Science of the Total Environment 612 (2018) 405–414
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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv
Estimation of spatial distribution of quorum sensing signaling in sequencing batch biofilm reactor (SBBR) biofilms Jinfeng Wang, Lili Ding, Kan Li, Hui Huang, Haidong Hu, Jinju Geng, Ke Xu, Hongqiang Ren ⁎ State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210023, Jiangsu, PR China
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Biofilms has been divided into three different fractions by centrifugal forces; • SEPS and LB EPS concentration in different fractions of biofilms displayed significant positive relationship with the distribution of C12-HSL; • Biofilm adhesion and compliance was the strongest in the tightly-bound biofilm, the weakest in the supernatant/ surface biofilm; • QS and QQ bacteria have been recognized in the wastewater treatment biofilms.
a r t i c l e
i n f o
Article history: Received 7 June 2017 Received in revised form 31 July 2017 Accepted 31 July 2017 Available online xxxx Editor: D. Barcelo Keywords: Biofilms Quorum sensing (QS) Quorum quenching (QQ) Extracellular polymeric substances (EPS) Mechanical properties Acylated homoserine lactones (AHLs)
a b s t r a c t Quorum sensing (QS) signaling, plays a significant role in regulating formation of biofilms in the nature; however, little information about the occurrence and distribution of quorum sensing molecular in the biofilm of carriers has been reported. In this study, distribution of QS signaling molecules (the acylated homoserine lactonesAHLs, and AI-2), extracellular polymeric substances (EPS) and the mechanical properties in sequencing batch biofilm reactor (SBBR) biofilms have been investigated. Using increased centrifugal force, the biofilms were detached into different fractions. The AHLs ranged from 5.2 ng/g to 98.3 ng/g in different fractions of biofilms, and N-decanoyl-DL-homoserine lactone (C10-HSL) and N-dodecanoyl-DL-homoserine lactone (C12-HSL) in the biofilms obtained at various centrifugal forces displayed significant differences (p b 0.01). Interspecies communication signal autoinducer-2(AI-2) in the biofilms ranged from 79.2 ng/g to 98.3 ng/g. Soluble EPS and loosely bound EPS content in the different fractions of biofilms displayed significant positive relationship with the distribution of C12-HSL (r = 0.86, p b 0.05). Furthermore, 49.62% of bacteria in the biofilms were positively related with AHLs with 22.76% was significantly positively (p b 0.05) related with AHLs. Biofilm adhesion and compliance was the strongest in the tightly-bound biofilm, the weakest in the supernatant/surface biofilm, which was in accordance with the distribution of C12 HSL(r = 0.77, p b 0.05) and C10-HSL(r = 0.75, p b 0.05), respectively. This study addressed on better understanding of possible methods for the improvement of wastewater bio-treatment through biofilm application. © 2017 Elsevier B.V. All rights reserved.
1. Introduction
⁎ Corresponding author. E-mail address:
[email protected] (H. Ren).
http://dx.doi.org/10.1016/j.scitotenv.2017.07.277 0048-9697/© 2017 Elsevier B.V. All rights reserved.
In recent years, biofilm, in terms of dense and diverse microbial communities encased in a secreted polymer matrix, has attracted researchers' growing interests for its high efficiency of pollutants
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removal, to upgrade the capacity in Anaeroxic-Anoxic-Oxic (A2O) process in wastewater treatment systems (Bassin et al., 2012; Nadell et al., 2016; Rikmann et al., 2017). Key characteristics of biofilm used for wastewater treatment are thickness (Torresi et al., 2016), extracellular polymeric substances (EPS) (Flemming and Wingender, 2010), tolerance (Lewis, 2006), mechanical properties (Persat et al., 2015). Interestingly, chemical signals which are also used for intercellular communication, i.e., quorum sensing (QS), a mechanism controls biofilm development and the growth of bacterial populations (Williams et al., 2007), has drawn the attention of scientists and has been investigated in the special forms of biofilm, i.e., sludge (Tan et al., 2015; Tan et al., 2014). Quorum sensing communities also perform nitrogen shortcut technologies by Pseudomonas organism-like biofilms and autotrophic bacteria offering significant cost savings over traditional biological nitrogen removal (Zekker et al., 2015; Zekker et al., 2016). Furthermore, some function microbial, such as Pseudomonas (Daija et al., 2016), which uses quorum-sensing signaling systems (Chugani et al., 2001), was used as seed for many water treatment systems (Raudkivi et al., 2017; Zekker et al., 2016). However, the mechanism by which QS regulated formation of biofilm in wastewater treatment process, and the complex relation among QS, biofilm thickness and bacterial community composition needs further investigation. Biofilm formation and microbial attachment on the bio-carriers are multiple-step processes. Physicochemical forces (hydrodynamic force, gravity force, etc.) and biological forces (production of extracellular polymer, growth of bacteria clusters, etc.) play significant roles in these processes (Boelee et al., 2011). A good understanding of the mechanical forces of biofilms which provide mechanical stability of microorganisms requires additional structural and compositional analysis of the samples (Persat et al., 2015). Mechanical properties of different types of wastewater treatment biofilms have been studied under various modes of loading as given in a comprehensive review by Safari et al. (2015). The significant variations in the reported data were not surprising as different bacterial species produced various types of EPS under different environmental conditions (Williams and Bloebaum, 2010). EPS immobilize microbes in biofilms and keep them in close proximity, thus providing a continuous on–off mechanism for modifying the concentration of these molecules in the biofilm matrix. The matrix are consisting of polysaccharides, proteins, lipids and extracellular DNA(eDNA), released by microbes and adhered onto the cell surfaces of activated sludge, granular sludge, and biofilms in wastewater treatment (Flemming and Wingender, 2010; Yu et al., 2008). PellicerNàcher et al. (2013) assayed EPS in sludge flocs, and their findings indicated that EPS were composed of soluble EPS (SEPS) and bound EPS, which can be further classified into loosely bound extracellular polymeric substances (LB-EPS) and tightly