Elsevier

Water Research

Volume 177, 15 June 2020, 115756
Water Research

Analysis of N-Acy-L-homoserine lactones (AHLs) in wastewater treatment systems using SPE-LLE with LC-MS/MS

https://doi.org/10.1016/j.watres.2020.115756Get rights and content

Highlights

  • SPE-LLE is a breakthrough for AHLs quantification in complex wastewater.

  • SPE-LLE process alleviates matrix effects effectively compared to single SPE.

  • Matrix-matched calibration curve is not required for accurate analysis of AHLs.

  • LODs of the AHLs were below 5 ng/L for domestic and industrial wastewater.

  • Wide spectrum (C4 to C14) of AHLs were detected in 5 wastewater treatment systems.

Abstract

The occurrence and distribution of N-acyl homoserine lactones (AHLs) in membrane bioreactors (MBRs) treating wastewater has garnered much attention as they have been shown to play critical role in biofouling. There is a need to develop a single method capable of analysing AHLs in various wastewater with comparable and reliable performance. A novel and robust method was proposed for trace analysis of 11 AHLs in wastewater treatment systems treating domestic and industrial wastewater. This method utilised solid phase extraction (SPE) to extract AHLs from wastewater followed by liquid-liquid extraction (LLE) to extract AHLs from the SPE eluant, and used N-heptanoyl-dl-homoserine lactone (C7-HSL) as an internal standard. There was no need to prepare matrix-matched calibration curve for accurate quantification of AHLs in the liquid chromatography tandem mass chromatography (LC-MS/MS) analysis. The developed method was validated with six different types of domestic and industrial wastewater with regard to AHLs recoveries and matrix effects. For treated domestic and industrial wastewater, the relative recoveries ranged from 75% to 130% and the matrix effects ranged from 89% to 122%. This method exhibited remarkable improvement compared with single SPE. The results also indicated that inclusion of LLE after SPE could effectively alleviate matrix effects, which may be because of the removal of relatively hydrophilic interferences by using dichloromethane to extract AHLs from the SPE eluant composing of methanol and water. The limits of detection of the AHLs were all below 5 ng/L for the tested wastewater samples. The developed method of SPE-LLE with LC-MS/MS was applied to analyse AHLs in four lab-scale and one pilot-scale wastewater treatment systems. Wide spectrum of AHLs with alkanoyl chains ranging from C4 to C14 were detected with concentrations ranging from 2.7 to 299.2 ng/L. This method is capable of identifying and quantifying trace levels of AHLs in various wastewater treatment systems and can help us better understand the mechanisms of AHL-mediated quroum sensing in various wastewater treatment systems.

Introduction

Membrane bioreactor (MBR) technology is an integration of a physical separation process (i.e., the membrane) into the activated sludge process for solids and liquid separation. Despite the increasing popularity in its application for both industrial and domestic wastewater treatment in the past decade, membrane fouling in MBRs remains a largely unsolved impediment towards greater adoption of this technology. Membrane fouling in MBRs treating wastewaters is dominated by biofilm formation on the membrane’s surface and is a natural process. It was proposed that biological methods would sustain any fouling alleviation strategy. Quorum quenching (QQ) is a method to block the naturally occurring quorum sensing (QS) process, which is a form of bacterial communication between cells in which bacteria secrete and sense specific chemicals. N-acyl homoserine lactones (AHLs) were found to be the main signalling compound in Gram-negative bacteria, which dominate the bacterial population in biological wastewater treatment systems (Huang et al., 2016). AHL molecules are composed of a hydrophilic lactonized homoserine moiety with a hydrophobic acyl chain that varies between 4 and 18 carbons, usually in increments of 2 carbon units (Churchill and Chen, 2011). Importantly, these AHL-based QS systems are responsible for biofilm formation, swarming motility (Labbate et al., 2004) and the production of microbial by-products, which provide architectural support for biofilms in MBRs (Tan et al., 2014). The application of QQ in MBRs has shown to retard membrane fouling by slowing growth in transmembrane pressures by up to 500% (Lee et al., 2016).

