Cell adhesion, ammonia removal and granulation of autotrophic nitrifying sludge facilitated by N-acyl-homoserine lactones

Bioresource Technology 196 (2015) 550–558

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Cell adhesion, ammonia removal and granulation of autotrophic nitrifying sludge facilitated by N-acyl-homoserine lactones An-jie Li ⇑, Bao-lian Hou, Mei-xi Li Key Laboratory of Water and Sediment Sciences of Ministry of Education/State Key Laboratory of Water Environment Simulation, School of Environment, Beijing Normal University, Beijing 100875, PR China

h i g h l i g h t s
AHLs addition enhanced granulation of autotrophic nitrifying sludge.
AHLs improved microbial attachment of nitrifying sludge and ammonia degradation.
Efficiencies of cell adhesion and ammonia removal related to AHLs chemical structure.
AHLs increased biomass growth rate, microbial activity and extracellular protein.

a r t i c l e

i n f o

Article history: Received 15 June 2015 Received in revised form 5 August 2015 Accepted 8 August 2015 Available online 14 August 2015 Keywords: N-Acyl-homoserine lactone (AHL) Autotrophic nitrification Cell adhesion Sludge granulation Ammonia-rich inorganic wastewater

a b s t r a c t In this study, six N-acyl-homoserine lactone (AHL) molecules (C6-HSL, C8-HSL, C10-HSL, 3-oxo-C6-HSL, 3oxo-C8-HSL and 3-oxo-C10-HSL) were each dosed into a bioreactor and seeded using autotrophic nitrifying sludge (ANS). The effects of the AHLs on cell adhesion, nitrification and sludge granulation were investigated. The results indicated that the efficiencies of cell adhesion and ammonia removal both had a close correlation with the side chain length and b position substituent group of the AHLs. The best-performing AHL in terms of accelerating bacterial attached-growth was 3-oxo-C6-HSL, whereas C6-HSL outperformed the others in terms of the ammonia degradation rate. The addition of 3-oxo-C6HSL or C6-HSL increased the biomass growth rate, microbial activity, extracellular proteins and nitrifying bacteria, which can accelerate the formation of nitrifying granules. Consequently, selecting AHL molecules that could improve bacteria in attached-growth mode and nitrification efficiency simultaneously will most likely facilitate the rapid granulation of nitrifying sludge. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Ammonium, which is abundant in many industrial and agricultural wastewaters, must be removed to prevent the oxygen depletion and eutrophication of surface water. Biological nitrogen removal (BNR) is highly promising for ammonium removal due to its various advantages, which include its high economic efficiency, lack of secondary pollution and facile operation. The nitrification process is normally the rate-limiting step of BNR due to the low proportion and slow growth of nitrifying bacteria. The granulation of nitrifying activated sludge can aid in avoiding the loss of nitrifying bacteria from suspended sludge and enable the reactor to have a high concentration of nitrifying bacteria even ⇑ Corresponding author at: School of Environment, Beijing Normal University, No. 19, Xinjiekouwai Street, Haidian District, Beijing 100875, PR China. Tel.: +86 10 58805853. E-mail address: [email protected] (A.-j. Li). http://dx.doi.org/10.1016/j.biortech.2015.08.022 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.

under short hydraulic retention times. However, sludge granulation is rather difficult to achieve in nitrification due to the slow growth rates of nitrifying bacteria (Belmonte et al., 2009). In several of the successful cases reported to date, a long cultivation time of 2 months or more was required to achieve nitrifying granular sludge, particularly for concentrated ammonia feeds with several organic substrates (Liu et al., 2008; Shi et al., 2010; Hosseini et al., 2014). Therefore, a method to achieve the rapid granulation of nitrifying activated sludge must be developed. Otherwise, it will hinder the actual application of granular sludge for nitrification or BNR on a large scale. By exchanging chemical signals, a bacterial community may regulate its actions to coordinate the response of bacteria to environmental challenges. This phenomenon can be denoted as quorum sensing (QS) (Raina et al., 2010). Previous studies have shown that QS plays a significant role in coordinating biofilm formation, extracellular polymeric substance (EPS) production and microbial community structure (Valle et al., 2004; Wang et al.,

