and protein-like substances, hydrophilic acids and humic-like acids (Luo et al., 2014). The compact granules in R2 with an SRT of 6 days contained more tryptophan and protein-like substances with a peak intensity of 7403.22 during the maintenance phase. On the other hand, the peak intensity of humic-like acids was lower in stable granules of R2 at 166.83 during the maintenance phase. By contrast, the loose granules in R3 with an SRT of 12 days contained less tryptophan and protein-like substances with a peak intensity of 4657.17, and more humic-like acids with a peak intensity of 1077.51 during the maintenance phase. Results showed that the granular sludge with an SRT of 6 days has the ability to enhance the formation of tryptophan and protein-like substances and repress the formation of humic-like acid substances. --------------------------Figure 2-------------------------3.3 Variation of AHLs during sludge granulation The AHL content was measured at the operation periods of 6 days, 20 days and 48 days, representing the initiation, maturation and maintenance phases, respectively. Four main AHLs, C4-HSL, C8-HSL, 3OC8-HSL and 3OHC12-HSL, were detected in the reactors. Fig. 3 shows that almost all AHLs were abundant during operation in all reactors. Therein, C8-HSL accumulated in R2 at the highest concentration of 2150 ng/L during the initial phase and remained more concentrated in R2 throughout the
whole operation. The C8-HSL concentration in R2 was significantly higher than that in R3, and the corresponding p values were less than 0.05 and 0.01 for the operation of 20 days and 48 days, respectively. 3OHC8-HSL was only detected in R2 at the operation of 6 days and had the highest concentrations in the effluent of R2 at the operation of 20 days and 48 days, 1150 and 1325 ng/L, respectively. Moreover, the concentration of 3OHC12-HSL in R2 was significantly higher than that in R3 at the operation of 20 days and 48 days. The concentration of C4-HSL in R2 was lower (1120 ng/L, 1850 ng/L and 425 ng/L at the operation of 6 days, 20 days and 48 days, respectively) than those in the other reactors. It seemed that C8-HSL, 3OHC8-HSL and 3OHC12-HSL were concentrated in the reactor with the SRT of 6 days and maybe enhance the aerobic sludge granulation. --------------------------Figure 3-------------------------3.4 Analysis of the sludge microbial community AHLs are produced and sensed by bacteria in activated sludge, and several microbes have the function to secrete EPS and AHLs, which leads to the EPS content increasing during microbial aggregation. Family-level microbial diversity analyses at the operation of 20 days and 50 days were carried out to investigate the effect of quorum sensing on aerobic sludge granulation. As shown in Fig. 4, microbes with the ability to produce AHLs, i.e., Rhodobacteraceae and Xanthomonadaceae, were enriched in R2, with relative abundances of 18.6% and 13.1% respectively at the operation of 20 days. Xanthomonadaceae especially, with the function of exopolysaccharides secretion as well, was enriched in R2 compared to R1 and R3. On the other hand, Rhodobacteraceae was the main denitrification bacteria in this period (Huang et al., 2013; Tan et al., 2014). Along with the aerobic sludge granulation, Rhodobacteraceae enriched gradually in R2 more than in the other reactors, and its relative abundance
increased to 28.6%. Xanthomonadaceae was also enriched in R2, with the relative abundance increasing to 18.6%. Other microbes with function of denitrification, i.e., Rhodospirillaceae and Hyphomonadaceae, were gradually detected and enriched in R2. Among them, Hyphomonadaceae is the main microorganism for extracellular protein secretion (Jakob et al., 2016). It seemed that shorter SRTs (6 days) could enrich the microorganisms with function of AHL production and EPS secretion during the initial phase of sludge granulation, and more microbes with function of denitrification, EPS secretion and AHL production were enriched during the maintenance phase of sludge granulation. --------------------------Figure 4-------------------------4. Discussion 4.1 Correlation of AHLs and EPS components in sludge granulation Statistical analysis was implemented to illuminate the role of AHLs in EPS secretion and to identify the main AHLs that promote the EPS secretion. As shown in Fig.3 e-f, at the operation of 20 days 3OHC8-HSL is the only AHL strongly correlated with tryptophan and protein-like substances (p<0.01) and hydrophilic acid (p<0.05), with Pearson correlation coefficients of 0.84 and 0.61 respectively. Following the sludge granulation, C8-HSL and 3OHC8-HS were significantly positively correlated with tryptophan and protein-like substances, with Pearson correlation coefficients of 0.84 (p<0.01) and 0.70 (p<0.001) respectively. By contrast, C8-HSL, 3OHC8-HSL and 3OHC12-HSL were negative correlated with the peak intensity of humic-like acid substances in all operation, and C4-HSL was positively correlated with the peak intensity of humic-like acid substances, with Pearson correlation coefficients of 0.95 and 0.98 at the operation of 20 and 48 days respectively. The matrix of biofilm EPS can form a rigid net for microorganisms (Flemming and Wingender, 2010). According
to the related results, tryptophan and protein-like substances and hydrophilic acids, especially tryptophan and protein-like substances, were the main EPS components in stable granules (Fig. 2), and C8-HSL and 3OHC8-HSL were positively significantly correlated with tryptophan and protein-like substances. It was speculated that AHLs especially with 8-carbon sidechains, could enhanced the aerobic sludge granulation by promoting the secretion of tryptophan and protein-like substances in sludge EPS components according to the quorum sensing theory described previously (Dandekar et al., 2012; Li et al., 2014; Song et al., 2014; Hu et al., 2016). The AHL add-back studies were carried out to further prove the speculation that some AHLs in aerobic granular sludge system promoted the specific EPS secretion and enhanced aerobic sludge granulation. Tryptophan and protein-like substances and hydrophilic acids were the main EPS