Detection, characterisation and quantification of AHL molecules in the MBRs are essential to achieve the targeted effect from the QQ process. Ma et al. (2018) detected four types of AHLs between C6-HSL and C12-HSL in aerobic granulation bioreactors and differed depending on environmental condition. In contrast, AHLs of C10-HSL and longer were detected from various MBRs (Oh et al., 2012; Yeon et al., 2009). However, Yu et al. (2016b) detected only C6-HSL and C8-HSL AHLs in their MBRs and the concentrations differed with solids retention time. These results indicated that while AHLs were universal signalling molecules in Gram-negative bacteria, the composition of AHLs was dependent on the bacterial community and environmental conditions of the systems. Therefore, a clear profile of the AHL composition specific to each system must be understood to achieve maximum impact from the QQ process. As such, standardisation of characterisation methods and improvements their detection limits must be achieved to accurately determine the AHL profiles.

Various methods have been used to extract and identify AHLs from pure cultures and environmental samples. Liquid-liquid extraction (LLE) (Brelles-Mariño and Bedmar, 2001) and solid-phase extraction (SPE) (Gould et al., 2006) were routinely applied to samples to isolate and concentrate AHLs such that artefacts from the bacterial supernatants interfering downstream analyses may be reduced or removed. Bacterial sensors (Steindler and Venturi, 2007) were able detect femtomole levels of AHLs but require tailor-made strains for each AHL molecule and were time consuming. In contrast, thin layer chromatography (Shaw et al., 1997) was comparatively faster but hampered by the poor resolution. High performance liquid chromatography (HPLC) was, thus, the most commonly used method for characterising and quantifying AHLs. The further integration of mass spectrometry (MS) to HPLC or gas chromatography (GC) has been shown to enhance detection sensitivity and the spectrum of AHLs detected (Sun et al., 2018).

Since multiple processes, namely, extraction and analysis, are required for the complete characterisation and quantification of AHLs, optimization of the entire flow is crucial to develop suitable conditions for accuracy and reproducibility. Studies to optimise SPE with HPLC (Li et al., 2006) and SPE with LC-MS (Wang et al., 2017) have only been recently reported. It should be noted that low sample volumes (e.g., 2 mL and 10 mL) of spiked samples with high concentrations of AHLs (i.e., 8–15 μg/L) were extracted to evaluate the extraction efficiencies of the developed SPE methods in previous studies (Li et al., 2006; Wang et al., 2017). As such, large sample volume (e.g., 50 mL) is needed to accurately quantify AHLs that are mostly at ng/L levels in wastewater. To date, the study on analysis of AHLs using GC-MS is very limited. One study developed a GC-MS method for analysis of N-butyryl-l-homoserine lactone (C4-HSL) in wastewater through LLE, in which a high concentration of C4-HSL (i.e., 12.5 mg/L) was used to evaluate the extraction efficiency (Bakaraki et al., 2016). Much understanding is still lacking in the conditions required in the processes involved in SPE or LLE with GC-MS for AHL detection. Accurate characterisation is further hampered by the low concentration of AHLs naturally occurring in environmental samples (Wang et al., 2011). Moreover, AHLs loss due to adsorption by biomass or materials and to biological action was found to be significant (Tan et al., 2015). As such, extraction and concentration methods must be properly assessed for subsequent analyses to be conducted.

A further compounding problem in biological and environmental samples for the analyses with LC-MS or GC-MS is the matrix effect from environmental samples. Matrix effect is known to cause detrimental impacts on the analyses in terms of the detection and quantification limits and the accuracy and precision of results (Trufelli et al., 2011). Matrix effects can lead to either a suppression or enhancement in response (Van Eeckhaut et al., 2009), and is not known how they affect each type of AHLs. These effects may also be exerted by co-eluting components of the extraction processes (Van Eeckhaut et al., 2009). For example, metabolic by-products from bacteria supressed the detection response of long-chain AHLs, such as C12-HSL and C14-HSL (Morin et al., 2003). While Morin et al. (2003) developed an LC-MS method to detect and quantify AHLs up to picomolar concentrations in pure bacterial cultures with standard deviations of less than 10%. Environmental samples such as those from MBRs are known to have lower AHL concentrations and more complex matrices than pure culture samples, and few studies have conducted comprehensive analyses on matrix effect. The use of internal standards has been proposed to overcome matrix complexity. Isotopically labelled analogue internal standards were introduced into the samples to compensate for matrix effects (Vanderford and Snyder, 2006). However, these deuterated AHLs are neither readily available nor, to the best of our knowledge, produced by any commercial companies. As such, C7-HSL are proposed as the alternative internal standard since bacteria are not reported to produce this AHL. Feasibility studies must therefore be conducted to determine if C7-HSL is suitable as the internal standard in real wastewater samples. These characteristics necessitate in-depth studies to develop suitable extraction and analysis methods.