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2012), thus providing a new perspective for facilitating the adhesion and aggregation of nitrifying bacteria and reducing the loss thereof. N-Acyl-homoserine lactone (AHL) is a type of QS chemical, and many Gram-negative bacteria are able to produce AHLs. Nitrosomonas europaea, known as an ammonia oxidation bacterium (AOB), can produce AHL molecules, including 3-oxo-C6-HSL, C6HSL, C8-HSL and C10-HSL, in wastewater treatment systems (Batchelor et al., 1997; Burton et al., 2005). Additionally, dosing with AHL molecules (e.g., 3-oxo-C6-HSL, C8-HSL) can increase the biomass of nitrifying biofilm and accelerate the recovery of damaged biofilm (Batchelor et al., 1997; Gamage et al., 2011). Although the QS effect of pure species of nitrifying bacteria on cell adhesion and biofilm formation has been characterized, the role of AHLs on the attached-growth and nitrification efficiency of activated sludge with a complex microbial community remains unknown. The AHLs with appropriate molecular structures will likely improve the microbial activity of nitrifying sludge, regulate the yield of EPSs for cell adhesion and strengthen the formation of nitrifying granular sludge, thus improving nitrification performance. QS signal chemicals are often referred to as auto-inducers and can be classified based upon their molecular structures. All AHLs contain an acyl-homoserine lactonic ring, which can have different acylation branched chains in terms of the lengths of side chains and b position substituent groups (i.e., hydrogen, carbonyl or hydroxyl) (Fuqua et al., 1996). In this study, six AHL chemical molecules with different molecular structures (C6-HSL, C8-HSL, C10-HSL, 3-oxo-C6-HSL, 3-oxo-C8-HSL and 3-oxo-C10-HSL) were added to an autotrophic nitrifying sludge system to be tested separately. The influence of the AHLs on cell adhesion, nitrification efficiency and the granulation process of the sludge was investigated. In addition, the distribution of EPSs and the microbial community structure of nitrifying granules dosed with different AHLs were examined. The aims of the experimental study were to gauge the importance of AHL chemicals in the granulation of autotrophic nitrifying sludge and provide a strategy for achieving rapid sludge granulation. 2. Methods 2.1. AHL reagents for dosing N-Hexanoyl-L-homoserine lactone (C6-HSL), N-octanoyl-Lhomoserine lactone (C8-HSL), N-decanoyl-L-homoserine lactone (C10-HSL), N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6HSL), N-(3-oxooctanoyl)-L-homoserine lactone (3-oxo-C8-HSL) and N-(3-oxodecanoyl)-L-homoserine lactone (3-oxo-C10-HSL) were purchased from Sigma–Aldrich (mainland China) and stored at 20 °C.

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50 min of sludge settling and 4 min of effluent withdrawal from the middle port of the column. Aeration was conducted at an airflow rate of 1.0 L/min during the aeration phase. The experiment was performed at room temperature, and the water temperature was 20–22 °C. NaHCO3 was dosed into the feed solution to maintain the pH of the reactor in the range between 7.5 and 8.0. The activated sludge from a full-scale sewage treatment plant (Xiaojia River, Beijing, China) was used as the seed sludge. Initially, the ammonia degradation efficiency was only approximately 80% at the initial low feeding load of 50 mg NH+4-N/L. Then, the concentration of ammonia nitrogen in the feeding wastewater was gradually increased to 500 mg/L during the 300 days of operation. The ratio of nitrifying bacteria in the cultured sludge increased, and nitrification gradually improved. Finally, the efficiency of the ammonia degradation was approximately 100% for the cultured nitrifying sludge. 2.3. Adhesion test of autotrophic nitrifying sludge dosing with different AHLs The effect of the AHLs on the adhesion ability of autotrophic nitrifying sludge was investigated following a cell adhesion test protocol used in the literature (Gamage et al., 2011; Lv et al., 2014). First, the sludge was collected from the SBR and homogenized at 10,000 rpm for 30 s. Then, the sludge suspension was diluted with fresh phosphate buffer solution (PBS), and 5 mL of sludge suspension with an OD600 value of approximately 0.2 and a NH+4-N concentration of 100 mg/L was pipetted into each well of a 6-well flat-bottomed plastic plate (/ 34.8 mm  D 20 mm). Six AHLs (C6-HSL, C8-HSL, C10-HSL, 3-oxo-C6-HSL, 3-oxo-C8-HSL and 3-oxo-C10-HSL) were tested in the plates using a concentration of 2 lM. Six replicate wells were used for each analysis for each of the six AHLs. The negative control well did not contain AHL. All plates were statically incubated at 30 °C. The attached biomass in the well was quantified after incubation under two different conditions: (1) incubation for 12 h and (2) incubation for 24 h. Ammonia and AHLs were added to the well again simultaneously after the first 12 h for the 24-h sample. The attached biomass was quantified according to the method reported by Stepanovic et al. (2000) and Taweechaisupapong et al. (2005). Each well of the 6-well plates was washed with PBS repeatedly to remove all non-adherent cells. The plates were dried at 50 °C. A 5-mL aliquot of 99% methanol was used in each well to fix the remaining attached bacteria for 15 min. Then, the plates were emptied and dried again. A 3-mL aliquot of 2% Hucker crystal violet was added to each well for Gram staining and incubated for 30 min. Excess dye liquor was then removed under running tap water, and the plates were dried. Finally, 5 mL of 33% (v/v) glacial acetic acid was dosed into each well to dissolve the dye bound to the attached bacteria for 1 h, and the OD value was measured at 600 nm with an enzyme immunosorbent assay reader (OD600).