components in activated sludge analyzed by 3DEEM followed by PARAFAC. As shown in Fig.5, the peak intensity of tryptophan and protein-like substances increased when C8-HSL, 3OHC8-HSL and 3OHC12-HSL were added in either concentration. When C8-HSL was added, the highest peak intensity of 745.32 was reached compared to the control group (456.25), while the flasks with only C4-HSL added had a slightly higher concentration of tryptophan and protein-like substance secretion compared with the control group (The peak intensity increased by 55.31). By contrast, the peak intensity of hydrophilic acid increased slightly in the flasks with 3OHC8-HSL and 3OHC12-HSL (The peak intensity increased by 33.97 and 39.04, respectively), and increased scarcely when C4-HSL and C8-HSL were added compared with the control group. Recent studies have suggested that tryptophan and protein-like substances and hydrophilic acids were abundant in the EPS of mature granules (Luo et al., 2014). Humic acid, mainly derived from adsorption of wastewater components and hydrolysis of other biopolymers, such as
protein (Zhang et al., 2015), was the main component of membrane foulants in MBRs with high hydrophilicity (Al-Halbouni et al., 2008). In light of the change in peak intensity and statistical results during sludge granulation, tryptophan and protein-like substances played an important role in the aerobic sludge granulation under the mode of SRT regulation. AHLs with 8-carbon sidechains promoted the secretion of tryptophan and protein-like substances and then enhanced the aerobic sludge granulation. --------------------------Figure 5-------------------------4.2 Enhancement of AHL production during sludge granulation via SRT control We demonstrated that the SRT-controlled strategy enhanced the enrichment of microorganisms with the ability of AHLs production and EPS secretion, which facilitated the formation of granular sludge with excellent stability and high pollutants removal performance. The sludge EPS contents in the reactor with an SRT of 6 days increased more than that in the reactor with an SRT of 12 days during the initial phase (shown in Appendix A). The microbes that function in AHL production and EPS secretion were enriched in the reactor with an SRT of 6 days at the same time (Fig.4). The granular size distribution curves were similar among the three reactors (Fig.1 c). This means that the shorter SRT (6 days) could promote the enrichment of functional microorganism (group I), i.e., Rhodobacteraceae with the ability for AHL production and denitrification, and microorganism (group II), i.e., Xanthomonadaceae with the ability for AHL production and EPS secretion during the initial phase of sludge granulation (Fig. 6). Related researches showed that the heterotrophic bacteria mentioned above with a fast growth rate could proliferate under shorter SRTs (Moussa et al., 2005; Xie et al., 2012), and the microorganisms with the ability for AHL production could promote their own adhesion and metabolism. On the other
hand, these microbes regulate the EPS secretion and metabolism of other microorganisms with QS activity. After the enrichment of microorganisms (group I and II), microorganism (group III), i.e., Hyphomonadaceae with the ability for EPS secretion and denitrification, was detected and enriched at high relative abundance. As described by the AHL add-back study, the microorganisms with function of EPS secretion were regulated by AHLs (mainly C8-HSL and 3OHC8-HSL) to secret more EPS especially tryptophan and protein-like substances, which contributed to microbial aggregation. Mature granules further formed and provided a suitable ecological niche for denitrification. According to the results of the high-throughput sequencing, tryptophan and protein-like substances might be mainly secreted by Hyphomonadaceae (Show et al., 2012; Zhang et al., 2015). In this study, the EPS secretion and granular formation were explained from the aspect of AHL production among the functional microorganism in aerobic granules. --------------------------Figure 6-------------------------4. Conclusion Aerobic granular sludge with a stable structure and high pollutants removal performance was formed in the reactor operated at an SRT of 6 days. C8-HSL and 3OHC8-HSL were positively correlated with the major EPS components of tryptophan and protein-like substances. AHLs especially those with 8-carbon sidechains, play an important role in extracellular protein secretion and microbial aggregation, demonstrating a possible regulation strategy of aerobic sludge granulation via QS mechanism. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (No. 51478416), the Major Science and Technology Program for Water
Pollution Control and Treatment (2017ZX07201003), “Three Agricultures and Six Departments” Agricultural Science and Technology Cooperation Project of Zhejiang Province (CTZB-F170623LWZ-SNY1-30), and Science and Technology Project of Shaoxing (2017B70007). Appendix A. Supplementary data References Ahmed, Z., Cho, J., Lim, B.R., Song, K.G., Ahn, K.H., 2007. Effects of sludge retention time on membrane fouling and microbial community structure in a membrane bioreactor. J. Membrane Sci. 287, 211-218. Al-Halbouni, D., Traber, J., Lyko, S., Wintgens, T., Melin, T., Tacke, D., Janot, A., Dott, W., Hollender, J., 2008. Correlation of EPS content in activated sludge at different sludge retention times with membrane fouling phenomena. Water Res. 42, 1475-1488. APHA, 2005. Standard methods for the examination of water and wastewater, Washington, DC, USA. Bourven, I., Joussein, E., Guibaud, G., 2011. Characterisation of the mineral fraction in extracellular polymeric substances (EPS) from activated sludges extracted by eight different methods. Bioresour. Technol. 102, 7124-7130. Corsino, S.F., Capodici, M., Morici, C., Torregrossa, M., Viviani, G., 2016. Simultaneous nitritation-denitritation for the treatment of high-strength nitrogen in hypersaline wastewater by aerobic granular sludge. Water Res. 88, 329-336. Dandekar, A.A., Chugani, S., Greenberg, E.P., 2012. Bacterial quorum sensing and metabolic incentives to cooperate. Science 338, 264-266.