We conducted a holistic and systematic study to optimise the characterisation and quantification of AHLs, with further validation of the method from real environmental samples. Firstly, SPE and LLE processes were optimized to achieve the maximum recoveries of AHLs from aqueous sample. Subsequently, the performance of the developed method which combined the optimized SEP and LLE was evaluated using six types of domestic wastewater and industrial wastewater. The feasibility of using C7-HSL as internal standard also was studied. Finally, method was applied to detect the AHLs distribution of five wastewater treatment systems.

Section snippets

Chemicals and materials

Seven N-acyl-lactones and four N-3-oxoacylhomoserine lactones were used in this study. Synthetic AHLs (>97%) including N-butyryl-dl-homoserine lactone (C4-HSL), N-hexanoyl-dl-homoserine lactone (C6-HSL), N-(3-Oxohexanoyl-dl-homoserine lactone (3-oxo-C6-HSL), N-heptanoyl-dl-homoserine lactone (C7-HSL), N-octanoyl-dl-homoserine lactone (C8-HSL), N-(3-Oxooctanoyl)-l-homoserine lactone (3-oxo-C8-HSL), N-decanoyl-dl-homoserine lactone (C10-HSL), N-dodecanoyl-dl-homoserine lactone (C12-HSL),

SPE of AHLs

SPE with HLB or C18 cartridges have been applied to extract AHLs from aqueous samples (Hu et al., 2016; Li et al., 2006; Wang et al., 2017; Yu et al., 2016b). Previous studies suggested that HLB cartridge could recover short- to long-chain AHLs, while C18 cartridge only showed acceptable performance for medium-chain AHLs (Li et al., 2006; Wang et al., 2017). Thus, HLB is a promising sorbent for the extraction of AHLs with a wide range of polarities due to its capability of retaining hydrophilic

Conclusions

In this study, a novel and robust method was developed and validated to identify and quantify 11 types of AHLs in complex wastewater. The developed method employed the SPE to extract AHLs from wastewater and LLE to extract AHLs from the SPE eluant in sequence. Trace levels of AHLs could be quantified accurately using LC-MS/MS without inclusion of matrix-matched calibration curve. The main conclusions of this study are as follows:

  • 1.

    The excess matrices in SPE eluant could be greatly removed by

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

This research was supported by Singapore National Research Foundation under NRF-CRP17-2017- 01 (R-284-000-165-281).

References (37)

  • A. Van Eeckhaut et al.

    Validation of bioanalytical LC–MS/MS assays: evaluation of matrix effects

    J. Chromatogr. B

    (2009)
  • J. Wang et al.

    Development of an extraction method and LC–MS analysis for N-acylated-L-homoserine lactones (AHLs) in wastewater treatment biofilms

    J. Chromatogr. B: Anal. Technol. Biomed. Life Sci.

    (2017)
  • H. Yu et al.

    Core-shell structured quorum quenching beads for more sustainable anti-biofouling in membrane bioreactors

    Water Res.

    (2019)
  • X. Yue et al.

    Effects of Dissolved Organic Matters (DOMs) on Membrane Fouling in Anaerobic Ceramic Membrane Bioreactors (AnCMBRs) Treating Domestic Wastewater

    (2015)
  • H. Zhang et al.

    Multi-residue analysis of legacy POPs and emerging organic contaminants in Singapore’s coastal waters using gas chromatography–triple quadrupole tandem mass spectrometry

    Sci. Total Environ.

    (2015)
  • N. Bakaraki et al.

    Development of a sensitive liquid–liquid extraction method for the determination of N-butyryl-l-homoserine lactone produced in a submerged membrane bioreactor by gas chromatography mass spectrometry and deuterated anthracene as the internal standard

    Anal. Methods

    (2016)
  • M.E.A. Churchill et al.

    Structural basis of acyl-homoserine lactone-dependent signaling

    Chem. Rev.

    (2011)
  • A.W. Decho et al.

    Chemical challenges to bacterial AHL signaling in the environment

    Chem. Rev.

    (2011)
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    1

    Co-first authors. These authors contributed equally to this work.

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