2.2. Cultivation of autotrophic nitrifying sludge The nitrifying activated sludge was cultivated in the sequencing batch reactor (SBR) for 300 days. The influent into the reactors was a synthetic inorganic nitrogen wastewater prepared with NH4Cl and other nutrients without any organic substrates added. The compositions of the main nutrients and micronutrients in the synthetic wastewater were as follows (per liter): 4.89 mg of MgSO4, 7.5 mg of CaCl22H2O, 2.40 mg of FeCl3, 8.75 mg of KH2PO4, 26.84 mg of Na2HPO412H2O, 12.5 lg of H3BO3, 15.85 lg of CuCl22H2O, 12.5 lg of MnSO4H2O, 12.5 lg of (NH4)6Mo74H2O, 12.5 lg of AlCl3, 12.5 lg of CoCl26H2O and 12.5 lg of NiCl2. The SBR column was 6 cm in diameter and 85 cm in height with a working volume of 2.4 L, which was operated in a fixed sequential mode for a 4-h cycle with 4 min of feeding, 182 min of aeration,

2.4. Determination of the nitrification ability of cultured sludge dosing with different AHLs To evaluate the effect of AHLs on the nitrification ability of the sludge, 100 mL of sludge suspension with an OD600 of 0.2 and a concentration of 120 mg/L NH+4-N was dosed into a 250-mL Erlenmeyer flask and then incubated at 130 rpm in an orbital shaker at 30 °C. One of the six AHLs (C6-HSL, C8-HSL, C10-HSL, 3-oxoC6-HSL, 3-oxo-C8-HSL and 3-oxo-C10-HSL), each with a concentration of 2 lM, was also added to one of six separate flasks. The experiments were performed in duplicate. The sludge mixture was sampled at various time intervals, and the concentrations of  nitrogen in various forms (i.e., NH+4-N, NO 3 -N and NO2 -N) in the liquid phase of the sludge were measured. The mass-balance

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equation for NH+4-N in the reactor may be written as (dS/dt) V = QS0  QS + rV, where S is the concentration of NH+4-N, S0 is its initial concentration, t is time and r is the rate of ammonia removal. Because Q = 0 for a batch reactor and a first-order correlation r = kS may be assumed for the early phase of NH+4-N uptake where k is the NH+4-N uptake rate coefficient, the rate of NH+4-N removal may be approximated by dS/dt = kS. The maximum NH+4-N degradation rate coefficient of the sludge can be estimated based on the linear regression of ln(S0/S) versus t. The coefficient data were evaluated using an analysis of variance, and the results were considered to be significant at p < 0.05. 2.5. Granulation test of autotrophic nitrifying sludge dosing with different AHLs Three identical columns (H 16 cm  D 4 cm with a working volume of 200 mL) were used as batch reactors for the sludge granulation tests. Nitrifying activated sludge from the SBR was washed with PBS and inoculated into the three batch reactors at an initial MLSS concentration of 2.359 ± 0.091 g/L and a mean size of 168.5 lm. The sequential operating cycle for each reactor was 24 h, and the ammonia nitrogen loading was 0.5 kg/m3 d. Air was supplied from the bottom of the reactors at a flow rate of 1.0 L/ min during the aeration phase. After a predetermined period of sedimentation, e.g., 10 min, the effluent and slow-settling sludge were withdrawn from each column, resulting in a volumetric exchange ratio of 85% per cycle for each reactor. By adjusting the sedimentation time, the ratio of sludge discharge from each reactor was maintained at approximately 3% every day. NaHCO3 was added to the feed to maintain the solution pH in the reactors in the range of 7.0–8.0. The reactors were operated at room temperature and a water temperature of 23–25 °C. No AHL reagent was dosed into one of the three reactors, which was used as the control. The main difference between the other two reactors lay in the addition of AHLs. The AHL reagents C6-HSL and 3-oxo-C6-HSL were dosed into separate reactors once a day at a concentration of 2 lM. 2.6. Analytical methods The sludge MLSS, MLVSS and ESS were measured according to the Standard Methods (APHA, 2005). The concentrations of ammonia, nitrate and nitrite were determined by the Nesslerization, ultraviolet, and colorimetric spectrophotometric methods, respectively. Sludge morphology was examined with a microscope (Olympus B41) equipped with a digital camera (Olympus C-5060 Wide Zoom). DO and pH were monitored with dissolved oxygen meters (FG4, METTLER TOLEDO) and a pH meter (PHBJ-260, Leici), respectively. The particle size distribution (PSD) of the sludge was measured using a laser particle size analyzer (Microtrac S3500, USA). The EPSs of the sludge were obtained by the heat extraction method, and the EPS extraction was used for the quantitative analysis of polysaccharides and proteins (Li et al., 2011). Moreover, Fourier transform infrared spectroscopy (FTIR) was used to analyze the functional groups of EPSs (Si et al., 2014). A microbial community analysis was performed by Illumina MiSeq sequencing. The granular sludge in the three batch reactors was collected and concentrated by centrifugation (13,400g, 5 min). The supernatant was then discarded, and the cell pellets were stored at 80 °C until further processing. Microbial DNA was extracted from sludge samples using the E.Z.N.A.Ò Bacteria DNA Kit (Omega Bio-Tek, Norcross, GA, U.S.) according to the manufacturer’s protocols. The V3–V4 region of the bacteria 16S ribosomal RNA gene was amplified by PCR (95 °C for 2 min, followed by 25 cycles at 95 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s and a final extension at 72 °C for 5 min) using primers 338F 50 -barcode-ACT