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Fig.1 Physical characteristic of aerobic granular sludge in different reactors: a. MLSS; b. SVI5; c. Sludge size cumulative distribution during initial phase; d. Sludge size cumulative distribution during maintenance phase Fig.2 Peak intensity changes of the PARAFAC-derived components in EPS during aerobic sludge granulation process Fig.3 Variation of AHLs concentration in different reactors and correlation between AHLs and EPS components: a. C4-HSL; b. C8-HSL; c. 3OHC8-HSL; d. 3OHC12HSL; e-f: Pearson correlations between AHLs and EPS components in the operation period of 14 days and 48 days, respectively Fig.4 Microbial diversity analysis in different reactors during maintenance phase Fig.5 Peak intensity changes of the PARAFAC-derived components in EPS of activated slduge in AHLs add-back study Fig.6 The mechanism of Aerobic sludge granulation regulated by SRT through enhanced quorum sensing
Figure 1 Physical characteristic of aerobic granular sludge in different reactors: a. MLSS; b. SVI5; c. Sludge size density distribution during initiation phase; d. Sludge size density distribution during maintenance phase; e. Integrity coefficient of aerobic granular sludge during maintenance phase
Tryptophan and protein-like
Hydropholic acids
Humic acid like Figure 2 Peak intensity changes of the PARAFAC-derived components in EPS during aerobic sludge granulation process
Figure 3 Variation of AHLs concentration in different reactors and correlation between AHLs and EPS components: a. C4-HSL; b. C8-HSL; c. 3OHC8-HSL; d. 3OHC12-HSL; e-f: Pearson correlations between AHLs and EPS components in the operation period of 14 days and 48 days, respectively # not significance; * p<0.05; ** p<0.01; *** p<0.001
Figure 4 Microbial diversity analysis in different reactors during maintenance phase: a. 20 day during operation; b. 50 day during operation
Tryptophan and protein-like
Hydropholic acids Figure 5 Peak intensity changes of the PARAFAC-derived components in EPS of activated slduge in AHLs add-back study
Figure 6 The mechanism of Aerobic sludge granulation regulated by SRT through enhanced quorum sensing Highlights SRT is controlled to enhance the microbial richness and QS in aerobic granule sludge. Typical AHLs are quantified to study the relation between QS and granular property.
Granules with 6-day SRT enrich microbes involving AHL production and EPS secretion. 8-carbon-sidechain AHLs promote protein secretion and granules formation.
S0960-8524(18)31448-2 https://doi.org/10.1016/j.biortech.2018.10.027 BITE 20591
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Bioresource Technology
Received Date: Revised Date: Accepted Date:
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Please cite this article as: Zhang, Z., Yu, Z., Wang, Z., Ma, K., Xu, X., Alvarezc, P.J.J., Zhu, L., Understanding of aerobic sludge granulation enhanced by sludge retention time in the aspect of quorum sensing, Bioresource Technology (2018), doi: https://doi.org/10.1016/j.biortech.2018.10.027
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Understanding of aerobic sludge granulation enhanced by sludge retention time in the aspect of quorum sensing Zhiming Zhanga, Zhuodong Yua, Zihao Wanga, Ke Maa, Xiangyang Xua,b,c, Pedro J.J. Alvarezcd, Liang Zhua,b,c* a. Institute of Environmental Pollution Control and Treatment, Zhejiang University, Hangzhou 310058, China b. Zhejiang Province Key Laboratory for Water Pollution Control and Environmental Safety, Hangzhou 310058, China c. Zhejiang Provincial Engineering Laboratory of Water Pollution Control, 388 Yuhangtang Road, Hangzhou 310058, China d. Department of Civil and Environmental Engineering, Rice University, 6100 Main St., Houston, TX 77005, USA *Corresponding
author: Liang Zhu
Address: Department of Environmental Engineering, Zhejiang University, No. 866 Yuhangtang Road, Hangzhou 310058, PR China. Tel (fax): +86 571 88982343 E-mail address: [email protected]. Abstract Aerobic granular sludge (AGS) reactors with different sludge retention times (SRTs) were established for enhanced functional microorganism enrichment and granular formation. Results showed that higher total nitrogen (TN) removal efficiency and compact granules were achieved in the 6-day-SRT reactor. Also, Xanthomonadaceae, Rhodobacteraceae and Hyphomonadaceae with AHL-producing and EPS-secreting functions also enriched under 6-day SRT. For investigating the enhanced mechanism of sludge granulation, typical quorum sensing signals of acylated-homoserine-lactones
(AHLs) and extracellular polymeric substances (EPS) were analyzed. Tryptophanand-protein-like substances were major EPS components in granules formed at 6-day SRT. Meanwhile, most detected AHLs, i.e. C8-HSL and 3OHC8-HSL, were correlated positively with contents of tryptophan-and-protein-like substances. Accroding to AHLs add-back test, AHLs especially those with 8-carbon sidechains, played important roles in aerobic sludge granulation via secreting special extracellular proteins by functional microbes enrichment. Keywords: Aerobic granular sludge; sludge retention time (SRT); acylated homoserine lactones (AHLs); extracellular polymeric substance (EPS); quorum sensing (QS) 1. Introduction Aerobic granular sludge has been investigated extensively in the past two decades because of its excellent settling ability, enhanced pollutants removal efficiency and high biomass retention (de Kreuk et al., 2007; Show et al., 2012; Liu et al., 2015; Derlon et al., 2016; Li et al., 2017), and it has been widely applied to industrial and municipal wastewater treatment (Su and Yu, 2005; Pronk et al., 2015; Corsino et al., 2016; Derlon et al., 2016). The stability of aerobic granular sludge is essential for long-term aerobic granule processes. However, granular disintegration occurs in both lab-scale and pilot-scale reactors, which impedes the optimization and application of this technology. As is well known, the seeding sludge, type and loading of the substrate, reactor operating mode, and sludge retention time (SRT) are the main factors affecting the granular disintegration, related to variations in the microbial community and extracellular polymeric substances (EPS) of sludge (de Kreuk et al., 2007; Show et al., 2012). Therein, many papers showed that the activated sludge of laboratory-scale sequencing batch bioreactors (SBR) has higher diversity at shorter