bound EPS (TB-EPS). The adhesion of SEPS to cells is weak; as a result, SEPS are often dissolved in solution. Furthermore, TB-EPS have a certain morphology while LBEPS have no distinct boundary (Liu and Fang, 2002). Despite the complexity of biofilm matrix and communities, cell–cell interactions and communication have played significant roles in biofilm spatial structuring (Battin et al., 2016). Biofilm formation enables effective intercellular communication, either using chemical or electrical signals, even in habitats where signaling molecules are not contained by the biofilm would be readily diffused (Flemming et al., 2016). It has been suggested that the production of QS signal chemicals from biofilms induces the gene expression of bacteria in suspensions to enable attached growth rather than suspended growth (Ren et al., 2010). Numerous QS signal molecules and circuits involved in acylated homoserine lactones (AHLs) have been identified and genes regulated by QS have been defined in a diverse group of bacteria genera, which are closely related with EPS production in natural and manmade systems (Tan et al., 2014). Furthermore, quorum quenching (QQ) bacteria, which is possible to degrade the QS signal by secretion of certain enzymes, has drawn close attention. QQ bacterial were found to be distributed in the sludge environment (Tan et al., 2015). Indeed, the complex
relationship between QS and QQ needs further investigation. However, the majority of previous research studies have mainly focuses on the effect of QS on biofilm attachment on pure cultures or mechanism of bacteria granulation at bench scale, rather than in the real packing (Hu et al., 2016; Tan et al., 2014). Moreover, whether wastewater treatment or bioremediation can be enhanced and optimized by promoting specific QS signaling pathways for beneficial biofilms, is still unknown and requires further research, especially for the relationship among QS signal distribution, biofilm structure and energy efficiency. Up to now, the studies of mature biofilms have been focused on the whole character, but the heterogeneity of biofilms makes it complex and hard to evaluate different properties of different parts of the biofilm. A systemic investigation was necessary to present the inside world of biofilm and to bring a better understanding of wastewater treatment biofilms. In this study, the SBBR biofilm have employed and detached into different fractions. We aimed to investigate the distribution of signals (acylated homoserine lactones-AHLs, and autoinducer-2-AI-2) in the fractions of biofilm. And the role of QS signaling in the production of EPS, communities assemble and further highly complex biofilm mechanical properties has been investigated. 2. Material and methods 2.1. The sequencing batch biofilm reactor (SBBR) operation The bio-carriers had a 0.95 g/cm3 density, a 10 × 25 mm dimension and a specific area for biofilm growth of 460 m2/m3 (Jiangsu Yulong Environment Protection Co., Ltd., China) (Zhu et al., 2015). The bio-carriers were used for the SBBR experiments. Cylindrical SBBR had an effective volume of 9 L (45 cm height, 16 cm diameter). The cycle time of the reactor was 12 h, and the wastewater cycle was comprised of the following phases: 15 min feeding, 11 h 30 min aeration, and 15 min drainage. No settling phase was needed since the biomass was attached to plastic carriers (Zhu et al., 2015). The amount of carriers corresponded to a volume fraction of 35% (Vsupport/Vreactor). Moreover, an aerator was placed at one side of the bottom of the tank. The SBBR was initially fed with a synthetic medium and was inoculated with aerobic activated sludge collected from the secondary sedimentation tank of cyclic activated sludge technology (CAST) in Xianlin municipal WWTP in Nanjing, China. After 24 h, the inoculated sludge was discharged from the reactor when the microorganism of sludge adhered to the surface of carriers adequately. The reactor operated with synthetic wastewater and a detailed list of components is in Table 1. For the biofilm formation process, the concentration of 500 mg/L COD were selected as the higher concentration of municipal wastewater (Zhu et al., 2015), and shock resistance was conducted by a higher concentration of 1000 mg/L COD. For the SBBRs, The dissolved oxygen (DO) concentration was varied along the operational cycle, around 4.0–5.0 mg/L. The temperature was kept at 25 ± 2 °C, pH was maintained between 7.2 and 7.5 (Zhu et al., 2015). 2.2. Removal of the biofilm from the substratum at three different centrifugal speeds After 100-day reactor operation, the bio-carriers were harvested and used for further study. In order to examine the inside of biofilms, Table 1 The specific components of synthetic wastewater. Components
Concentration (mg/L)
Components
Concentration (mg/L)
Glucose NH4Cl KH2PO4 Na2CO3 FeCl3·3H2O CaCl2·2H2O
500.0 200.0 19.0 53.6 2.42 0.37
MgSO4·7H2O MnCl2·4H2O ZnSO4·7H2O CuSO4·5H2O CoCl2·6H2O Na2MoO4·2H2O
5.08 0.28 0.44 0.39 0.42 1.26
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centrifugal method was employed which was originated from Luo et al. (Luo et al., 2015). A different centrifugal force with a range from 500 to 12,000 × g (Avanti JXN-26, JS-13.1, Beckman Coulter, Inc., USA) was used to detach the biofilm into different fractions. Furthermore, time taken for the biofilm detachment process ranged from 1 to 10 min. Centrifuge tubes (Corning, PP, 50 mL, USA) with a diameter of 27 mm were used in this study and the schematic diagram for the biofilm detachment process was presented in Fig. S1. It is worth noting that biofilm were developed in the inner ‘pore’ of the bio-carriers, for the biofilm of the outer areas were detached by hydraulic shear and intercollision. At last, optimal time of 5 min and three centrifugal speeds have employed for the biofilm removal process. Furthermore, the biocarriers taken from bioreactor for centrifugal experiment need put into centrifuge tubes and centrifugal immediately. The samples, comprising of liquid and bio-carriers were centrifuged at 2000 × g for 5 min. The detached biomass were considered supernatant/surfacebiofilm. Then, the loosely attached biomass were detached from the bio-carriers by centrifugal speed of 5000 × g for 5 min and labeled with loosely-bound biofilm. Finally, the left biomass in the bio-carriers were detached with centrifugal speed of 10,000 × g for 5 min, and labeled with tightly-bound biofilm. Biomass weight and thickness of detached biofilm was presented in Fig. 1.