CCTACGGGAGGCAGCA)-30 and 806R 50 -GGACTACHV-GGGTWTCT AAT-30 , in which the barcode is an eight-base sequence unique to each sample. PCR reactions were performed in triplicate using a 20-lL reaction mixture containing 4 lL of 5 Fastpfu Buffer, 2 lL of 2.5 mM dNTPs, 0.8 lL of each primer (5 lM), 0.4 lL of Fastpfu Polymerase, and 10 ng of template DNA. Amplicons were extracted from 2% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, U.S.) according to the manufacturer’s instructions and quantified using QuantiFluorTM-ST (Promega, U.S.). Purified amplicons were pooled in equimolar and paired-end sequenced (2  250) on an Illumina MiSeq PE300 platform according to the standard protocols by Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd., Raw fastq files were demultiplexed and quality-filtered using QIIME (version 1.17) with the following criteria: (i) the 250-bp reads were truncated at any site receiving an average quality score of <20 over a 10-bp sliding window, discarding the truncated reads that were shorter than 50 bp. (ii) Exact barcode matching, 2 nucleotide mismatch in primer matching, and reads containing ambiguous characters were removed. (iii) Only sequences that overlap longer than 10 bp were assembled according to their overlap sequence. Reads that could not be assembled were discarded. Operational Units (OTUs) were clustered with a 97% similarity cutoff using UPARSE (version 7.1 http://drive5.com/uparse/), and chimeric sequences were identified and removed using UCHIME. The phylogenetic affiliation of each 16S rRNA gene sequence was analyzed by an RDP Classifier (http://rdp.cme.msu.edu/) against the silva (SSU115)16S rRNA database using a confidence threshold of 70% (Amato et al., 2013).

3. Results and discussion 3.1. Effect of AHLs on the attached growth of autotrophic nitrifying sludge After dosing with six AHL molecules, the densities of nitrifying sludge attached-growth on the plastic plates were all significantly higher than that of the control, which indicated that the addition of signal molecules has a positive effect on adhesion growth of autotrophic nitrifying sludge (Fig. 1). The 12-h cultivation of the sludge demonstrated that the addition of any AHL molecule increased the biomass with the attached-growth model by 0.44–0.63 times, with the exception of 3-oxo-C8-HSL, which had only a slight promotional effect. The 24-h cultivation period demonstrated that the attached-growth ability of the sludge could be further enhanced. Dosing with 3-oxo-C6-HSL yielded the highest biomass density on the plastic plate, and the attached cells were 2.56 times that of the control. The addition of C10-HSL, 3-oxo-C8-HSL and 3-oxoC10-HSL could also promote the adhesion of autotrophic nitrifying sludge to a certain degree, and the attached biomass was approximately 2 times that of the control. Both C8-HSL and C6-HSL had a slight positive influence, and the attached biomass was only 1.47 and 1.55 times that of the control, respectively. In summary, dosing with 3-oxo-C6-HSL had the most significant enhancement for the transformation of the autotrophic nitrifying sludge from the suspended-growth mode to the attached-growth mode. In the case of dosing with AHL molecules without b-position substituent groups (including C6-HSL, C8-HSL and C10-HSL), as the length of the N-group side chain increases, it further enhances the adhesion growth of the sludge. In contrast, if the b-position substituent group of AHL added is a carbonyl group (including 3oxo-C6-HSL, 3-oxo-C8-HSL and 3-oxo-C10-HSL), as the length of the N-group side chain decreases, the adhesion growth ability of the sludge grows stronger.

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C -HSL 6

C -HSL 8

C -HSL 10

3-oxo-C -HSL 3-oxo-C -HSL 3-oxo-C -HSL 6 10 8

Fig. 1. AHLs addition enhanced the adhesion of autotrophic nitrifying sludge.

