Bioresource Technology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Quorum quenching is responsible for the underestimated quorum sensing effects in biological wastewater treatment reactors Xiang-Ning Song a, Yuan-Yuan Cheng a,⇑, Wen-Wei Li a, Bing-Bing Li a, Guo-Ping Sheng a, Cai-Yun Fang a, Yun-Kun Wang a, Xiao-Yan Li b, Han-Qing Yu a a b
CAS Key Laboratory of Urban Pollutant Conversion, Department of Chemistry, University of Science & Technology of China, Hefei 230026, China Environmental Engineering Research Centre, Department of Civil Engineering, The University of Hong Kong, Hong Kong
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
Quorum quenching (QQ) activity was
evaluated in situ in an activated sludge reactor. The coexistence of quorum sensing (QS) and QQ in activated sludge was confirmed. The developed method could be used to monitor QQ in other biosystems.
a r t i c l e
i n f o
Article history: Received 4 June 2014 Received in revised form 2 August 2014 Accepted 5 August 2014 Available online xxxx Keywords: Activated sludge Acyl homoserine lactones (AHL) AHL-degrading enzymes Quorum quenching Quorum sensing
a b s t r a c t Quorum sensing (QS) and quorum quenching (QQ) are two antagonistic processes coexisting in various bacterial communities in bioreactors, e.g., activated sludge for biological wastewater treatment. Although QS signal molecules are detected in activated sludge reactors and known to affect sludge properties and reactor performance, there has been no direct evidence to prove the endogenous existence of QQ effects in activated sludge. In this study, for the first time, acyl homoserine lactones-degrading enzymatic activity, a typical QQ effect, was discovered in activated sludge and found to considerably affect the QS detection results. The coexistence of QS and QQ bacteria in activated sludge was further confirmed by bacterial screening and denaturing gradient gel electrophoresis analysis. The method developed in this study could also be used to evaluate QQ activities in bioreactors, and a possible way is provided to tune bioreactor performance through balancing the QS and QQ processes. Ó 2014 Published by Elsevier Ltd.
1. Introduction Bacterial communities can coordinate their behaviors as a group through a process called quorum sensing (QS), in which bacteria secrete small-molecular signal chemicals, i.e., autoinducers, ⇑ Corresponding author. E-mail addresses: [email protected] (Y.-Y. Cheng), [email protected] (H.-Q. Yu).
sense them, and consequently modulate cellular physiology as a feedback to population density (Miller and Bassler, 2001). Several types of autoinducers have been identified, among which acyl homoserine lactones (AHLs) are a major class of autoinducers commonly secreted and sensed by many bacterial species (Miller and Bassler, 2001). In biological wastewater treatment systems there are complex microbial communities, thus AHLs are considered to play an important role in adjusting these bacterial communities. So far, AHLs have been detected in various wastewater treatment
http://dx.doi.org/10.1016/j.biortech.2014.08.027 0960-8524/Ó 2014 Published by Elsevier Ltd.
Please cite this article in press as: Song, X.-N., et al. Quorum quenching is responsible for the underestimated quorum sensing effects in biological wastewater treatment reactors. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.08.027
X.-N. Song et al. / Bioresource Technology xxx (2014) xxx–xxx
bioreactors, which contain large quantities of dense microbial consortia in the form of flocs, granules or biofilm, and are found to affect activated sludge properties and reactor performance (Shrout and Nerenberg, 2012). Specifically, the microbial communities and pollutant degradation ability of activated sludge (Valle et al., 2004), the granulation of sludge (Tan et al., 2014), and the biofilm formation processes (Ren et al., 2013) are all affected by dosing AHLs. An antagonistic process of QS is quorum quenching (QQ), in which the autoinducers such as AHLs are degraded or the receptors of AHLs are inhibited (Dong and Zhang, 2005). Several AHL-hydrolyzing enzymes including acylase have been identified (Dong and Zhang, 2005). The acylase, or bacteria capable of secreting these hydrolyzing enzymes, have been used in wastewater treatment bioreactors to mitigate biofouling through hydrolyzing AHLs (Oh et al., 2012). These studies indicate that the performance of wastewater treatment bioreactor can be tuned through selectively manipulating either QS or QQ, i.e., raising or lowering AHL activity. Thus, a deep understanding about these processes is essential. Until now, most studies on QQ effect in bioreactors were conducted by exogenously dosing AHL-degrading enzymes or bacteira into reactors (Oh et al., 2012; Yeon et al., 2009). Several AHLsproducing bacteria, including Acinetobacter spp., Aeromonas spp. and Pseudomonas spp., have been identified in wastewater treatment bioreactors (Kämpfer et al., 1996). Meanwhile, some bacteria with QQ activity have been isolated from bioreactors (Cheong et al., 2013). By producing enzymes, which could cleavage of amide or lactone bonds (Uroz et al., 2009), these QQ bacteria, together with QS bacteria, control the concentration of AHLs and exert consequent biological effects. Although external dose of these bacteria was found to affect the QS and bioreactor performance, there is no direct evidence to support the existence of endogenous QQ in wastewater treatment bioreactors yet. QQ and QS, as the response of microbial community to environmental conditions, are thus very sensitive to the fluctuation of these conditions. Operational conditions, wastewater compositions, microbial diversity, etc., can affect the occurrence and balance of QQ and QS caused by diverse bacteria in bioreactors. In previous studies regarding QQ in bioreactors, QQ bacteria are usually isolated from the mixed cultures in bioreactors and then explored in terms of their QQ roles. Such a manner inevitably changes the status of the QQ bacteria, and thus it is impossible to judge whether these bacteria really play their QQ roles in bioreactors or not. So far, there is no report to directly reveal the native and endogenous QQ activity in situ. In other words, there is no clear information about the QQ in bioreactors, as well as whether QS and endogenous QQ would synergistically regulate the behaviors of microbial consortia in wastewater treatment bioreactors. Therefore, the main objective of this study was to examine whether there was an coexistence of endogenous QQ and QS activity in activated sludge process, with a membrane bioreactor (MBR) as an example, and explore the feasibility to improve reactor performance through regulating endogenous QQ activity.