SRTs (de Kreuk et al., 2007; Liebana et al., 2016). As described by Ahmed et al. (2007), the bound-EPS per unit of biomass increased with decreased SRT as the microorganisms shifted. Additionally, EPS is the matrix for granular sludge, and it is secreted by bacteria in the system to promote microorganism enrichment and provide a suitable ecological niche in stable aerobic granules, but the mechanism of EPS secretion in aerobic granular sludge is still vague (Flemming and Wingender, 2010; Sheng et al., 2010; Xuan et al., 2010; Ju and Zhang, 2014; Sasikumar et al., 2017). Quorum sensing (QS) is a cell-to-cell communication process that mostly occurs among microorganisms and is biomass density dependent (Fuqua et al., 1996; Papenfort and Bassler, 2016; Donato et al., 2017). QS was studied extensively as an important factor for EPS secretion and microbial aggregation, and can control many bacterial behaviors such as biofilm formation, bioluminescence, and public good production (Fuqua and Greenberg, 2002; Dandekar et al., 2012). Lots of studied showed that bacteria with function of nitrogen and phosphate removal and EPS secretion, such as Rhodobacter spp., Pseudomonas spp. and Xanthomonadaceae, are related to AHL-mediated QS, and these bacteria are enriched at biological wastewater treatment (Zhang et al., 2012; Tan et al., 2014; Duan et al., 2018; Figdore et al., 2018). It has been extensively reported that acyl-homoserine lactone (AHL)-mediated QS is related to aerobic granular sludge (Fuqua and Greenberg, 2002; Dandekar et al., 2012; Li et al., 2014; Donato et al., 2017), According to Liu et al. (2016), the starvation period during SBR cycle could enhance QS in aerobic granular sludge system, leading to EPS secretion and granular stability. Li et al. (2014) found that inactivation of AHLs led to reduction of EPS component and deteriorate granular structure. Tan et al. (2014) also highlighted that low concentrations of AHLs in aerobic granular sludge was a common feature to coordinate community behavior. It
seemed that QS was the key factor for aerobic sludge granulation. Nevertheless, the effect of AHLs-based QS mechanism on EPS-component secretion and functional microbes selective enrichment in aerobic granular sludge is still not clear. According to the effect of SRT on the selection of functional microbes, aerobic sludge granulation reactors with different sludge retention times (SRTs) were established in this study. The objectives are as follows: 1) the SRT is controlled to enhance the microbial richness and QS system in aerobic granular sludge; 2) AHLs are quantified to investigate the relationship between QS and the properties of granular sludge; and 3) AHLs are added back to investigate the effect of the enhancement of QS on EPS-component secretion and the stability of aerobic granular sludge. 2. Materials and methods 2.1 Reactor and operation Three identical sequencing batch reactors (50 cm in height and 10 cm in diameter with a corking volume of 4 L in each reactor) were operated in parallel with different SRTs: uncontrolled (R1), 6 days (R2), and 12 days (R3). Aeration was set at an airflow rate of 8.5 L/min. The SBR cycle was 4 h with a volume exchange ratio of 50% for all the reactors. The operation modes, including feeding, aeration, settling, and decanting, are shown in Appendix A. The SRT was maintained by discharging excess sludge from the middle of the reactor at the end of aeration in R2 and R3. The reactors were inoculated with 4.0 ± 0.1 g/L seeding sludge from a Sewage Treatment plant in Hangzhou. Synthetic wastewater used as feeding water, comprised the following (mg/L): sodium acetate, 1008.3; NH4Cl, 185.35; KH2PO4, 27.17; K2HPO4•5H2O, 34.72; yeast, 16.15; peptone, 24.2; CaCl2, 80; MgSO4, 30; and a trace element solution composed of the following components: H3BO3, 0.05; CuSO4•5H2O,
0.05; ZuSO4•7H2O, 0.05; AlCl3, 0.09; CoCl2, 0.05; MnSO4•H2O, 0.05; (NH4)2Mo7O24, 0.05; NiCl2•6 H2O, 0.09; and FeSO4•7H2O, 0.05. The ambient temperature in the laboratory was 25±2°C. 2.2 Experiments of AHL add-back test Batch experiments were conducted to further investigate the effect of AHLs on EPS secretion based on the results from the reactors. A total of 100 mL of activated sludge collected from the Sewage Treatment plant in Hangzhou was washed three times with phosphate buffer solution (PBS) and added into 250 mL flasks with an MLSS of 4000 ±500 mg/L. The main AHLs, i.e., C4-HSL, C8-HSL, 3OHC8-HSL and 3OHC12-HSL, were added into the flasks at a final concentration of 10 or 1000 μg/L. The higher concentration of AHLs was added to reduce the complex effect from environmental factors during long-term operation and observing the significant changes of sludge in short time (Tan et al., 2014; Ding et al., 2015; Li et al., 2015).Furthermore, the shorter reaction times in this study could ensure that the AHLs added and the EPS component secreted were not metabolized by the microbial community as substrate. Methanol was added into the flask as a negative control. All flasks were incubated at 150 rpm for 2 h at 25°C. The sludge was taken from the flasks at the end of batch experiment for EPS analysis. 2.3 Analytical methods 2.3.1 Water quality Total nitrogen (TN) concentrations were analyzed by sampling the effluent three times every week after it was filtered through a 0.45 μm cellulose filter. Their concentrations were determined following the standard methods (APHA, 2005). 2.3.2 Analysis of sludge physical properties The sludge physical properties included the mixed liquor suspended solids (MLSS),