2.3. Signaling molecule (AHLs and AI-2) extraction and analysis 2.3.1. AHLs extraction and UPLC-MS/MS analysis 5 g of biofilm sample was detached from bio-carriers under a centrifugal force of 10,000 × g for 5 min. Biofilm AHLs extraction was performed using ultrasonic extraction method with Ethyl acetate under temperature of 30 °C, power of 150 W and for 60 min, and the extracting solution of almost 30 mL was concentrated to almost 5 mL by rotary evaporation (RV 10 control FLEX auto, IKA, Germany) and then purified by HLB columns (Waters, America) (Wang et al., 2016). 3 mL of methyl alcohol and 3 mL ultrapure water were added to each column separately, then almost 5 mL sample was added to column. After the washing step, the column was eluted with methanol of 5 mL and then diluted to 1 mL.
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Solid-phase extracts (SPE) were analyzed by UPLC-MS/MS (Xevo TQ-XS,Waters, American). All samples were analyzed by UPLC (BEH C18 column, 1.7 μm, 2.1 × 50 mm) at a flow rate of 0.2 mL/min. The mobile phase consisted of a linear gradient of solvent B (methanol with 0.1% formic acid) and solvent A (25 mM ammonium formate with 0.1% formic acid). Effluents were ionized by electrospray ionization under positive mode and detected using the multiple reaction monitoring approaches (Tan et al., 2014). Effluent was ionized by electrospray ionization (ESI) and detected in the positive ion mode (Decho et al., 2009; Morin et al., 2003), with in-source collision-induced dissociation (CID) detailed in Table S1, and the condition as follows: sheath gas (nitrogen, N2) 500 L/h; capillary voltage 3000 V; ion source temperature 150 °C. Selectivity was based on the MS/MS fragmentation of the molecular [M + H]+ ions, and on their relative intensities (Decho et al., 2009). Mass spectrometry was conducted in multiple reaction monitoring (MRM) mode using two characteristic transitions for each AHL as listed in Supplementary Table S1. Specific UPLC retention time, characteristic of precursor ion m/z and quantitation fragment ions were chosen as the reference for identifying various AHLs, which were listed in Table S1. A quantitation transition ion was employed to construct the standard curves with analyte peak areas integrated using MassLynx™ software (Waters Corporation, USA). For AHL quantification, matrixmatched standard curves, ranging from 0.5 to 200 μM, were constructed. Limits of detection (LOD) and quantification (LOQ) for all AHLs were calculated with a signal-to-noise ratio of 3 and 10 respectively. One day and day-to-day RSDs were conducted with the standard samples (50 μg/L). Furthermore, triplicate blank injections were performed between sample injections to avoid sample carryover. 2.3.2. AI-2 extraction and HPLC-FLD analysis Biofilms peeled off from bio-carriers were homogenated in NaCl (0.9%) solution and filtered through polytetrafluoroethylene (PTFE) membranes (Anpel Co., Shanghai, China) with a pore size of 0.22 μm. 400 μL pretreated liquid were transferred to a 2 mL autosampler vial (Agilent Inc., USA) containing an equal volume of DAN solution (200 mg/L with 0.1 M HCl). The samples were thoroughly mixed for 2 min and then incubated in water at 90 °C for 40 min. After derivatization, the products were cooled down, and analyzed by HPLC. AI-2 was determined by modified HPLC-FLD method as proposed by Song et al. (Song et al., 2014) revision. Briefly, 1200 HPLC system was employed with a reverse-phase column ZORBAX Eclipse XDB-C18 (250 mm × 4.6 mm, 5 μm). The detailed operation conditions followed Song et al. (Song et al., 2014). 2.4. EPS extraction and analysis Since the EPS matrix was shear sensitive, and centrifugation and ultrasound could be applied to selectively remove the different EPS layers (Eriksson et al., 1992; Yu et al., 2008). The EPS was divided soluble EPS, loosely bound extracellular polymeric substances (LB-EPS) and tightly bound EPS (TB-EPS), which was extracted from biofilms conducted under the same protocol as that established by Yu et al. (2008) with a minor modifications. Briefly, the biofilms were homogenated, and then the mixture settled for 1.5 h at 4 °C, after which the bulk solution comprising the supernatant was carefully collected by a siphon. The following detailed extraction procedures were in accordance with Derlon et al. (2008). Polytetrafluoroethylene (PTFE) membranes (Anpel Co., Shanghai, China) with a pore size of 0.45 μm were used to remove the particulates present in the supernatant, S-EPS, LB-EPS, and TB-EPS solutions.
Fig. 1. The detachment of weight and biofilm thickness under various centrifugal forces. 2000, 5000 and 10,000 represented the three different centrifugal forces, which presented the S, LB and TB biofilm, respectively. The data are presented as the means ± SD from six times for each determination. ANOVA is significant at p b 0.05 and p b 0.01 separately. Different capital letters indicate significantly different values among centrifugal forces at significant at p b 0.05, and different case letters indicate significantly different values among centrifugal forces at significant p b 0.01.