2. Experimental 2.1. Chemicals and bacterial strains N-(3-oxooxtanoyl)-L-homoserine lactone (3-oxo-C8-HSL) was purchased from Sigma–Aldrich Co., USA. 5-Bromo-4-chloro-3indolyl-beta-D-galactopy-ranoside (X-gal), phenylmethanesulfonyl fluoride (PMSF) and o-nitrophenyl-b-D-galactopyranoside (ONPG) were purchased from Sangon Co., China. AHLs were detected by Agrobacterium tumefaciens KYC55, which shows a sensitive response to a wide spectrum of AHLs (Joelsson and Zhu, 2006).
Another reporter strain Aeromonas sp. (pBB–LuxR) expressing green fluorescent protein (GFP) under induction of AHLs was used to in situ detect AHLs in the activated sludge (Chong et al., 2012).
2.2. Sludge extracts and autoclaved sludge extracts MBR operation and sludge characteristics are described in Supplementary Materials. Aliquots of 4 ml sludge samples from an MBR were centrifuged at 5000g for 10 min. The pelleted sludge was washed three times using equal volume of phosphate buffered saline (PBS) solution containing 1.0 mM PMSF to inhibit proteases, and then resuspended in 1 ml PBS-PMSF. The sludge incubated in an ice-water bath was subjected to ultrasonic disruption (80 w power output, 300 times, each time for 3 s followed by 3-s interval) using an ultrasonic cell disruptor (Ningbo Scientz Biotechnology Co., China) to lyse cells, followed by centrifugation at 12,000g for 20 min. The extracted supernatants were filtered through a 0.22 lm filter. The filtrates were used to analyze AHL-degrading activity and autoclaved at 105 °C for 20 min to inactivate the AHL-hydrolyzing or AHL-modifying enzymes when necessary.
2.3. AHL degradation tests The sludge extracts and autoclaved ones were mixed with equal volume of PBS solution containing 2 lM AHL. Here, 3-oxo-C8-HSL, which is widely utilized by a variety of bacteria (Barnard and Salmond, 2007; Horng et al., 2002), was chosen as a model AHL. The corresponding mixed solutions without AHL were used as two negative controls, and 1-lM AHL PBS solution was used as positive control. These five mixtures were incubated at 30 °C. Specified volumes of liquid samples were taken out every 2 h, and then incubated in a water bath at 90 °C for 10 min to inactivate enzymes. The as-obtained liquid samples were stored at 20 °C before analyzing the residual 3-oxo-C8-HSL. The protein contents of the five mixtures were determined by the bicinchoninic acid assay using protein assay kit (Sangon Co., China). The relative content of 3-oxo-C8-HSL in the samples was determined following the method described by Joelsson and Zhu (2006). The assay of b-galactosidase activity was conducted according to Miller and the specific activity was expressed in Miller Units (Miller, 1972).
AHL+PBS AHL+autoclaved sludge extract AHL+sludge extract PBS+autoclaved sludge extract PBS+sludge extract
300
-galactosidase activity /(MILLER UNITS)
2
200
100
0 0
3
6
Time (h) Fig. 1. Residual AHLs detected by A. tumefaciens KYC55 after 6-h incubation. bGalactosidase activity of AHL + PBS, AHL + autoclaved sludge extract, AHL + sludge extract, PBS + autoclaved sludge extract, and PBS + sludge extract.
Please cite this article in press as: Song, X.-N., et al. Quorum quenching is responsible for the underestimated quorum sensing effects in biological wastewater treatment reactors. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.08.027
X.-N. Song et al. / Bioresource Technology xxx (2014) xxx–xxx
2.4. In situ detection of AHLs by confocal laser scanning microscopy (CLSM) AHL was in situ detected by CLSM following the method of Chong et al. (2012) with slight modification. Overnight cultures of AHLs reporter Aeromonas sp. (pBB–LuxR) were washed and diluted (1:5) with autoclaved synthetic wastewater (see Supporting Information, SI). Sludge samples from the MBR were washed and resuspended in equal volume of autoclaved synthetic wastewater. Then, 100 ll of sludge suspension was mixed with 100 ll of Aeromonas sp. (pBB–LuxR) for incubation. After 5-h incubation,
3
green fluorescence was observed using CLSM (FV1000-IX81, Olympus Co., Japan). Autoclaved sludge samples were used as negative controls. 3. Results and discussion 3.1. AHL degradation by sludge extracts In this study, the AHLs degradation activity of the sludge extracts was examined. Fig. 1 shows the quenching effect of the sludge extracts on AHL during 6-h incubation period. The initial
Fig. 2. CLSM images of the activated sludge sample without the AHL reporter strain (A), with the reporter strain (B), and autoclaved sludge sample with the reporter strain (C).