AOB in nitrifying activated sludge has been reported to secrete the AHL molecules C6-HSL, C8-HSL and C10-HSL (Burton et al., 2005). Furthermore, the biofilm formation of AOB was regulated by the AHL quorum sensing system (Gao et al., 2014; Batchelor et al., 1997). Additionally, several researchers have shown that the addition of 3-oxo-C6-HSL, C6-HSL, 3-oxo-C8-HSL or C8-HSL molecules could improve adhesion growth and biofilm formation of several Gram-negative bacteria (Morohoshi et al., 2007; Liu et al., 2011). Consequently, it is reasonable that the addition of these six AHLs, particularly 3-oxo-C6-HSL, could enhance the attached-growth of nitrifying activated sludge and prevent the loss of nitrifying bacteria from the bioreactor. Then, the biomass accumulation of autotrophic nitrifying sludge would contribute to improving the nitrification performance. 3.2. Effect of AHLs on the nitrification process of autotrophic nitrifying sludge Adding AHL signal molecules could also improve the nitritation (ammonia oxidation to nitrite) efficiency of autotrophic nitrifying sludge (Fig. 2). Compared with the control, the addition of the signal molecules C6-HSL, 3-oxo-C6-HSL, C8-HSL, 3-oxo-C8-HSL and C10-HSL all accelerated ammonia degradation (Fig. 2a) and the accumulation of nitrite in a short period (Fig. 2b), indicating that the five signal molecules played a clear role in enhancing ammoxidation. Ammonia degraded rapidly from the initial concentration of 120 mg/L to below 30 mg/L after 3 h of cultivation in all bioreactors. However, the maximum ammonia degradation rate coefficients of the five signal molecules were 0.704/h (C6-HSL), 0.652/h (3-oxo-C6-HSL), 0.568/h (C8-HSL), 0.549/h (3-oxo-C8-HSL) and 0.539/h (C10-HSL); all of which was higher than that of the control (0.510/h) using the same biomass concentration (Fig. 2d). The maximum ammonia degradation rate coefficient upon the addition of 3-oxo-C10-HSL was only 0.476/h, which showed that the addition of 3-oxo-C10-HSL has no positive impact on the ammonia

degradation of the sludge. Moreover, following the degradation of ammonia, the nitrite concentration in each bioreactor rose gradually and reached the maximum value in the period from 2 to 3 h. One possible reason for this phenomenon might be related to the DO concentration in the bioreactor. Nitrite-oxidizing bacteria (NOB) are more sensitive than AOB with the DO limitation during the nitrification process. During the first 3 h, oxygen was mainly used to oxidize ammonia to nitrite by AOB. Shown in Fig. 2a, the ammonia concentration decreased from approximately 120– 30 mg/L during the first 3 h. During this stage, it is likely that no sufficient amount of oxygen could be utilized by NOB to oxidize nitrite to nitrate. Therefore, nitrite accumulated until the ammonia concentration decreased to a low level after 3 h and NOB outcompeted AOB to obtain ample oxygen to fulfill the total nitrification. In addition, the nitrite concentration of the bioreactors that contained signal molecules was considerably higher than that of the control, and the signal molecules with nitrite concentrations listed from high to low in each bioreactor are C6-HSL, 3-oxo-C6-HSL, C8HSL, 3-oxo-C8-HSL, C10-HSL and 3-oxo-C10-HSL. This result also indicated that the addition of signal molecules improved ammoxidation. The rapid recovery of ammoxidation for the N. europaea biofilm might be due to the production and accumulation of 3oxo-C6-HSL (Batchelor et al., 1997). Moreover, ammonia degradation efficiency has a closer correlation with the chemical structure of the AHLs added to the bioreactors. As the length of the AHL Nside chain decreases, its contribution in improving ammonia degradation increases. The AHLs without the b-position substituent group had a greater effect on the promotion of ammonia degradation compared to the AHLs of the same side chain length but with a carbonyl as the substituent group. There was only a slight difference in the nitrate concentration of each bioreactor that contained signal molecules and the control bioreactor (Fig. 2c), which suggested that the six signal molecules did not affect nitratation (nitrite being oxidized to nitrate) of the autotrophic nitrifying sludge.

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 Fig. 2. AHLs addition improved nitrification process of autotrophic nitrifying sludge: (a) NH+4-N concentration; (b) NO 2 -N concentration; (c) NO3 -N concentration; (d) the maximum ammonia degradation rate coefficient. Asterisks indicate statistical differences (p < 0.05) from the control.

3.3. Effect of AHL addition on the characteristics of the nitrifying granular sludge Based on the above experimental results, 3-oxo-C6-HSL had the most significant effect on enhancing the attached growth of autotrophic nitrifying sludge, whereas C6-HSL contributed the most to increasing the ammonia degradation efficiency. Therefore, these two signal molecules were selected for the following sludge granulation test. Either 3-oxo-C6-HSL or C6-HSL was added to the bioreactor during the granulation process, and the effects of the two AHL molecules on the formation and biochemical characteristics of nitrifying granular sludge were investigated. 3.3.1. Morphology and size The formation of nitrifying granular sludge can be promoted through dosing with AHL signal molecules (Fig. S1,

Supplementary Materials). Although the conditions in the three batch reactors were otherwise identical in terms of the ammonia loading, seed biomass and sludge discharge ratio, two different AHL molecules were added to two different reactors, and the third reactor that did not contain AHL was used as the control. The sludge size produced with AHL addition was significantly larger than that of the control after 30 days (Fig. S2, Supplementary Materials). As a result, granulation was achieved at rather different rates in the three reactors. As shown in Fig. S1, the sludges in the three batch reactors were all aggregated to a certain extent after 30 days compared to the loose flocs of seed sludge (with a mean size of approximately 168.5 lm). As the selective sludge discharge is adopted, loose and slow-settling flocs were discharged selectively every day during operation. The mean size of the sludge without the addition of the signal molecule only reached 208.1 ± 5.2 lm after 30 days. In contrast, granular sludge with a

Table 1 The characteristics of autotrophic nitrifying sludge in the three batch reactors after running 30 days.