2.5. Lipopolysaccharide (LPS) extraction and analysis Biofilms peeled from bio-carriers were homogenated. 0.5 mL pretreatment mixtures and 0.5 mL 0.025 N HCl solutions were placed into autosampler vials (Agilent Inc., USA) and vortexed gently for 60 min.
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After acidification of the samples, solutions were filtered with polytetrafluoroethylene (PTFE) membranes (Anpel Co., Shanghai, China) with a pore size of 0.22 μm. 1,2-Diamino-4,5methylenedioxybenzene (MDB) (Nakamura et al., 1987) was used as derivating agent to determine LPS concentration in biosamples(Narita et al., 2005). 0.5 mL MDB solution (Hara et al., 1987) with a concentration of 0.6 mg/mL and 0.5 mL pretreated samples were mixed uniformly and kept in water at 80 °C for 180 min. After derivatization, the cooled down products were analyzed by HPLC. 10 μL samples were injected for analysis using a 1200 HPLC system equipped with a fluorescence detector (Agilent Inc., USA). Separation was achieved on an Agilent ZORBAX Eclipse XDB-C18 reverse-phase column (250 mm × 4.6 mm, 5 μm) set at 30 °C. The mobile phase contained methanol, water and acetonitrile with a proportion of 21:70:9 and at a flow rate of 1.0 mL/min. The excitation and emission wavelengths of the fluorescence detector were set at 367 nm and 446 nm, respectively. 2.6. Shear rheometry and atomic force microscopy (AFM) Kinexus ultra+ rheometer (Malvern, England) was used to perform creep experiments on biofilm samples. This type of rheometer is typically used to analyze the time dependent behavior of materials and thus is used for non-Newtonian fluids or plastic solids. Creep tests were conducted at a constant shear stress of 1 Pa for a period of 300 s, where the bob rotational speed was set at 0.5 rpm (a linear speed of 7.5 mm min−1). A gap size of 1 mm was used in the experiments (Safari et al., 2015). Atomic force microscopy (AFM) was used to characterize microbial surface in terms of structure and function with higher resolution, surface features and force measurements (Zhu et al., 2015). On the 100th day, the biofilm samples were observed using AFM after dried naturally. The adhesion force between tips and cells was analyzed by forcedistance measurement (Zhu et al., 2015). Images and force distance curves of the biofilm were recorded using an AFM (Multimode 8, Bruker Inc., Germany) and scanned in a contact mode using a silicon nitride cantilever with a spring constant of 0.2113 N/m at speed of 1.99 Hz with 256 × 256 resolutions (Ma et al., 2016). The entire process was repeated six times for each sample. 2.7. 16S rRNA gene amplicon sequencing The microbial community structures were investigated using MiSeq platform. Biofilm fractions for the 16S rRNA gene sequencing amplicon were processed as follows: DNA extraction, 16S rRNA gene PCR amplification and PCR products purification (Zhu et al., 2015). To amplify and sequence the V1V2 hypervariable region of the 16S rRNA gene (Pandrea et al., 2016; Salipante et al., 2014), forward primer (50AGAGTTTGATYMTGGCTCAG-30) and the reverse primer (50-TGCTGC CTCCCGTAGGAGT-30) were selected and different 8-base barcodes and a guanine were linked to the 50 end of each primer. Then the purified products were sent for sequencing using Illumina sequencing platform (MiSeq 2500, Illumina Inc., USA). The acquired data was processed by the software of Sickle and Mothur to remove the low quality sequences and reduce noises. Lastly, the filtered sequences were assigned to a taxon by an RDP classifier. 2.8. Other analysis methods Method used for pretreatment of biofilms for analyzing volatile solids (VS) was followed by Zhu et al. (2015). Chemical oxygen demand (COD), NH+ 4 -N and VS were determined according to standard methods (APHA, 2012). Polysaccharide was determined by phenol sulfuric acid method, and protein content was determined by the Bradford assay with bovine serum albumin (BSA) as a standard (Ma et al., 2016). Dissolved Oxygen (DO) and pH were determined by a portable water
quality determination meter (METTLER TOLEDO, IP67, Switzerland). Fluorescence excitation-emission matrix spectroscopy (EEMs) were measured using the method described in Huang et al. (2014). The morphology of mature biofilms was observed through an inverted microscope (Nikon Ti2000, Japan) with a 2× objective lens and recorded via a CCD camera. Furthermore, Environmental scanning electron microscopy (ESEM) (FEI, Quanta 200FEG, Netherlands) and Energy dispersive spectrometer (EDS) were employed to observe and identify composition of the peeled biofilm (Delatolla et al., 2009). Laser Raman spectrometer (RM) recorded Raman spectra along with JY HR800 (JOBIN YVON, France) (Walrafen, 1975). 2.9. Data analysis Means and standard errors of three replicates were calculated. All data presented are expressed as the mean ± standard deviation (SD). SPSS 19.0 was used for the statistical evaluation of the results. Oneway analysis of variance (ANOVA) and the least significant differences (LSDs) test were performed to determine the significant difference among different centrifugal forces. Significant differences in centrifugal forces were observed using independent sample tests within the SPSS software. Microbial abundance at the genus level was mapped using heat map modules in “R” statistical packages. The specific methods and data analysis are available in the supporting material. 3. Results 3.1. Biofilm reactor operation and general observation The operator operated for 100 days and the biofilms matured after 20 days. Data showing the operation performance of the SBBR were displayed in Fig. S2. The reactor operated at COD concentration of 500 mg/L for 50 days and then raised the concentration to 1000 mg/L. Removal rate of COD remained above 90% during 60–120 days period of testing (Fig. S2, a). At the same time, TN and NH4-N removal rates increased during 50–120 days, with the highest removal rate of 60.1% and 68.2% separately (Fig. S2, b and c). During the period, COD loading is 2.0 kg COD·m−3·day−1 and N loading is 0.06 kg N·m−3·day−1 with the removal is 1.8 kg COD •m−3•day−1 and 0.10 kg N·m− 3·day−1. The content of polysaccharide (PS) and protein (PN) indicated that biofilm in carriers was kept at biofilm formation and peeling after a period of 30 days (Fig. S2,d). The ultimate carriers were harvested in the 100th day. Three different centrifugal force under