Please cite this article in press as: Song, X.-N., et al. Quorum quenching is responsible for the underestimated quorum sensing effects in biological wastewater treatment reactors. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.08.027
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X.-N. Song et al. / Bioresource Technology xxx (2014) xxx–xxx
protein concentration in all the reaction mixtures was 728 ± 21 lg/ ml. The 3-oxo-C8-HSL after 6-h incubation with the sludge extracts hardly induced the b-galactosidase expressed by reporter A. tumefaciens KYC55, suggesting the degradation of 3-oxo-C8-HSL. While the b-galactosidase activities had no significant difference between the samples incubated with medium only or with autoclaved sludge. This result suggests that some thermally unstable substances existed in the sludge extracts, which could degrade AHLs or inhibit their activity, and thus became inactivated after autoclaving. Similar results were also obtained for the sludge from a granular sludge reactor operated at our laboratory (Figure S1). These results confirm the existence of QQ activity in the activated sludge process. 3.2. Presence of AHLs around flocs The presence of AHLs in the activated sludge was investigated, firstly by in situ detection. Fig. 2 shows the response of the AHL reporter strain Aeromonas sp. (pBB–LuxR) to sludge samples after 5-h incubation. The sludge without dose of the reporter bacterium showed no green fluorescence (Fig. 2A). In comparison, only light fluorescence was detected for the autoclaved sludge sample when incubated with the reporter (Fig. 2C), while strong fluorescence was observed for the activated sludge sample with dose of the reporter (Fig. 2B). A comparison between the fluorescence image and the phase-contrast image reveals that the reporter cells with green fluorescence were mainly attached on the surface of sludge flocs, rather than in the bulk solution. Such a result implies that the AHLs concentration was sufficiently high at the sludge floc surface to induce the fluorescence of the reporter bacterium, but was not sufficient to reach a relevant concentration level in the solution. Furthermore, efforts were made to concentrate and detect AHLs ex situ from the bulk solution of the bioreactors. Several methods, including bioreporters and high-performance liquid chromatography with tandem mass spectrometric detection, were used in this study (see Supplementary Materials). Surprisingly, none of them showed the presence of AHLs. Taking into account this result and the QS in situ detection result together, it is assumed that AHLs might have been secreted by the QS bacteria and then ingested or degraded by adjacent bacteria within a short time period. The local concentration of AHLs might be sufficiently high to trigger biological events for surrounding bacteria, but their content would become undetectable levels at reactor scale. This is reasonable from an energy and carbon source consumption point of view, because it would be uneconomical for the microbial community, which accounts for a small part of volume in bioreactor, to synthesize a large quantity of AHLs to fill the bulk solution. Moreover, the extracellular polymeric substances (EPS) of sludge are constituted by many macromolecules, such as polysaccharides, proteins, nucleic acids, and lipids, etc. These macromolecules contain diverse charged groups (e.g., carboxyl, phosphoric, sulfhydryl, phenolic and hydroxyl groups) and apolar groups (e.g., aromatics, aliphatics in proteins, and hydrophobic regions in carbohydrates). AHLs contain carbonyl and amide groups, which might form hydrogen bond with hydroxyl groups. Besides, the alkyl side chain of AHL might form complex with the apolar groups of EPS through hydrophobic interactions. Thus, EPS have a potential to hinder the diffusion of AHLs through group interactions. Therefore, no AHLs could be detected ex situ in the reactors. Nevertheless, the local concentration of AHLs at floc surface was still sufficiently high to induce reporter gene expression. This suggests that such a level of AHLs might play a role in regulating physiological and biological activities of neighboring cells. Our results also imply that, due to such an endogenous QQ effect, the role of QS in activated sludge reactors might have been underestimated in previous studies.
3.3. Presence of both AHLs-producing and -quenching bacteria If the above assumption that QQ bacteria lead to the failure of AHL ex situ detection is true, such QQ bacteria should be found in activated sludge samples. Thus, bacterial screening (see Supplementary Materials) was conducted to explore the microbial communities of the activated sludge. Both AHLs-producing and AHLs-degrading bacteria were isolated from the activated sludge. One of the isolates was Bacillus thruingiensis, which has been widely reported to harbor aiiA gene for encoding a lactone-degrading enzyme (Dong et al., 2002; Lee et al., 2002). Another isolate was Aeromonas hydrophila, which utilizes two types of AHLs as autoinducer signals (Lynch et al., 2002). These bacteria have been widely found in wastewater treatment bioreactors. Denaturing gradient gel electrophoresis (DGGE) analysis (see Supplementary Materials and further sequencing confirm the presence of these isolates in the activated sludge (Fig. 3). This result also shows that the QS and QQ effects occurred simultaneously in activated sludge reactors. The physiological–biological activities of activated sludge induced by either QS or QQ alone can significantly affect the performance of wastewater treatment bioreactors (Shrout and Nerenberg, 2012). However, the coexistence of QS and QQ in activated sludge systems has not been fully elucidated so far. In previous studies, it was found that Agrobacterium tumefaciens could produce 3-oxo-C8-HSL and modulate its level via two lactonases encoded by attM and aiiB (Haudecoeur et al., 2009). The QS pathways and the QQ gene of A. tumefaciens coordinate to express virulence functions of A. tumefaciens optimally (Haudecoeur et al., 2009). In addition, two rhizosphere microbes, Acinetobacter and Burkholderia, also possess both AHL-dependent QS and QQ activities and can regulate the whole rhizosphere bacterial community to attenuate toxicity to plants (Chan et al., 2011). In addition to lactonase and acylase, both of which are identified in diverse bacteria, oxido-reductase and oxidase are recently identified and far less common. Moreover, oxido-reductase and oxidase are able to change AHLs, rather than hydrolyzing AHLs (Uroz
Fig. 3. DGGE of 16S rRNA gene sequence from (1) Bacillus thruingiensis; (2) Aeromonas hydrophila; and (3) activated sludge samples.