MLSS/(g L1) MLVSS/(g L1) VSS/SS Mean size(lm) Average ESS/(gSS L1 d1) Average growth rate/(gSS L1 d1)

Control

C6-HSL

3-Oxo-C6-HSL

2.57 ± 0.06 1.39 ± 0.03 0.541 208.1 ± 5.2 0.08 0.126

3.52 ± 0.07 2.26 ± 0.03 0.644 309.3 ± 10.5 0.075 0.260

3.57 ± 0.14 2.68 + 0.21 0.751 356.7 ± 7.6 0.07 0.227

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regular surface and compact structure was apparent in the bioreactors to which C6-HSL or 3-oxo-C6-HSL was added, with mean sizes of 309.3 ± 10.5 and 356.7 ± 7.6 lm, respectively (Table 1). Considering that the ratio of sludge discharge from each reactor remained the same (approximately 3% every day), dosing with AHLs clearly facilitated the formation of granular sludge. Selective sludge discharge through controlling sludge settling time removes loose sludge flocs in suspended-growth mode and enhances the feeding of substrates to the biomass of attached growth, leading to granulation (Li and Li, 2009). However, the strategy of enhanced sludge washout is not suitable for the granulation of nitrifying sludge, particularly for treating ammonia inorganic wastewater. Due to slow growth rate, nitrifying bacteria cannot be accumulated in a bioreactor with enhanced sludge washout because it would lead to nitrification failure. The sludge discharge ratio must be kept at a low level for promising nitrification performance (i.e., a controlled sludge discharge ratio of 3% was used in the present study), which leads to slower nitrifying granule formation, as reported previously (Wei et al., 2014). In this study, dosing with the AHLs was effective in accelerating the granulation process of autotrophic nitrifying sludge. The AHL signal chemicals had the ability to induce the gene expression of the bacteria for attached growth, which in turn facilitated the granulation of nitrifying sludge and the maintenance of granular structures. The increase of 3-oxo-C6-HSL, C6-HSL and 3-oxo-C8-HSL signals could accelerate the formation of biofilm and achieve rapid sludge granulation (Wang et al., 2014). Moreover, dosing with the cellular substance extracted from granular sludge that contains AHLs could accelerate the granulation process (Ren et al., 2010, 2013). Thus, the granulation of autotrophic nitrifying sludge can be facilitated through the addition of AHLs. The granulation process of nitrifying sludge can be greatly enhanced by increasing the intensity of the AHL signals in the early startup stage. The experimental results show that the addition of 3-oxo-C6HSL was more efficient than C6-HSL in accelerating the granulation process. This provided further evidence that 3-oxo-C6-HSL, which was advantageous in increasing both cell adhesion and ammonia degradation, was more conducive in the granulation of nitrifying sludge, followed by C6-HSL, which was only advantageous in terms of improving ammonia removal. In short, the addition of the AHL molecules that had the ability to facilitate cell attachment was the most efficient strategy for granulation. However, the AHL molecule with the function of accelerating ammonia degradation also contributed to the rapid growth of nitrifying bacteria in sludge aggregates and played a positive role in the formation of granular sludge. Selecting the AHL molecules able to improve bacteria in the attached-growth mode and nitrification efficiency will most likely achieve rapid granulation of autotrophic nitrifying sludge. 3.3.2. Growth rate and microbial activity The addition of AHL signal molecules significantly promoted the biomass accumulation of the autotrophic nitrifying sludge (Table 1). The concentration of the initial seed sludge was highly similar in the three reactors and approximately 2.36 ± 0.09 g/L, but the MLSS of the two reactors dosed with C6-HSL and 3-oxoC6-HSL reached 3.52 ± 0.07 and 3.57 ± 0.14 g/L, respectively. These values were both higher than that of the control reactor, which was 2.57 ± 0.06 g/L after 30 days of operation. The daily effluent sludge concentration of the three reactors during sequencing batch operation was generally maintained at approximately 0.075 g SS/L, indicating that the reactors with the added signal molecules contain more biomass after running for 30 days compared to the control reactor. Using calculations based on the daily controlled sludge discharge of each reactor, the mean growth rate of biomass dosing with C6-HSL and 3-oxo-C6-HSL was 0.260 and 0.227 g SS/L d, respectively, approximately twice that of the