detachment time of 5 min were employed, 2000 ×g, 5000 ×g, and 10,000 ×g, which contributed to supernatant/surface biofilm, loosely-bound biofilm and tightlybound biofilm, respectively (Fig. S3). Briefly, the carriers were centrifuged under 2000 × g and the detached biofilms were collected. After the 2000 × g detached carriers, it was used to detach biofilms under 5000 × g followed by 10,000 × g centrifugal force. Morphology of the biofilm under different centrifugal forces was recorded by ESEM and NikonTi 2000 (Fig. S4). Different centrifugal force exposed different fractions of the biofilms, thus various morphologies could be observed. Filamentous bacteria were presented in skeleton structure. Complex and compact layer constructures of filamentous bacteria were observed in the supernatant/surface biofilm (Karizmeh, 2012) while sphaerophorus presented in the tightly-bound biofilm. In addition, “porous” in the various fractions of biofilm were observed (Fig. S4, A). Meanwhile, the biofilms obtained under various forces show that the supernatant/surface biofilms of biofilm could be detached by lower centrifugal force (2000 ×g) and the tightly-bound biofilm required higher centrifugal force (10,000 ×g) to peel from bio-carriers (Fig. S4). The supernatant/surface biofilm of biofilms presented higher thickness and weight obtained by lower centrifugal forces, furthermore, a significant difference (p b 0.05) among the thickness and weight of biofilms detached by various centrifugal forces were obtained (Fig. 1), Different
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centrifugal forces could divide the natural wastewater treatment biofilms into different fractions which presented various morphologies in the biofilms (Fig. 1). 3.2. AHLs and AI-2 distribution in different fractions of the biofilm Quorum sensing (QS) signal molecules of AHLs and AI-2 in the biofilms under three different centrifugal forces were investigated (Fig. 2). The concentration of AHLs ranged from 5.2 ng/g to 98.3 ng/g in the different fractions of biofilms. Inner biofilms detached at higher centrifugal forces, present higher AHLs concentrations, with C10-HSL and C12-HSL in the biofilms obtained at various centrifugal forces (2000, 5000 and 10,000 × g) displaying significant differences (p b 0.01). Nevertheless, 3OC12-HSL presented an opposite trends with
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other AHLs, but the AHLs in 2000 ×g detached biofilms presented significant differences (p b 0.01) with the AHLs in 5000 and 10,000 × g detached biofilms. Furthermore, the residual AHLs in the biofilms presented similar distribution trends with the highest AHLs in the biofilms detached by 10,000 × g followed by 2000 × g and 5000 × g (Fig. 2,a). AHLs in the biofilms detached under different centrifugal forces presented the following discipline: middle chain AHLs presented the highest content followed by the AHLs content of the short chain and long chain. Interspecies communication signal AI-2 in the biofilm samples ranged from 79.2 ng/g to 98.3 ng/g (Fig. 2, b). The biofilms obtained by 2000 ×g contained the highest AI-2 concentration followed by AI-2 in 10,000 × g and 5000 × g detached biofilms. A significant difference (p b 0.01) was observed between the biofilms detached under 2000 ×g and 5000 ×g, and significant difference (p b 0.05) of AI-2 was also obtained between the biofilms under 5000 ×g and 10,000 ×g. 3.3. Community composition among different fractions of biofilm Biofilms obtained by various centrifugal forces presented different bacterial community compositions (Fig. S5). The microbial community at phylum level (2000, 5000 and 10,000 centrifugal forces) dominantly consisted of Proteobacteria (40.32%, 40.41% and 38.76%), Actinobacteria (30.20%, 27.71% and 28.63%), Bacteroidetes (13.75%, 14.64% and 18.18%), Spirochaetes (4.42%, 4.62% and 3.75%), Chlorobi (3.81%, 3.98% and 0.9%) and Acidobacteria (3.60%, 4.45% and 6.07%). The other phylum were Candidatus, Saccharibacteria, Chloroflexi, Ignavibacteriae, Verrucomicrobia, Firmicutes, Armatimonadetes, Lentisphaerae, Thermotogae, Nitrospirae, Parcubacteria, Synergistetes, Latescibacteria, Cloacimonetes and Cyanobacteria/Chloroplast, Planctomycetes and Gemmatimonadetes. Biofilm in the inner and loosely-bound biofilm presented closer relationship than with supernatant/surface biofilm (Fig. S5, a). Relative abundance of genus with an abundance of above 5% in three fractions of biofilm were only Desulfovibrio, Desulfobuibus, Propionibacterium, Zoogloea, Acincetobacter, Brachymonas, Geothrix, Chlorobium (Fig. S5,b). Pearson correlation coefficients were calculated by comparing the top 50 most abundant community members with the concentration of individual. Data statistics indicated that 47.83% and 65.22% bacteria were positively correlated to AHLs (except for 3OC10-HSL) and AI-2 separately (Fig. S6). Furthermore, Actinomyces, Terrimonas, Nitrospira, Microlunatus, Ottowia, Paludibacter and Acinetobacterwere were significantly positively correlated to AHLs (r = 0.994, p b 0.05, minimum ‘r’ and maximum ‘p’). Flavobacterium, Bdellovibrio, Victivallis, Chthonomonas, Rhizomicrobium, Pleomorphomonas, Saccharibacteri, Chlorobium and Desulfobulbus were significantly negatively correlated to AHLs (r = 0.996, p b 0.05, minimum ‘r’ and maximum ‘p’). At the same time, only Spirochaeta significantly positively related to AI-2 (r = 0.997, p b 0.05) and Propionivibrio significantly negatively corresponding to AI-2. r = 0.996, p b 0.05). The correlation relationship between sequenced bacteria, AHLs and AI-2 indicated that 49.62% were positively related with AHLs with 22.76% significantly related with AHLs, and 40.60% were positively corresponding to AI-2 with 1.12% significantly related to AI-2 (Fig. S6). 3.4. Distribution of EPS and LPS, and its correlation with signaling distribution
Fig. 2. Signal molecules (AHLs and AI-2) distribution among the biofilms under the three different centrifugal forces. 2000 × g, 5000 ×g and 10,000 × g represented the three different centrifugal forces, which presented the S, LB and TB biofilm, respectively. ANOVA is significant at p b 0.05 and p b 0.01 separately. Different capital letters indicate significantly different values among centrifugal forces at significant at p b 0.05, and different case letters indicate significantly different values among centrifugal forces at significant p b 0.01.