Please cite this article in press as: Song, X.-N., et al. Quorum quenching is responsible for the underestimated quorum sensing effects in biological wastewater treatment reactors. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.08.027
X.-N. Song et al. / Bioresource Technology xxx (2014) xxx–xxx
et al., 2009). The modified AHL, although still functionally active, may fail to bind specifically to its receptors, thus causing disturbance to the activation of QS-mediated genes regulated by a particular AHL (Uroz et al., 2009). Therefore, the roles of both QQ and QS in activated sludge reactors should be given more attention. In addition, our study also suggests that more sensitive in situ analytical methods should be needed for further investigations into these processes. 4. Conclusions In this study, the existence of endogenous QQ effect in wastewater treatment bioreactors is confirmed in situ, and its roles in regulating QS are also identified. Our results clearly indicate that, due to such an endogenous QQ effect, the role of QS in activated sludge reactors might have been underestimated previously. Activated sludge properties and reactor performance could be tuned by appropriately balancing the endogenous QQ and QS in bioreactors. Furthermore, the developed method could be used to detect QQ activity in situ for monitoring QQ in other bioreactors. Acknowledgements The authors wish to thank the Natural Science Foundation of China (51129803), and the Program for Changjiang Scholars and Innovative Research Team in University, China for the partial support of this study. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2014. 08.027. References Barnard, A.M.L., Salmond, G.P.C., 2007. Quorum sensing in Erwinia species. Anal. Bioanal. Chem. 387, 415–423. Chan, K.G., Atkinson, S., Mathee, K., Sam, C.K., Chhabra, S.R., Camara, M., Koh, C.L., Williams, P., 2011. Characterization of N-acylhomoserine lactone-degrading bacteria associated with the Zingiber officinale (ginger) rhizosphere: coexistence of quorum quenching and quorum sensing in Acinetobacter and Burkholderia. BMC Microbiol. 11, 51. Cheong, W.S., Lee, C.H., Moon, Y.H., Oh, H.S., Kim, S.R., Lee, S.H., Lee, C.H., Lee, J.K., 2013. Isolation and identification of indigenous quorum quenching bacteria,
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Pseudomonas sp. 1A1, for biofouling control in MBR. Ind. Eng. Chem. Res. 52, 10554–10560. Chong, G., Kimyon, O., Rice, S.A., Kjelleberg, S., Manefield, M., 2012. The presence and role of bacterial quorum sensing in activated sludge. Microb. Biotechnol. 5, 621–633. Dong, Y.H., Gusti, A.R., Zhang, Q., Xu, J.L., Zhang, L.H., 2002. Identification of quorumquenching N-acyl homoserine lactonases from Bacillus species. Appl. Environ. Microb. 68, 1754–1759. Dong, Y.H., Zhang, L.H., 2005. Quorum sensing and quorum-quenching enzymes. J. Microbiol. 43, 101–109. Haudecoeur, E., Tannieres, M., Cirou, A., Raffoux, A., Dessaux, Y., Faure, D., 2009. Different regulation and roles of lactonases AiiB and AttM in Agrobacterium tumefaciens C58. Mol. Plant Microbe Interact. 22, 529–537. Horng, Y.T., Deng, S.C., Daykin, M., Soo, P.C., Wei, J.R., Luh, K.T., Ho, S.W., Swift, S., Lai, H.C., Williams, P., 2002. The LuxR family protein SpnR functions as a negative regulator of N-acylhomoserine lactone-dependent quorum sensing in Serratia marcescens. Mol. Microbiol. 45, 1655–1671. Joelsson, A.C., Zhu, J., 2006. LacZ-based detection of acyl-homoserine lactone quorum-sensing signals. Curr. Protoc. Microb., 1C. 2.1–1C. 2.9. Kämpfer, P., Erhart, R., Beimfohr, C., Böhringer, J., Wagner, M., Amann, R., 1996. Characterization of bacterial communities from activated sludge: culturedependent numerical identification versus in situ identification using groupand genus-specific rRNA-targeted oligonucleotide probes. Microb. Ecol. 32, 101–121. Lee, S.J., Park, S.Y., Lee, J.J., Yum, D.Y., Koo, B.T., Lee, J.K., 2002. Genes encoding the Nacyl homoserine lactone-degrading enzyme are widespread in many subspecies of Bacillus thuringiensis. Appl. Environ. Microb. 68, 3919–3924. Lynch, M.J., Swift, S., Kirke, D.F., Keevil, C.W., Dodd, C.E.R., Williams, P., 2002. The regulation of biofilm development by quorum sensing in Aeromonas hydrophila. Environ. Microbiol. 4, 18–28. Miller, J.H., 1972. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, N.Y.. Miller, M.B., Bassler, B.L., 2001. Quorum sensing in bacteria. Annu. Rev. Microbiol. 55, 165–199. Oh, H.S., Yeon, K.M., Yang, C.S., Kim, S.R., Lee, C.H., Park, S.Y., Han, J.Y., Lee, J.K., 2012. Control of membrane biofouling in MBR for wastewater treatment by quorum quenching bacteria encapsulated in microporous membrane. Environ. Sci. Technol. 