sludge volume index (SVI30), microstructure and morphology. MLSS and SVI30 were measured according to the standard methods (APHA, 2005) three times every week. The mean size and size density distribution of the aerobic granular sludge were measured by analyzers for particle size and shape (QICPIC, Sympatec, Germany) according to the international standards (ISO, 2001). The sludge microstructure was measured using microscopy (Leica DM300, Germany) and scanning electron microscopy (SEM). The sludge sample from reactors was fixed with 2.5% glutaraldehyde in phosphate buffer (pH 7.0) for more than 4 hours, and then was washed three times in phosphate buffer, post-fixed with 1% OsO4 in phosphate buffer (pH 7.0) for 1 hour and washed three times in phosphate buffer. The sludge sample was dehydrated by a graded series of ethanol (50%, 70%, 80%, 90%, 95% and 100%) for approximately 15 to 20 minutes at each step. Finally, the sample was dehydrated and coated with gold-palladium, then observed in the SEM (Hitachi Model SU8010FE, Japan). The granular strength was described as the integrity coefficient and measured according to Ghangrekar et al. (2005). Briefly, granules taken from reactors were added into flasks and diluted using distilled water to a final volume of 100 mL. The flasks were agitated using an orbital shaker at 200 rpm for 5 min, and then, the mixed solution was put into a 150 mL-measuring cylinder to separate ruptured granules. The dry weights of the settled granules and the residual granules in the supernatant were measured. The ratio of the solid in the supernatant to the total weight of the granular sludge was used for the granular strength measurement, which is expressed in percentages as an integrity coefficient. 2.3.3 Analysis of EPS content and components EPS was extracted from the sludge sample using a heating method (Bourven et al., 2011). The polysaccharide (PS) content in the EPS was then quantified using the
phenol-sulfuric acid method with glucose as the standard (Dubois et al., 1956). The protein (PN) content in the EPS was further determined by a modified Lowry colorimetric method with bovine albumin serum as the standard (Lowry et al., 1951). The excitation-emission matrix of the EPS extracted above was analyzed using a fluorospectrophotometer (Shimadzu F-4500) according to Luo et al. (2014) and as follows: Scanning emission spectra was obtained from 250 to 550 nm in 5-nm increments, and the excitation wavelength varied from 200 to 400 nm in 5-nm increments. Excitation and emission slits were kept at 5 nm and 10 nm, respectively, while the scanning speed was set at 1200 nm/min. A parallel factor (PARAFAC) analysis was employed to process the EEM data to obtain the variation in the EPS components. Raman scattering and Rayleigh scattering were eliminated according to Sheng et al. (2013). MatLab R2016a (MathWorks Inc., USA) software was employed for handling the EEM data. 2.3.4 AHL extraction and analysis Standard substances, N-butyryl-DL-homoserine lactone (C4-HSL), N-hexanoyl-DLhomoserine lactone (C6-HSL), N-(3-oxohexanoyl)-DL-homoserine lactone (3OC6HSL), N-octanoyl-DL-homoserine lactone (C8-HSL), N-(3-hydroxydodecanoyl)-DLhomoserine lactone (3OHC8-HSL), N-decanoyl-DL-homoserine lactone (C10-HSL), N-(3-oxodecanoyl)-L-homoserine lactone (3OC10-HSL), N-dodecanoyl-DLhomoserine lactone (C12-HSL), N-(3-oxododecanoyl)-L-homoserine lactone (3OC12-HSL), and N-(3-hydroxydodecanoyl)-DL-homoserine lactone (3OHC12HSL), were purchased from Sigma-Aldrich. The effluents from the reactors were used for AHL extraction after filtration with 0.45 μm cellulose filters according to Shaw et al. (1997) with slight modifications. Briefly, 100 mL of filtered effluents was extracted three times with one volume of
dichloromethane. All dichloromethane extracts were concentrated by rotary evaporators (Buchi R210) at 30 °C and resuspended in 100 μL of methanol:water (1:1 v/v). The resuspended solution was analyzed by HPLC-MS/MS (Agilent 1290/6460). The chromatographic column used for HPLC was XR-ODS C18, and the samples were chromatographed at a flow rate of 0.3 mL/min. The mobile phase consisted of a linear gradient (40%-90%) of solvent A (2 mM ammonium acetate with 0.1% formic acid) and solvent B (acetonitrile). Effluents were ionized by electrospray positive ionization (ESI+) and scanned by multiple reaction monitoring (MRM). For AHLs quantification, matrix-matched standard curves, ranging from 20 to 2000 ng/L, were constructed. The samples were tested three times for the data credibility. 2.3.5 Microbial community analysis The genomic DNA of the biomass in granule samples was extracted following the protocol of the Power Soil DNA extraction kit (MO BIO Laboratories Inc.). The total DNA extracted from the samples was used as a template, and the V3-V4 region of the bacterial 16S rRNA was amplified with the primers (338F 5'ACTCCTACGGGAGGCAGCAG-3'; 806R 5'-GGACTACHVGGGTWTCTAAT-3'). All reactions were carried out in 25 µL (total volume) mixtures containing approximately 25 ng of genomic DNA extract, 12.5 µL of PCR Premix, 2.5 µL of each primer, and PCR-grade water to adjust the volume. PCR reactions were performed in a Master Cycler Gradient thermocycler (Eppendorf, Hamburg, Germany) set to the following conditions: initial denaturation at 98 °C for 30 seconds; 35 cycles of denaturation at 98 °C for 10 seconds, annealing at 53 °C for 30 seconds, and extension at 72 °C for 45 seconds; and final extension at 72 °C for 10 minutes. The PCR products of the samples were sequenced with the Illumina MiSeq platform (PE300, CA, USA).