Main composition of EPS (PN and PS) presented a similar increase in trend with the increase of centrifugal forces, with PN ranging from 0.60 to 3.92 mg/g and PS ranging from 4.62 to 10.50 mg/g (Fig. 4,a,b). The significant differences (p b 0.05) in PN content and PS distribution belonging to S, LB and TB EPS could be attributed to different centrifugal forces. In general, the contents of PN and PS presented increasing trends among S, LB and TB EPS. The concentration of EPS presented positive relationship (r = 0.998, p = 0.036 b 0.05) with the increase of centrifugal force.
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In order to verify the components of different types of EPS, RM and EEM were employed. Similar qualitative Raman spectra of SEPS, LBEPS and TBEPS were obtained (Fig. S7). However, some differences among samples peeled by different centrifugal force were detected. The tryptophan group was presented in 5000 and 10,000 ×g SEPS but not in 2000 × g SEPS, and the same phenomena occurred in the TBEPS. However, tryptophan group was only detected in the 10,000 ×g LBEPS, which was absent in 2000 and 5000 ×g LBEPS. The C\\N stretching vibrations of proteins presented a similar tendency as a tryptophan group. The chain C_C stretching vibrations of carotenoids were detected in all samples except for the loosely-bound biofilm's LBEPS. At last, a group of amide VI and amylopectin were absence in the LBEPS of loosely-bound biofilm. Finally, the EEM of EPS was obtained from different centrifugal force of biofilms (Fig. S8). All the EEM displayed a similar distribution. Two characteristic peaks (A and B) which belong to proteins were observed. Meanwhile, EPS obtained from biofilm at 2000 ×g displayed UV-fulviclike (C) and visible fulvic-like (D) characteristic peaks. Furthermore, the distribution of LPS in biofilm under different centrifugal force ranged from 0.024 to 1.121 mg/g (Fig. 4,c) which increased with increasing centrifugal force and presented a significant difference (p b 0.01). Furthermore, the results indicated that LPS are more concentrated in the tightly-bound biofilm or microorganisms with LPS (i.e., Gram-negative bacteria). They were also hard to detach from mature biofilms with lower centrifugal force. Pearson correlation analysis between signal molecules (AHLs and AI-2) and EPS fractions (including LPS) indicated that 3OC12-HSL were negatively correlated to EPS fractions, while the residue AHLs positively related to EPS fractions (Fig. 4, d). C12-HSL were significantly corresponding to LB-PS/PN and S-PS/PN (r ≥ 0.998, p b 0.05; minimum ‘r’). At the same time, AI-2 did not present obvious corresponding relationship with EPS. In addition, EPS fractions could be divided into three groups, i.e., S-PS/PN and LB-PS/PN could be put into one group and the other groups were LPS and TB-PS/PN. 3.5. Mechanical properties of biofilm Macroscale compression and microscale indentation of mature biofilms obtained at various centrifugal forces (2000, 5000 and 10,000 × g) has been studied (Fig. 5). The biofilm fractions detached by 10,000 ×g presented the highest creep compliance, followed by biofilm detached by a centrifugal force of 5000 ×g, and then 2000 ×g (Fig. 5, a). The fitting curves of biofilms detached by centrifugal forces are listed as follows: 2000 ×g, Creep compliance = 0.00512–0.00389e(−T/37.0084), R2 = 0.9012, p b 0.01; 5000 ×g, Creep compliance = 0.01853– 0.01549e(−T/13.05), R2 = 0.927, p b 0.01; 10,000 ×g, Creep compliance = 0.06419–0.06403e(−T/13.0.2951), R2 = 0.994, p b 0.01. The fitted equations coefficients were listed as follows: Co2000 = − 0.004, Co5000 = −0.015 and Co10000 = − 0.064, which indicated that the biofilms detached by 10,000 ×g presented the highest absolute value followed by centrifugal forces of 5000 ×g and 2000 ×g detached biofilms. Furthermore, the energy dispersive spectroscopy (EDS) (Fig. S4, A) results indicated that there are no significant differences on the major components (elements) of the biofilms. The force of different layer biofilms obtained by AFM ranged from 4.12 nN to 6.31 nN (Fig. 5, b).The outer biofilms presented the lowest adhesive force and the inner layer showed the highest adhesive force. However, the loosely-bound biofilm presented a large-scale of adhesive force ranging from 3.65 to 6.28 nN, which covered the lowest and highest force obtained from samples under 2000 and 10,000 ×g. At the same time, the roughness of the biofilms, which were obtained from AFM (Fig. S4(c)) indicated that a higher centrifugal force can be attributed to a smoother surface. 4. Discussion The formation of EPS matrix leads to an establishment of stable gradients that provide different localized habitats at a small scale.