46, 4877–4884. Ren, T.T., Li, X.Y., Yu, H.Q., 2013. Effect of N-acy-L-homoserine lactones-like molecules from aerobic granules on biofilm formation by Escherichia coli K12. Bioresour. Technol. 129, 655–658. Shrout, J.D., Nerenberg, R., 2012. Monitoring bacterial twitter: does quorum sensing determine the behavior of water and wastewater treatment biofilms? Environ. Sci. Technol. 46, 1995–2005. Tan, C.H., Koh, K.S., Xie, C., Tay, M., Zhou, Y., Williams, R., Ng, W.J., Rice, S.A., Kjelleberg, S., 2014. The role of quorum sensing signalling in EPS production and the assembly of a sludge community into aerobic granules. ISME J. 8, 1186– 1197. Uroz, S., Dessaux, Y., Oger, P., 2009. Quorum sensing and quorum quenching: the Yin and Yang of bacterial communication. ChemBioChem 10, 205–216. Valle, A., Bailey, M.J., Whiteley, A.S., Manefield, M., 2004. N-acyl-L-homoserine lactones (AHLs) affect microbial community composition and function in activated sludge. Environ. Microbiol. 6, 424–433. Yeon, K.M., Cheong, W.S., Oh, H.S., Lee, W.N., Hwang, B.K., Lee, C.H., Beyenal, H., Lewandowski, Z., 2009. Quorum sensing: a new biofouling control paradigm in a membrane bioreactor for advanced wastewater treatment. Environ. Sci. Technol. 43, 380–385.
Please cite this article in press as: Song, X.-N., et al. Quorum quenching is responsible for the underestimated quorum sensing effects in biological wastewater treatment reactors. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.08.027
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Quorum quenching is responsible for the underestimated quorum sensing effects in biological wastewater treatment reactors Xiang-Ning Song a, Yuan-Yuan Cheng a,⇑, Wen-Wei Li a, Bing-Bing Li a, Guo-Ping Sheng a, Cai-Yun Fang a, Yun-Kun Wang a, Xiao-Yan Li b, Han-Qing Yu a a b
CAS Key Laboratory of Urban Pollutant Conversion, Department of Chemistry, University of Science & Technology of China, Hefei 230026, China Environmental Engineering Research Centre, Department of Civil Engineering, The University of Hong Kong, Hong Kong
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
Quorum quenching (QQ) activity was
evaluated in situ in an activated sludge reactor. The coexistence of quorum sensing (QS) and QQ in activated sludge was confirmed. The developed method could be used to monitor QQ in other biosystems.
a r t i c l e
i n f o
Article history: Received 4 June 2014 Received in revised form 2 August 2014 Accepted 5 August 2014 Available online xxxx Keywords: Activated sludge Acyl homoserine lactones (AHL) AHL-degrading enzymes Quorum quenching Quorum sensing
a b s t r a c t Quorum sensing (QS) and quorum quenching (QQ) are two antagonistic processes coexisting in various bacterial communities in bioreactors, e.g., activated sludge for biological wastewater treatment. Although QS signal molecules are detected in activated sludge reactors and known to affect sludge properties and reactor performance, there has been no direct evidence to prove the endogenous existence of QQ effects in activated sludge. In this study, for the first time, acyl homoserine lactones-degrading enzymatic activity, a typical QQ effect, was discovered in activated sludge and found to considerably affect the QS detection results. The coexistence of QS and QQ bacteria in activated sludge was further confirmed by bacterial screening and denaturing gradient gel electrophoresis analysis. The method developed in this study could also be used to evaluate QQ activities in bioreactors, and a possible way is provided to tune bioreactor performance through balancing the QS and QQ processes. Ó 2014 Published by Elsevier Ltd.
1. Introduction Bacterial communities can coordinate their behaviors as a group through a process called quorum sensing (QS), in which bacteria secrete small-molecular signal chemicals, i.e., autoinducers, ⇑ Corresponding author. E-mail addresses: [email protected] (Y.-Y. Cheng), [email protected] (H.-Q. Yu).
sense them, and consequently modulate cellular physiology as a feedback to population density (Miller and Bassler, 2001). Several types of autoinducers have been identified, among which acyl homoserine lactones (AHLs) are a major class of autoinducers commonly secreted and sensed by many bacterial species (Miller and Bassler, 2001). In biological wastewater treatment systems there are complex microbial communities, thus AHLs are considered to play an important role in adjusting these bacterial communities. So far, AHLs have been detected in various wastewater treatment
http://dx.doi.org/10.1016/j.biortech.2014.08.027 0960-8524/Ó 2014 Published by Elsevier Ltd.