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control (0.126 g SS/L d). The mean growth rate of the biomass with added C6-HSL was higher than that with added 3-oxo-C6-HSL, which is consistent with the result that the maximum ammonia degradation rate coefficient of C6-HSL addition was higher than that of 3-oxo-C6-HSL in the nitrification efficiency test (Fig. 2d). Moreover, the addition of signal molecules contributed to the improvement of the microbial activity of the granular sludge. The VSS/SS ratio of the granular sludge with C6-HSL and 3-oxo-C6HSL was 0.644 and 0.751, respectively; both of which were higher than the control value of 0.541, and the microbial activity of the granular sludge with 3-oxo-C6-HSL was the highest. Therefore, the addition of AHLs remarkably improved the growth rate and microbial activity of the autotrophic nitrifying sludge, enabling sludge aggregate or granular precursors to rapidly grow and provide a positive driving force for the formation of nitrifying granular sludge. 3.3.3. Microbial EPSs Nitrifying granular sludge dosing with AHLs (C6-HSL or 3-oxoC6-HSL) contained a larger amount of extracellular proteins than the sludge without AHLs. This was unlike the content of the polysaccharides in EPSs, which were similar regardless of whether AHLs were added (Fig. 3), suggesting that AHLs were positively correlated with the protein production of microorganisms in autotrophic nitrifying sludge. The EPSs of the sludge in the three reactors produced a similar change trend during the granulation process (Fig. 3). The extracellular proteins of the sludge in the three reactors increased from 15.9 ± 3.6 to 31.2 ± 3.3 (control), 74.6 ± 6.2 (dosing with C6-HSL) and 63.4 ± 3.9 (dosing with 3-oxo-C6-HSL) mg/g SS, respectively, after running for 30 days. The extracellular polysaccharides of sludge decreased from 47.2 ± 8.8 mg/g SS to a similar level of approximately 20 mg/g SS. The extracellular polysaccharides were considerably higher than the extracellular proteins in the seed sludge, and conversely, EPSs showed a higher protein content than polysaccharide content in the granular sludge in all three reactors. Proteins have been reported as the core EPSs constitutes of aerobic granules, and proteins are believed to be important building materials for the internal structure of granules (Chen et al., 2007). High concentrations of extracellular proteins can also be used by microorganisms in aerobic starvation to increase cell surface hydrophobicity (McSwain et al., 2004; Wang et al., 2005). Moreover, AHL-mediated QS has been reported to enhance the microbial attachment of granular sludge through the regulation of extracellular proteins (Lv et al., 2014). In fact, nitrifying granular sludge could be achieved more rapidly and could have a larger size when AHLs were added (Table 1). Therefore, AHL quorum sensing favored the production of more extracellular proteins from autotrophic nitrifying sludge, which enhanced the formation and structure stability of nitrifying granules. The above study indicated that the addition of AHLs and the granulation process resulted in a change in the EPS matrix. Thus, the effects of AHL addition and the granulation process on the chemical functional groups of EPS were further investigated using FTIR (Fig. 4). For the seed and granular sludges with no AHLs and for those dosed with AHLs, the FTIR spectra of EPS exhibited the same characteristic bands representing several functional groups, including the CH3 asymmetric stretch, the CH2 asymmetric stretch, the protein C@O stretch (Amide I), the CH2 bend, the COO symmetric stretch, the PO2 asymmetric stretch, the PO2 symmetric stretch and glycogen (Garip et al., 2009). These functional groups mainly represent lipids, polysaccharides, proteins and fatty acids. However, a new band appeared at 3423 cm1, which could be related to the stretching vibration of O–H from the polysaccharides or proteins that appeared after sludge granulation in all three reactors. These results are similar to the FTIR spectra for EPSs extracted from microbial granules (D’Abzac et al., 2010), indicating that the

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polysaccharides

proteins

EPS content (mg/g MLSS)

80

60

40

20

0 seed sludge

C -HSL 6

Control

3-oxo-C -HSL 6

Fig. 3. Extracellular polysaccharides and proteins of seed sludge and nitrifying granular sludge dosing with AHLs for 30 days.

100

Control

C6-HSL

3-oxo-C6-HSL

Seed Sludge

Transmittance (%)

80 1233

60

2960

1234 2929

1404

1074

1547 1238

2932 2962

40 3421

1406

1640

1075

2961 2930 1407

20

1077

1235

1634

3424 2960

1636

2932

3424

0 3500

1406

1639

3000

2500

2000

1076

1500

1000

wavenumber (cm-1) Fig. 4. The effects of AHLs addition and granulation process on the chemical functional groups of EPS after 30 days.