Organisms are stratified according to oxygen availability, nutrient consumption and the other environmental variables (Flemming et al., 2016). Most cells in multilayered biofilms experience cell-to-cell contact, either in surface-attached biofilms, in which only one layer is in direct contact with the substratum, or in flocs (Flemming et al., 2016). Indeed, three various centrifugal forces have been employed to separate the biofilms into different fractions by physical detachment method in this study. The obvious morphology and roughness difference in various fractions at 10 μm and 50 μm scale were obtained by ESEM and AFM (Fig. S4). The distribution of bacteria (Fig. S5), dissolved oxygen (DO) (Lee et al., 2007), concentration of organic matter may contribute to the morphology change in different fraction of biofilm. Filamentous bacteria constructed the framework structure of the biofilms and spherical bacteria attached on the skeleton (Fig. S4,b). At the same time, porous in the biofilm has been recognized, which presents small size and high density of the supernatant/surface biofilm of the biofilm and rather big and sparse in the inner layer. The hole connected to the bulk solution and allowed the bulk solution to enter and exit freely (Chen et al., 2013), which promote the nutrients and signal transmission timely. QS has been paid great attention to, in recent years especially in high diversity communities, such in activated sludge and granular sludge. Nevertheless, Tan et al. indicated that quorum quenching (QQ) widely existed in natural and manmade ecosystems (Tan et al., 2015), and floccular sludge community showed a high rate of QQ activity based on AHLs. Li et al. (2016) indicated that it is impossible to directly detect the AHL-producing ability of a bacterium by using polymerase chain reaction (PCR) or rRNA probing technologies for the heterogeneous of the AHL synthetase genes. However, plenty of work has been done in this field to identify the QS and QQ bacteria in granules sludge, which provide insight for us to understand the complex process in species-rich environment. Here, we addressed the community level QS and QQ signaling and responses using a moving bed biofilm reactor system. Interspecific signal molecules (AI-2) and intraspecific signal molecules (AHLs) have been investigated in the different fractions of biofilm. AI-2 could induce the expression of related genes to strengthen the communication among interspecific bacteria in the sludge system and guide sludge cells to switch from planktonic growth to attached growth (Zhang et al., 2011; Feng et al., 2014). Among the different fractions, AI2 presented the highest concentration in the supernatant/surface biofilm and the lowest concentration in the loosely-bound biofilm. Spirochaeta may be an AI-2 producer, which presents high percentage in supernatant/surface biofilm. Nevertheless, Propionivibrio, which was significantly negatively related to AI-2 distribution, may quench AI-2 and present lower concentration in middle and inner layer of biofilm. Most AHLs present the opposite trend compared to AI-2, which presents the highest concentration in the tightly-bound biofilm and the lowest concentration in the supernatant/surface biofilm (Fig. 2, a). Bacteria in cluster 1 (Fig. 3, a) may be contributors to QS signal pool, and in cluster 2 may be quorum quenching contributors. A relative balanced distribution between QS and QQ in 50 most abundant community members were obtained in Fig. 3. Tan et al. (2015) and Li et al. (2016) have reported quite a large number of bacteria connected with QS and QQ in aerobic granular sludge. In the QS bacteria, Acinetobacter presented negatively related to 3OC12-HSL, which was in accordance with Tan et al. (2015). But at the same time, Acinetobacter was significantly positively related to 3OC8-HSL (r = 0.999, p b 0.01), which indicated that Acinetobacter may have both QS and QQ function. In addition, Actinomyces, Terrimonas, Nitrospira, Microlunatus, Ottowia and Paludibacter recognized in this study which were significantly positively related with AHLs have not been reported in previous study, which need pure culture and further identification. Furthermore, Flavobacterium has been found to be negatively related to C4-HSL while no obvious negative or positive relationship with 3OC12-HSL (r = −0.146, p N 0.05) was presented, which were significantly negatively related with 3OC12-HSL reported by Tan et al. (2015). At the same time, Bdellovibrio, Victivallis, Chthonomonas,
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Fig. 3. (a) The Kinexus ultra + based creep compliance and (b) the AFM based force curve. The curve fitting has been employed in (a) with a regular equation Y = y0 + A1*e(x/t1) (R2 N 0.90). 2000 ×g, 5000 ×g and 10,000 ×g represented the three different centrifugal forces, which presented the S, LB and TB biofilm, respectively. Pearson correlation analysis between presented significant p b 0.01. signal molecules (AHLs and AI-2) and force properties of biofilms(c). presented significant p b 0.05 and
Rhizomicrobium, Pleomorphomonas, Saccharibacteria, Chlorobium and Desulfobulbus recognized in this study which were significantly negatively related with AHLs have not been reported in previous study, which also need further identification. EPS immobilizes biofilm cells and keep them in close proximity, thus allowing for intense interactions, including cell–cell communication (Flemming and Wingender, 2010). Quorum sensing is a concentration-sensing mechanism that plays a vital role in biofilm EPS formation (Tan et al., 2015). EPS have a significant influence on the physicochemical properties of microbial aggregates, including structure, surface charge, adsorption ability (Sheng et al., 2010b) and viscosity (Freitas et al., 2011). Proteins and polysaccharides were major components as identified from Raman spectra and EEM analyses (Figs. S7 and S8). The results in Fig. 4 suggest that the extracellular polysaccharides were mainly presented in the TB-EPS fraction, which were in agreement with the finding of Liang et al. (2010). Furthermore, Liu and Fang (2002) found that the LB-EPS played a positive role in sludge aggregations. Similarly, Sheng et al. (2010a) determined that LB-EPS had a strong binding and flocculating capacity, which indicated that EPS has mechanical properties. Also, results in Fig. 4(d) indicated that