Please cite this article in press as: Song, X.-N., et al. Quorum quenching is responsible for the underestimated quorum sensing effects in biological wastewater treatment reactors. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.08.027
X.-N. Song et al. / Bioresource Technology xxx (2014) xxx–xxx
bioreactors, which contain large quantities of dense microbial consortia in the form of flocs, granules or biofilm, and are found to affect activated sludge properties and reactor performance (Shrout and Nerenberg, 2012). Specifically, the microbial communities and pollutant degradation ability of activated sludge (Valle et al., 2004), the granulation of sludge (Tan et al., 2014), and the biofilm formation processes (Ren et al., 2013) are all affected by dosing AHLs. An antagonistic process of QS is quorum quenching (QQ), in which the autoinducers such as AHLs are degraded or the receptors of AHLs are inhibited (Dong and Zhang, 2005). Several AHL-hydrolyzing enzymes including acylase have been identified (Dong and Zhang, 2005). The acylase, or bacteria capable of secreting these hydrolyzing enzymes, have been used in wastewater treatment bioreactors to mitigate biofouling through hydrolyzing AHLs (Oh et al., 2012). These studies indicate that the performance of wastewater treatment bioreactor can be tuned through selectively manipulating either QS or QQ, i.e., raising or lowering AHL activity. Thus, a deep understanding about these processes is essential. Until now, most studies on QQ effect in bioreactors were conducted by exogenously dosing AHL-degrading enzymes or bacteira into reactors (Oh et al., 2012; Yeon et al., 2009). Several AHLsproducing bacteria, including Acinetobacter spp., Aeromonas spp. and Pseudomonas spp., have been identified in wastewater treatment bioreactors (Kämpfer et al., 1996). Meanwhile, some bacteria with QQ activity have been isolated from bioreactors (Cheong et al., 2013). By producing enzymes, which could cleavage of amide or lactone bonds (Uroz et al., 2009), these QQ bacteria, together with QS bacteria, control the concentration of AHLs and exert consequent biological effects. Although external dose of these bacteria was found to affect the QS and bioreactor performance, there is no direct evidence to support the existence of endogenous QQ in wastewater treatment bioreactors yet. QQ and QS, as the response of microbial community to environmental conditions, are thus very sensitive to the fluctuation of these conditions. Operational conditions, wastewater compositions, microbial diversity, etc., can affect the occurrence and balance of QQ and QS caused by diverse bacteria in bioreactors. In previous studies regarding QQ in bioreactors, QQ bacteria are usually isolated from the mixed cultures in bioreactors and then explored in terms of their QQ roles. Such a manner inevitably changes the status of the QQ bacteria, and thus it is impossible to judge whether these bacteria really play their QQ roles in bioreactors or not. So far, there is no report to directly reveal the native and endogenous QQ activity in situ. In other words, there is no clear information about the QQ in bioreactors, as well as whether QS and endogenous QQ would synergistically regulate the behaviors of microbial consortia in wastewater treatment bioreactors. Therefore, the main objective of this study was to examine whether there was an coexistence of endogenous QQ and QS activity in activated sludge process, with a membrane bioreactor (MBR) as an example, and explore the feasibility to improve reactor performance through regulating endogenous QQ activity.
2. Experimental 2.1. Chemicals and bacterial strains N-(3-oxooxtanoyl)-L-homoserine lactone (3-oxo-C8-HSL) was purchased from Sigma–Aldrich Co., USA. 5-Bromo-4-chloro-3indolyl-beta-D-galactopy-ranoside (X-gal), phenylmethanesulfonyl fluoride (PMSF) and o-nitrophenyl-b-D-galactopyranoside (ONPG) were purchased from Sangon Co., China. AHLs were detected by Agrobacterium tumefaciens KYC55, which shows a sensitive response to a wide spectrum of AHLs (Joelsson and Zhu, 2006).
Another reporter strain Aeromonas sp. (pBB–LuxR) expressing green fluorescent protein (GFP) under induction of AHLs was used to in situ detect AHLs in the activated sludge (Chong et al., 2012).
2.2. Sludge extracts and autoclaved sludge extracts MBR operation and sludge characteristics are described in Supplementary Materials. Aliquots of 4 ml sludge samples from an MBR were centrifuged at 5000g for 10 min. The pelleted sludge was washed three times using equal volume of phosphate buffered saline (PBS) solution containing 1.0 mM PMSF to inhibit proteases, and then resuspended in 1 ml PBS-PMSF. The sludge incubated in an ice-water bath was subjected to ultrasonic disruption (80 w power output, 300 times, each time for 3 s followed by 3-s interval) using an ultrasonic cell disruptor (Ningbo Scientz Biotechnology Co., China) to lyse cells, followed by centrifugation at 12,000g for 20 min. The extracted supernatants were filtered through a 0.22 lm filter. The filtrates were used to analyze AHL-degrading activity and autoclaved at 105 °C for 20 min to inactivate the AHL-hydrolyzing or AHL-modifying enzymes when necessary.