2.4 Statistical analysis Statistical analysis between all the variables was conducted by the t-test using SPSS (SPSS 17.0). p < 0.05 was considered to be statistically different, and p < 0.01 was considered to be significantly different. 3. Results 3.1 Reactor performance under different SRTs The operation of all the reactors was divided into three phases: phase I (days 1-15) was the initial phase when sludge flocs were converted to a granular shape, phase II (days 15-32) was the mature phase when more sludge flocs accumulated and granular sludge was formed gradually, and phase III (days 32-55) was the maintenance phase when mature granules were operated steadily. The changes in MLSS at different SRTs are shown in Fig. 1a. It is apparent that during the first 20 days of operation, the MLSS in the three reactors increase gradually from 3.5±0.5 g/L to 8.0±0.3 g/L, 4.0±0.2 g/L and 6.0±0.4 g/L in R1, R2 and R3, respectively. Although the MLSS increase from approximately 4 g/L to 6 g/L as the SRT increases from 6 days to 12 days, granules with excellent ability to settle were formed in R2 with an SRT of 6 days. The SVI5 in R2 decreased sharply to 60 mL/g, while the SVI5 in R3 (SRT=12 days) was maintained between 250 and 300 mL/g during phase I and phase II. The shorter SRT led to excellent granules. After 20 days of operation, granules appeared and the size distribution curves of the three reactors were similar. The mean granular sizes were 145.7, 184.2 and 160.1 μm in R1, R2 and R3, respectively (Fig. 1c). The granules gradually matured after 30 days of operation, and the size distribution curves of the three reactors during maintenance phase were distinctive, especially granules in R2 with a mean granular size of 561.3 μm, which is larger than those in R1 and R3 (Fig. 1d). The integrity
coefficients of aerobic granular sludge in R1 and R2 were 0.11 and 0.08, respectively, which were significantly lower than that in R3 (p<0.05) at 45 days of operation (Fig. 1e). SEM images of aerobic granular sludge in R1, R2 and R3 after 50 days of operation are shown in Appendix A. Granules with compact structures were observed in R1 and R2, while loose granules were observed in R3. Results showed that granular sludge with a controlled SRT could maintain large and compact structures, especially in the sludge with an SRT of 6 days. --------------------------Figure 1-------------------------As shown in Appendix A, the TN removal efficiency of the three reactors was approximately 58% when seeded with activated sludge, and gradually increased to 72%, 85% and 80% in R1, R2 and R3 respectively during the maintenance phase. The results showed that the TN removal efficiency increased with aerobic sludge granulation, and the stable granular sludge reactor with an SRT of 6 days had the granules with a larger size, more compact structure and higher TN removal efficiency (Fig. 1a-e). 3.2 Variation of the sludge EPS components The content of the sludge EPS in three reactors is shown in Fig. S3. It was observed that the EPS components (PS and PN) increased by 44.59 and 42.85 mg/g VSS in R1 (from 172.85 to 217.44 mg/g VSS) and R2 (from 193.18 to 236.03 mg/g VSS), respectively. These increases were higher than that in R3 of 20.59 mg/g VSS (from 200.86 to 221.45 mg/g VSS) during the first 20 days. On the other hand, the PS content of granules in R1 and R2 was 34.28 mg/g VSS and 38.45 mg/g VSS, respectively, at 20 days of operation. By contrast, the PS content of granules in R3 was 24.53 mg/g VSS, which was lower than that in R1 and R2. The sludge with the suitable SRT of 6 days can produce more EPS for adherent bacteria and granule
formation during the initial phase. The EPS contents and PN/PS ratios were similar (2.51, 2.56 and 2.32) for each of the three reactors after 20 days of operation, and it was necessary to further analyze the EPS components of granular sludge. 3D-EEM was used to confirm the main components of the EPS during slduge granulation, which were further analyzed by PARAFAC (Fig. 2). The results indicated that there were three major components in the 3D-EEM spectra, i.e., tryptophan and protein-like substances, hydrophilic acids and humic-like acids (Luo et al., 2014). The compact granules in R2 with an SRT of 6 days contained more tryptophan and protein-like substances with a peak intensity of 7403.22 during the maintenance phase. On the other hand, the peak intensity of humic-like acids was lower in stable granules of R2 at 166.83 during the maintenance phase. By contrast, the loose granules in R3 with an SRT of 12 days contained less tryptophan and protein-like substances with a peak intensity of 4657.17, and more humic-like acids with a peak intensity of 1077.51 during the maintenance phase. Results showed that the granular sludge with an SRT of 6 days has the ability to enhance the formation of tryptophan and protein-like substances and repress the formation of humic-like acid substances. --------------------------Figure 2-------------------------3.3 Variation of AHLs during sludge granulation The AHL content was measured at the operation periods of 6 days, 20 days and 48 days, representing the initiation, maturation and maintenance phases, respectively. Four main AHLs, C4-HSL, C8-HSL, 3OC8-HSL and 3OHC12-HSL, were detected in the reactors. Fig. 3 shows that almost all AHLs were abundant during operation in all reactors. Therein, C8-HSL accumulated in R2 at the highest concentration of 2150 ng/L during the initial phase and remained more concentrated in R2 throughout the