AHLs (C12-HSL), which presented higher concentration in the biofilm, plays significantly positive role with S and LB EPS. It indicated that AHLs transport form S-EPS to LB-EPS. Therefore, TB-EPS connected with target cells do not present a significant effect on AHLs. It is worth mentioning that AI-2 did not show significant correlation, either positive or negative, with EPS in the mature biofilms, but most AHLs presented significant relationship with EPS, which might indicate that AHLs contributed to EPS production process of mature biofilms. Understanding the mechanical properties of EPS helps verifying the mechanism of microbial attachment to biofilms and carriers. Microscale adhesion conducted by AFM and large-scale compliance conducted by kinexus ultra + rheometer indicated that the biofilms detached by 10,000 × g, i.e., the tightly-bound biofilm presented the highest force followed by the loosely-bound biofilm and supernatant/surface biofilm. The physical properties of the three fractions of biofilm may contribute to the various mechanical properties. At the same time, the t EPS content exhibited positive linear relationship with creeping compliance and adhere force of the biofilm at each layer (Fig. 5, c) (Flemming et al., 2007). The existing studies indicate that glucose and cellulose contributed to the high viscosity of biofilms (Freitas
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et al., 2011; Rehm, 2009), which was observed from RM in accordance with compliance, and the force conducted by AFM. Furthermore, because of the heterogeneity of biofilms, the large scale of mechanismrelated properties could present a closed character of biofilms (Safari et al., 2015). Fig. 5(c) indicated that the most abundant signals (C10HSL and C12-HSL) within the biofilm were significantly corresponding to compliance and the force conducted by AFM. At the same time, the high concentration of AHLs distribution in the biofilm were in accordance with LPS which presented the abundance of gram-negative bacteria. A relatively high concentration of AHLs in tightly-bound biofilm may contribute to the high amount of EPS, which may be helpful to heighten the mechanical properties. Although it is not yet possible to determine if QS signaling is a direct cause of mechanical properties, the observation here that the three fractions, EPS production, community composition and AHL concentrations, were all strongly correlated with each other suggests that they are important in the mechanical properties. Furthermore, the QS signals in mature biofilms have been deeply investigated here, and the next task is to study QS signals distribution in different fractions of biofilms during the entire biofilm formation process. Indeed, QS and QQ existing in the nature and artificial environment may play important role in community cooperative and in other behaviors. A better understanding of the relationship between QS and mechanical properties helps us verify the complex process of multiple biofilm formation and its detachment.
5. Conclusions Wastewater treatment biofilms detached into three different fractions under the centrifugal force of 2000, 5000 and 10,000 × g. AHLs ranged from 5.2 ng/g to 98.3 ng/g in the different fraction of biofilms, and the concentration of C10-HSL and C12-HSL, which presented the highest in the biofilm, displayed significant differences (p b 0.01) in the S, LB and TB of biofilm. The concentration of EPS was positively correlated (p b 0.05) with the increase of centrifugal force, and the concentration of with PN range from 0.60 to 3.92 mg/g and PS range from 4.62 to 10.50 mg/g. Furthermore, QS bactreia, such as Acinetobacter, QQ bacteria such as Flavobacterium, have been measured and confirmed within the biofilm. The biofilm adhesive and compliance corresponded to the distribution of AHLs (C10-HSL and C12-HSL), indicating that QS and QQ plays vital roles in the mechanical properties of biofilms, which brings new in sight of the aged biofilm excoriation and the initial bacterial pioneer the bio-carrier in wastewater treatment using biofilm.
Acknowledgements This research was supported by the National Science and Technology Major Project (2017ZX07204001), National Science & Technology Support Program of China (No. 2014BAC08B04) and National Natural Science Foundation of China (51608254). Appendix A. Supplementary data Supplementary material related to this article can be found in the online version http://doi.dx.org/10.1016/j.scitotenv.2017.07.277.
Fig. 4. PN, PS and LPS distribution of biofilms under different centrifugal forces. Pearson correlation analysis between signal molecules (AHLs and AI-2) and EPS fractions (d). 2000 × g, 5000 ×g and 10,000 × g represented the three different centrifugal forces, which presented the S, LB and TB biofilm, respectively.The data are presented as the means ± SD from six times for each determination. ANOVA is significant at p b 0.05 and p b 0.01, separately. Different capital letters indicate significantly different values among centrifugal forces as significant at p b 0.05, and different case letters indicating significantly different values among centrifugal forces at significant p b 0.01. presented presented significant p b 0.01. significant p b 0.05 and
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Fig. 5. (a) The Kinexus ultra + based creep compliance and (b) the AFM based force curve. The curve fitting has been employed in (a) with a regular eq. Y = y0+ A1*e (x/t1) (R2 N 0.90). 2000 ×g, 5000 ×g and 10,000 ×g represented the three different centrifugal forces, which presented the S, LB and TB biofilm, respectively. Pearson correlation analysis between EPS and force properties of biofilms(c) and pearson correlation analysis between signal molecules (AHLs and AI-2) and force properties of biofilms(d). presented significant p b 0.05 and presented significant p b 0.01.
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