2.3. AHL degradation tests The sludge extracts and autoclaved ones were mixed with equal volume of PBS solution containing 2 lM AHL. Here, 3-oxo-C8-HSL, which is widely utilized by a variety of bacteria (Barnard and Salmond, 2007; Horng et al., 2002), was chosen as a model AHL. The corresponding mixed solutions without AHL were used as two negative controls, and 1-lM AHL PBS solution was used as positive control. These five mixtures were incubated at 30 °C. Specified volumes of liquid samples were taken out every 2 h, and then incubated in a water bath at 90 °C for 10 min to inactivate enzymes. The as-obtained liquid samples were stored at 20 °C before analyzing the residual 3-oxo-C8-HSL. The protein contents of the five mixtures were determined by the bicinchoninic acid assay using protein assay kit (Sangon Co., China). The relative content of 3-oxo-C8-HSL in the samples was determined following the method described by Joelsson and Zhu (2006). The assay of b-galactosidase activity was conducted according to Miller and the specific activity was expressed in Miller Units (Miller, 1972).
AHL+PBS AHL+autoclaved sludge extract AHL+sludge extract PBS+autoclaved sludge extract PBS+sludge extract
300
-galactosidase activity /(MILLER UNITS)
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100
0 0
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Time (h) Fig. 1. Residual AHLs detected by A. tumefaciens KYC55 after 6-h incubation. bGalactosidase activity of AHL + PBS, AHL + autoclaved sludge extract, AHL + sludge extract, PBS + autoclaved sludge extract, and PBS + sludge extract.
Please cite this article in press as: Song, X.-N., et al. Quorum quenching is responsible for the underestimated quorum sensing effects in biological wastewater treatment reactors. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.08.027
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2.4. In situ detection of AHLs by confocal laser scanning microscopy (CLSM) AHL was in situ detected by CLSM following the method of Chong et al. (2012) with slight modification. Overnight cultures of AHLs reporter Aeromonas sp. (pBB–LuxR) were washed and diluted (1:5) with autoclaved synthetic wastewater (see Supporting Information, SI). Sludge samples from the MBR were washed and resuspended in equal volume of autoclaved synthetic wastewater. Then, 100 ll of sludge suspension was mixed with 100 ll of Aeromonas sp. (pBB–LuxR) for incubation. After 5-h incubation,
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green fluorescence was observed using CLSM (FV1000-IX81, Olympus Co., Japan). Autoclaved sludge samples were used as negative controls. 3. Results and discussion 3.1. AHL degradation by sludge extracts In this study, the AHLs degradation activity of the sludge extracts was examined. Fig. 1 shows the quenching effect of the sludge extracts on AHL during 6-h incubation period. The initial
Fig. 2. CLSM images of the activated sludge sample without the AHL reporter strain (A), with the reporter strain (B), and autoclaved sludge sample with the reporter strain (C).
Please cite this article in press as: Song, X.-N., et al. Quorum quenching is responsible for the underestimated quorum sensing effects in biological wastewater treatment reactors. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.08.027
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X.-N. Song et al. / Bioresource Technology xxx (2014) xxx–xxx
protein concentration in all the reaction mixtures was 728 ± 21 lg/ ml. The 3-oxo-C8-HSL after 6-h incubation with the sludge extracts hardly induced the b-galactosidase expressed by reporter A. tumefaciens KYC55, suggesting the degradation of 3-oxo-C8-HSL. While the b-galactosidase activities had no significant difference between the samples incubated with medium only or with autoclaved sludge. This result suggests that some thermally unstable substances existed in the sludge extracts, which could degrade AHLs or inhibit their activity, and thus became inactivated after autoclaving. Similar results were also obtained for the sludge from a granular sludge reactor operated at our laboratory (Figure S1). These results confirm the existence of QQ activity in the activated sludge process. 3.2. Presence of AHLs around flocs The presence of AHLs in the activated sludge was investigated, firstly by in situ detection. Fig. 2 shows the response of the AHL reporter strain Aeromonas sp. (pBB–LuxR) to sludge samples after 5-h incubation. The sludge without dose of the reporter bacterium showed no green fluorescence (Fig. 2A). In comparison, only light fluorescence was detected for the autoclaved sludge sample when incubated with the reporter (Fig. 2C), while strong fluorescence was observed for the activated sludge sample with dose of the reporter (Fig. 2B). A comparison between the fluorescence image and the phase-contrast image reveals that the reporter cells with green fluorescence were mainly attached on the surface of sludge flocs, rather than in the bulk solution. Such a result implies that the AHLs concentration was sufficiently high at the sludge floc surface to induce the fluorescence of the reporter bacterium, but was not sufficient to reach a relevant concentration level in the solution. Furthermore, efforts were made to concentrate and detect AHLs ex situ from the bulk solution of the bioreactors. Several methods, including bioreporters and high-performance liquid chromatography with tandem mass spectrometric detection, were used in this study (see Supplementary Materials). Surprisingly, none of them showed the presence of AHLs. Taking into account this result and the QS in situ detection result together, it is assumed that AHLs might have been secreted by the QS bacteria and then ingested or degraded by adjacent bacteria within a short time period. The local concentration of AHLs might be sufficiently high to trigger biological events for surrounding bacteria, but their content would become undetectable levels at reactor scale. This is reasonable from an energy and carbon source consumption point of view, because it would be uneconomical for the microbial community, which accounts for a small part of volume in bioreactor, to synthesize a large quantity of AHLs to fill the bulk solution. Moreover, the extracellular polymeric substances (EPS) of sludge are constituted by many macromolecules, such as polysaccharides, proteins, nucleic acids, and lipids, etc. These macromolecules contain diverse charged groups (e.g., carboxyl, phosphoric, sulfhydryl, phenolic and hydroxyl groups) and apolar groups (e.g., aromatics, aliphatics in proteins, and hydrophobic regions in carbohydrates). AHLs contain carbonyl and amide groups, which might form hydrogen bond with hydroxyl groups. Besides, the alkyl side chain of AHL might form complex with the apolar groups of EPS through hydrophobic interactions. Thus, EPS have a potential to hinder the diffusion of AHLs through group interactions. Therefore, no AHLs could be detected ex situ in the reactors. Nevertheless, the local concentration of AHLs at floc surface was still sufficiently high to induce reporter gene expression. This suggests that such a level of AHLs might play a role in regulating physiological and biological activities of neighboring cells. Our results also imply that, due to such an endogenous QQ effect, the role of QS in activated sludge reactors might have been underestimated in previous studies.