whole operation. The C8-HSL concentration in R2 was significantly higher than that in R3, and the corresponding p values were less than 0.05 and 0.01 for the operation of 20 days and 48 days, respectively. 3OHC8-HSL was only detected in R2 at the operation of 6 days and had the highest concentrations in the effluent of R2 at the operation of 20 days and 48 days, 1150 and 1325 ng/L, respectively. Moreover, the concentration of 3OHC12-HSL in R2 was significantly higher than that in R3 at the operation of 20 days and 48 days. The concentration of C4-HSL in R2 was lower (1120 ng/L, 1850 ng/L and 425 ng/L at the operation of 6 days, 20 days and 48 days, respectively) than those in the other reactors. It seemed that C8-HSL, 3OHC8-HSL and 3OHC12-HSL were concentrated in the reactor with the SRT of 6 days and maybe enhance the aerobic sludge granulation. --------------------------Figure 3-------------------------3.4 Analysis of the sludge microbial community AHLs are produced and sensed by bacteria in activated sludge, and several microbes have the function to secrete EPS and AHLs, which leads to the EPS content increasing during microbial aggregation. Family-level microbial diversity analyses at the operation of 20 days and 50 days were carried out to investigate the effect of quorum sensing on aerobic sludge granulation. As shown in Fig. 4, microbes with the ability to produce AHLs, i.e., Rhodobacteraceae and Xanthomonadaceae, were enriched in R2, with relative abundances of 18.6% and 13.1% respectively at the operation of 20 days. Xanthomonadaceae especially, with the function of exopolysaccharides secretion as well, was enriched in R2 compared to R1 and R3. On the other hand, Rhodobacteraceae was the main denitrification bacteria in this period (Huang et al., 2013; Tan et al., 2014). Along with the aerobic sludge granulation, Rhodobacteraceae enriched gradually in R2 more than in the other reactors, and its relative abundance
increased to 28.6%. Xanthomonadaceae was also enriched in R2, with the relative abundance increasing to 18.6%. Other microbes with function of denitrification, i.e., Rhodospirillaceae and Hyphomonadaceae, were gradually detected and enriched in R2. Among them, Hyphomonadaceae is the main microorganism for extracellular protein secretion (Jakob et al., 2016). It seemed that shorter SRTs (6 days) could enrich the microorganisms with function of AHL production and EPS secretion during the initial phase of sludge granulation, and more microbes with function of denitrification, EPS secretion and AHL production were enriched during the maintenance phase of sludge granulation. --------------------------Figure 4-------------------------4. Discussion 4.1 Correlation of AHLs and EPS components in sludge granulation Statistical analysis was implemented to illuminate the role of AHLs in EPS secretion and to identify the main AHLs that promote the EPS secretion. As shown in Fig.3 e-f, at the operation of 20 days 3OHC8-HSL is the only AHL strongly correlated with tryptophan and protein-like substances (p<0.01) and hydrophilic acid (p<0.05), with Pearson correlation coefficients of 0.84 and 0.61 respectively. Following the sludge granulation, C8-HSL and 3OHC8-HS were significantly positively correlated with tryptophan and protein-like substances, with Pearson correlation coefficients of 0.84 (p<0.01) and 0.70 (p<0.001) respectively. By contrast, C8-HSL, 3OHC8-HSL and 3OHC12-HSL were negative correlated with the peak intensity of humic-like acid substances in all operation, and C4-HSL was positively correlated with the peak intensity of humic-like acid substances, with Pearson correlation coefficients of 0.95 and 0.98 at the operation of 20 and 48 days respectively. The matrix of biofilm EPS can form a rigid net for microorganisms (Flemming and Wingender, 2010). According
to the related results, tryptophan and protein-like substances and hydrophilic acids, especially tryptophan and protein-like substances, were the main EPS components in stable granules (Fig. 2), and C8-HSL and 3OHC8-HSL were positively significantly correlated with tryptophan and protein-like substances. It was speculated that AHLs especially with 8-carbon sidechains, could enhanced the aerobic sludge granulation by promoting the secretion of tryptophan and protein-like substances in sludge EPS components according to the quorum sensing theory described previously (Dandekar et al., 2012; Li et al., 2014; Song et al., 2014; Hu et al., 2016). The AHL add-back studies were carried out to further prove the speculation that some AHLs in aerobic granular sludge system promoted the specific EPS secretion and enhanced aerobic sludge granulation. Tryptophan and protein-like substances and hydrophilic acids were the main EPS components in activated sludge analyzed by 3DEEM followed by PARAFAC. As shown in Fig.5, the peak intensity of tryptophan and protein-like substances increased when C8-HSL, 3OHC8-HSL and 3OHC12-HSL were added in either concentration. When C8-HSL was added, the highest peak intensity of 745.32 was reached compared to the control group (456.25), while the flasks with only C4-HSL added had a slightly higher concentration of tryptophan and protein-like substance secretion compared with the control group (The peak intensity increased by 55.31). By contrast, the peak intensity of hydrophilic acid increased slightly in the flasks with 3OHC8-HSL and 3OHC12-HSL (The peak intensity increased by 33.97 and 39.04, respectively), and increased scarcely when C4-HSL and C8-HSL were added compared with the control group. Recent studies have suggested that tryptophan and protein-like substances and hydrophilic acids were abundant in the EPS of mature granules (Luo et al., 2014). Humic acid, mainly derived from adsorption of wastewater components and hydrolysis of other biopolymers, such as