functional group at 3423 cm1 might be related to the granulation process and a result of the selective discharge of loose flocs. Additionally, a new band at 1547 cm1 was observed only in the granular sludge dosed with 3-oxo-C6-HSL. The band indicates the N–H bending and C–N stretching (Amide II) of proteins (Gomez et al., 2003), demonstrating that 3-oxo-C6-HSL dosing had a direct interaction with the extracellular proteins of autotrophic nitrifying sludge. Dosing 3-oxo-C6-HSL not only enhanced protein production but also changed the functional groups of the protein, which may likely contribute to the formation and maintenance of the nitrifying granules. 3.3.4. Microbial community structure The results of high-throughput sequencing showed that the addition of AHLs influenced the microbial population dynamics of nitrifying sludge during the granulation process (Fig. 5). Different dominant genera of Paracoccus, Hyphomicrobium and Methylophaga within the granule communities dosed with C6HSL, 3-oxo-C6-HSL or no ASL were confirmed after 30 days. The

proportion of Paracoccus in the granular sludges with C6-HSL and 3-oxo-C6-HSL was 14.9% and 13.2%, respectively, lower than that of the control of 18.1%. However, the proportion of Hyphomicrobium in granular sludges with C6-HSL and 3-oxo-C6HSL increased by 9.8% and 7.1%, respectively. The proportion of Methylophaga also increased greatly (by 2.7% and 7.6%, respectively) compared with that of the control. As Lemmer et al. (1997) suggested, Paracoccus favored aerobic conditions, whereas Hyphomicrobium favored anoxic conditions in the biofilm. Thus, Paracoccus might be easy to grow and become dominant in the comparatively loose granular precursor in the control reactor. Conversely, larger and tighter granules in the bioreactors with AHLs might exist in the anoxic environment of the inner granules, which likely benefitted the dominance of Hyphomicrobium. Hyphomicrobium and Methylophaga are denitrifying bacteria commonly found in biofilm (Auclair et al., 2012; Villeneuve et al., 2013); thus, it is possible that the two species can sustain the structure of granules and hence improve the formation and maturation of the granules. Dosing with C6-HSL was more conducive to

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100

Relative abundance (%)

80

60

40

20

0

Control

C -HSL 6

others

Bosea

Sandaracinaceae_norank

Alphaproteobacteria_unclassified

Sphingobium

Aeromicrobium

Saprospiraceae_uncultured

Comamonadaceae_unclassified

Methylophaga

Nitrobacter

Shinella

Bradyrhizobiaceae_unclassified

Gelidibacter

Chryseolinea

Leadbetterella

SM1A02

Methylobacteriaceae_uncultured

Phyllobacteriaceae_unclassified

Flavobacterium

Leifsonia

Brevundimonas

Hyphomicrobium

Rhodobacteraceae_unclassified

Nitrosomonas

Anaerolineaceae_uncultured

Paracoccus

3-oxo-C -HSL 6

Fig. 5. The effect of AHLs addition on the microbial population structure of granular sludge.

the growth of Hyphomicrobium, whereas the addition of 3-oxo-C6HSL was more beneficial to the enrichment of Methylophaga. Moreover, C6-HSL did not have a significant effect on the distribution of Chryseolinea in sludge, whereas 3-oxo-C6-HSL inhibited the growth and accumulation of Chryseolinea. Moreover, a dendrogram was produced to compare the microbial population structures between different sludge samples (Fig. S3, Supplementary Materials). Granules with two different AHL additions were more closely related to each other and were distinct from precursor granules in the control reactor. This suggested that AHL addition could change the microbial structure of sludge significantly. Consequently, addition of the AHLs with different chemical structures could lead to a different microbial community structure of granular sludge. Although the proportions of AOB Nitrosomonas in the three reactors were equivalent and reached approximately 13%, the Nitrosomonas biomass in the reactors with either C6-HSL or 3oxo-C6-HSL was both higher than that of the control reactor due to the higher total biomass with AHL addition (Table 1). Thus, AHL addition contributed to the growth and accumulation of AOB in reactors, which could improve the nitritation performance. The NOB Nitrobacter in the three reactors accounted for 3.5% (the control), 4.0% (3-oxo-C6-HSL) and 4.3% (C6-HSL), which is consistent with the result of the nitrification efficiency test of nitrifying sludge (Fig. 2). In the nitrification efficiency test, the nitrite concentration in solutions from high to low at the time of maximum nitrite enrichment were C6-HSL, 3-oxo-C6-HSL and the control, which further demonstrates that the two signal molecules could promote ammonia degradation and accumulate more nitrite in a short time as nutrition for Nitrobacter growth and accumulation. The operating conditions of the three reactors were the same with the exception of the fact that C6-HSL and 3-oxo-C6-HSL were added to two reactors during the nitrifying sludge granulation. AHL addition affected the microbial community structure of the nitrifying sludge. The addition of AHL molecules increased the biomass of AOB and NOB and improved the growth of several heterotrophic bacteria, which likely played an important role in the formation and growth of nitrifying granules and helped sustain the granular sludge structure in the bioreactors. 4. Conclusions AHL addition may improve the attached-growth of autotrophic nitrifying sludge and ammonia degradation. The efficiencies of cell adhesion and ammonia removal both had close correlations to the

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