3.3. Presence of both AHLs-producing and -quenching bacteria If the above assumption that QQ bacteria lead to the failure of AHL ex situ detection is true, such QQ bacteria should be found in activated sludge samples. Thus, bacterial screening (see Supplementary Materials) was conducted to explore the microbial communities of the activated sludge. Both AHLs-producing and AHLs-degrading bacteria were isolated from the activated sludge. One of the isolates was Bacillus thruingiensis, which has been widely reported to harbor aiiA gene for encoding a lactone-degrading enzyme (Dong et al., 2002; Lee et al., 2002). Another isolate was Aeromonas hydrophila, which utilizes two types of AHLs as autoinducer signals (Lynch et al., 2002). These bacteria have been widely found in wastewater treatment bioreactors. Denaturing gradient gel electrophoresis (DGGE) analysis (see Supplementary Materials and further sequencing confirm the presence of these isolates in the activated sludge (Fig. 3). This result also shows that the QS and QQ effects occurred simultaneously in activated sludge reactors. The physiological–biological activities of activated sludge induced by either QS or QQ alone can significantly affect the performance of wastewater treatment bioreactors (Shrout and Nerenberg, 2012). However, the coexistence of QS and QQ in activated sludge systems has not been fully elucidated so far. In previous studies, it was found that Agrobacterium tumefaciens could produce 3-oxo-C8-HSL and modulate its level via two lactonases encoded by attM and aiiB (Haudecoeur et al., 2009). The QS pathways and the QQ gene of A. tumefaciens coordinate to express virulence functions of A. tumefaciens optimally (Haudecoeur et al., 2009). In addition, two rhizosphere microbes, Acinetobacter and Burkholderia, also possess both AHL-dependent QS and QQ activities and can regulate the whole rhizosphere bacterial community to attenuate toxicity to plants (Chan et al., 2011). In addition to lactonase and acylase, both of which are identified in diverse bacteria, oxido-reductase and oxidase are recently identified and far less common. Moreover, oxido-reductase and oxidase are able to change AHLs, rather than hydrolyzing AHLs (Uroz
Fig. 3. DGGE of 16S rRNA gene sequence from (1) Bacillus thruingiensis; (2) Aeromonas hydrophila; and (3) activated sludge samples.
Please cite this article in press as: Song, X.-N., et al. Quorum quenching is responsible for the underestimated quorum sensing effects in biological wastewater treatment reactors. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.08.027
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et al., 2009). The modified AHL, although still functionally active, may fail to bind specifically to its receptors, thus causing disturbance to the activation of QS-mediated genes regulated by a particular AHL (Uroz et al., 2009). Therefore, the roles of both QQ and QS in activated sludge reactors should be given more attention. In addition, our study also suggests that more sensitive in situ analytical methods should be needed for further investigations into these processes. 4. Conclusions In this study, the existence of endogenous QQ effect in wastewater treatment bioreactors is confirmed in situ, and its roles in regulating QS are also identified. Our results clearly indicate that, due to such an endogenous QQ effect, the role of QS in activated sludge reactors might have been underestimated previously. Activated sludge properties and reactor performance could be tuned by appropriately balancing the endogenous QQ and QS in bioreactors. Furthermore, the developed method could be used to detect QQ activity in situ for monitoring QQ in other bioreactors. Acknowledgements The authors wish to thank the Natural Science Foundation of China (51129803), and the Program for Changjiang Scholars and Innovative Research Team in University, China for the partial support of this study. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2014. 08.027. References Barnard, A.M.L., Salmond, G.P.C., 2007. Quorum sensing in Erwinia species. Anal. Bioanal. Chem. 387, 415–423. Chan, K.G., Atkinson, S., Mathee, K., Sam, C.K., Chhabra, S.R., Camara, M., Koh, C.L., Williams, P., 2011. Characterization of N-acylhomoserine lactone-degrading bacteria associated with the Zingiber officinale (ginger) rhizosphere: coexistence of quorum quenching and quorum sensing in Acinetobacter and Burkholderia. BMC Microbiol. 11, 51. Cheong, W.S., Lee, C.H., Moon, Y.H., Oh, H.S., Kim, S.R., Lee, S.H., Lee, C.H., Lee, J.K., 2013. Isolation and identification of indigenous quorum quenching bacteria,
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