protein (Zhang et al., 2015), was the main component of membrane foulants in MBRs with high hydrophilicity (Al-Halbouni et al., 2008). In light of the change in peak intensity and statistical results during sludge granulation, tryptophan and protein-like substances played an important role in the aerobic sludge granulation under the mode of SRT regulation. AHLs with 8-carbon sidechains promoted the secretion of tryptophan and protein-like substances and then enhanced the aerobic sludge granulation. --------------------------Figure 5-------------------------4.2 Enhancement of AHL production during sludge granulation via SRT control We demonstrated that the SRT-controlled strategy enhanced the enrichment of microorganisms with the ability of AHLs production and EPS secretion, which facilitated the formation of granular sludge with excellent stability and high pollutants removal performance. The sludge EPS contents in the reactor with an SRT of 6 days increased more than that in the reactor with an SRT of 12 days during the initial phase (shown in Appendix A). The microbes that function in AHL production and EPS secretion were enriched in the reactor with an SRT of 6 days at the same time (Fig.4). The granular size distribution curves were similar among the three reactors (Fig.1 c). This means that the shorter SRT (6 days) could promote the enrichment of functional microorganism (group I), i.e., Rhodobacteraceae with the ability for AHL production and denitrification, and microorganism (group II), i.e., Xanthomonadaceae with the ability for AHL production and EPS secretion during the initial phase of sludge granulation (Fig. 6). Related researches showed that the heterotrophic bacteria mentioned above with a fast growth rate could proliferate under shorter SRTs (Moussa et al., 2005; Xie et al., 2012), and the microorganisms with the ability for AHL production could promote their own adhesion and metabolism. On the other
hand, these microbes regulate the EPS secretion and metabolism of other microorganisms with QS activity. After the enrichment of microorganisms (group I and II), microorganism (group III), i.e., Hyphomonadaceae with the ability for EPS secretion and denitrification, was detected and enriched at high relative abundance. As described by the AHL add-back study, the microorganisms with function of EPS secretion were regulated by AHLs (mainly C8-HSL and 3OHC8-HSL) to secret more EPS especially tryptophan and protein-like substances, which contributed to microbial aggregation. Mature granules further formed and provided a suitable ecological niche for denitrification. According to the results of the high-throughput sequencing, tryptophan and protein-like substances might be mainly secreted by Hyphomonadaceae (Show et al., 2012; Zhang et al., 2015). In this study, the EPS secretion and granular formation were explained from the aspect of AHL production among the functional microorganism in aerobic granules. --------------------------Figure 6-------------------------4. Conclusion Aerobic granular sludge with a stable structure and high pollutants removal performance was formed in the reactor operated at an SRT of 6 days. C8-HSL and 3OHC8-HSL were positively correlated with the major EPS components of tryptophan and protein-like substances. AHLs especially those with 8-carbon sidechains, play an important role in extracellular protein secretion and microbial aggregation, demonstrating a possible regulation strategy of aerobic sludge granulation via QS mechanism. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (No. 51478416), the Major Science and Technology Program for Water
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Fig.1 Physical characteristic of aerobic granular sludge in different reactors: a. MLSS; b. SVI5; c. Sludge size cumulative distribution during initial phase; d. Sludge size cumulative distribution during maintenance phase Fig.2 Peak intensity changes of the PARAFAC-derived components in EPS during aerobic sludge granulation process Fig.3 Variation of AHLs concentration in different reactors and correlation between AHLs and EPS components: a. C4-HSL; b. C8-HSL; c. 3OHC8-HSL; d. 3OHC12HSL; e-f: Pearson correlations between AHLs and EPS components in the operation period of 14 days and 48 days, respectively Fig.4 Microbial diversity analysis in different reactors during maintenance phase Fig.5 Peak intensity changes of the PARAFAC-derived components in EPS of activated slduge in AHLs add-back study Fig.6 The mechanism of Aerobic sludge granulation regulated by SRT through enhanced quorum sensing
Figure 1 Physical characteristic of aerobic granular sludge in different reactors: a. MLSS; b. SVI5; c. Sludge size density distribution during initiation phase; d. Sludge size density distribution during maintenance phase; e. Integrity coefficient of aerobic granular sludge during maintenance phase
Tryptophan and protein-like
Hydropholic acids
Humic acid like Figure 2 Peak intensity changes of the PARAFAC-derived components in EPS during aerobic sludge granulation process
Figure 3 Variation of AHLs concentration in different reactors and correlation between AHLs and EPS components: a. C4-HSL; b. C8-HSL; c. 3OHC8-HSL; d. 3OHC12-HSL; e-f: Pearson correlations between AHLs and EPS components in the operation period of 14 days and 48 days, respectively # not significance; * p<0.05; ** p<0.01; *** p<0.001
Figure 4 Microbial diversity analysis in different reactors during maintenance phase: a. 20 day during operation; b. 50 day during operation
Tryptophan and protein-like
Hydropholic acids Figure 5 Peak intensity changes of the PARAFAC-derived components in EPS of activated slduge in AHLs add-back study
Figure 6 The mechanism of Aerobic sludge granulation regulated by SRT through enhanced quorum sensing Highlights SRT is controlled to enhance the microbial richness and QS in aerobic granule sludge. Typical AHLs are quantified to study the relation between QS and granular property.
Granules with 6-day SRT enrich microbes involving AHL production and EPS secretion. 8-carbon-sidechain AHLs promote protein secretion and granules formation.