Isolation and Identification of Indigenous Quorum Quenching Bacteria

Fungal Quorum Quenching: A Paradigm Shift for Energy Savings in Membrane Bioreactor (MBR) for Wastewater Treatment. Kibaek Lee , Seonki Lee , Sang ...
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Isolation and Identification of Indigenous Quorum Quenching Bacteria, Pseudomonas sp. 1A1, for Biofouling Control in MBR Won-Suk Cheong,† Chi-Ho Lee,‡ Yun-Hee Moon,† Hyun-Suk Oh,† Sang-Ryoung Kim,† Sang H Lee,† Chung-Hak Lee,*,† and Jung-Kee Lee*,‡ †

School of Chemical and Biological Engineering, Seoul National University, Seoul 151-744, Korea Departments of Life Science and Genetic Engineering, Paichai University, Daejeon 302-735, Korea

‡

ABSTRACT: Recently, interspecies quorum quenching by bacterial cells has been reported as a novel approach to the biofouling control in a membrane bioreactor (MBR) for wastewater treatment. In this study, a novel quorum quenching (QQ) bacterium, Pseudomonas sp. 1A1, was isolated from lab-scale MBR and was encapsulated in a microbial vessel made of a hollow fiber microporous membrane for the biofouling control in MBR. Pseudomonas sp. 1A1 was most likely to produce AHL-acylase and degraded to a wide range of N-acyl homoserine lactones (AHLs), although there was a large difference in the degradation rate of each AHL. It has an extracellular QQ activity, i.e., it produces QQ enzymes, and excretes them out of the cell. The microbial vessel encapsulating Pseudomonas sp. 1A1 was applied to a lab-scale MBR and proved substantial inhibition of the membrane biofouling.

1. INTRODUCTION The market growth of MBR technology has been driven by the more widespread implementation of water reuse technologies that are promoted by a combination of increasing water scarcity and increasingly stringent legislation.1 However, membrane biofouling that causes severe flux decline, short membrane lifespan, and increase of energy consumption still remains one of the major research issues in this area, restricting widespread application of MBR technology. Recently, the concept of bacterial quorum sensing was introduced to a MBR as a new biofouling control paradigm.2 Microorganisms can use quorum sensing systems to coordinate their communal behaviors including biofilm formation. AHLacylase or AHL-lactonase, which degrade the quorum sensing signal molecules (i.e., quorum quenching) N-acyl homoserine lactone (AHL), has proven its potential to inhibit biofouling in MBR.3,4 Following the enzymatic quorum quenching (QQ), Oh et al.5 isolated bacteria (Rhodococcus sp. BH4) producing the QQ enzyme and developed a microbial vessel encapsulating the QQ bacteria. They observed that an internal submerged MBR equipped with the microbial vessel has a much lower biofouling tendency compared with a conventional MBR. Therefore, the bacterial QQ could be an attractive approach to the control of biofouling in MBR because it gives rise to no declining of treatment efficiency, no consumption of additional energy, and no production of peculiar byproducts. Although there might be many other QQ bacteria in nature,6−8 however, we still do not know which strain is more appropriate for a real application of QQ to MBR for wastewater treatment. Thus, continuous searching for novel QQ strains should be required for further development of the bacterial QQ to mitigate the biofouling in MBR. In this study, we isolated a novel QQ bacterium, Pseudomonas sp. 1A1 from the lab-scale MBRs in which sludge from real wastewater treatment plants were inoculated and investigated the characteristics of the strain, particularly © 2013 American Chemical Society

comparing it with the previously reported QQ bacterium, Rhodococcus sp. BH4. Various characteristics of Pseudomonas sp. 1A1 were examined in terms of degradation rates of various AHLs, location of the QQ enzyme, specific growth rates, survival competition with other microbes, etc. Finally, we prepared a microbial vessel encapsulated with Pseudomonas sp. 1A1 and tested its ability to inhibit the membrane biofouling in continuous MBR.

2. MATERIALS AND METHODS 2.1. Isolation of Indigenous QQ Bacteria. In order to isolate indigenous QQ bacteria, activated sludge was sampled from two lab-scale MBRs in which sludge taken from a real wastewater treatment plant (Sihwa or Tancheon, Korea) were inoculated. Each sample of mixed bacteria was separately seeded in a minimal medium containing AHL (2.5 mM, N(hexanoyl)-L-homoserine lactone (C6-HSL)) as the sole carbon source and incubated for 3 days. Then, 1 vol% of the culture medium was transferred to a new minimal medium containing AHL. For each sample, this transfer procedure was repeated three times to ensure the isolation of bacteria that can live with only AHL as the carbon source. Then the final culture was spread on the LB agar to isolate single colonies, and they were separately incubated again in the minimal medium containing AHL. The 16S rRNA genes of the isolated strains were amplified by PCR using two universal primers: H+ (5′-GAGTTTGATCCTGGCTCAG-3′) and E- (5′-AGAAAGGAGGTGATCCAGCC-3′). Then PCR denaturation was performed Special Issue: Enrico Drioli Festschrift Received: Revised: Accepted: Published: 10554

November 15, 2012 January 28, 2013 January 29, 2013 January 30, 2013 dx.doi.org/10.1021/ie303146f | Ind. Eng. Chem. Res. 2013, 52, 10554−10560

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30 °C with orbital shaking (200 rpm). The cell concentrations were determined by measuring OD600 at intervals during incubation. The specific growth rates (μ) of Pseudomonas sp. 1A1 and Rhodococcus sp. BH4 were determined from the logarithmic scale of growth curve of each.13 We also investigated the survival competition between Pseudomonas sp. 1A1 and Rhodococcus sp. BH4. A total of 500 μL of each strain (OD600 4.0) was mixed, and the mixture was inoculated in a sterilized volumetric flask with 100 mL of LB medium and incubated at 30 °C with orbital shaking (200 rpm). The Gram staining procedures14 were applied at the beginning and 36 h of incubation to differentiate Pseudomonas sp. 1A1 (Gram-negative) from Rhodococcus sp. BH4 (Grampositive). The following eight AHL molecules (Sigma-Aldrich, U.S.) were chosen to compare their degradation rates by the Pseudomonas sp. 1A1: C6-HSL, C8-HSL, C10-HSL, N(dodecanoyl)-DL-homoserine lactone (C12-HSL), N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6-HSL), N-(3-oxooctanoyl)-L-homoserine lactone (3-oxo-C8-HSL), N-(3-oxodecanoyl)-L-homoserine lactone (3-oxo-C10-HSL), and N-(3oxo-dodecanoyl)-L-homoserine lactone (3-oxo-C12-HSL). Equal volumes of a culture medium (OD600 1.0) and a solution containing 0.4 μM AHLs were mixed and incubated (30 °C, 200 rpm). The percentage of degraded AHL in 10 min was measured to determine the degradation rate of each AHL by strain 1A1. 2.6. Bioassay for Detecting AHL Molecules. The concentrations of AHL molecules were measured via luminescence method using the reporter strain of A. tumefaciens A136. First, the reporter strain and AHL sample were mixed and loaded on the microwell plate. The microwell plate loaded with the mixed solution was placed on the incubator to keep the temperature at 30 °C for 1.5 h, and then the Beta-Glo Assay System (Promega, U.S.) was added to the solution for the luminescent reaction with β-galactosidase produced by the reporter strain. After 30 min, the luminescence was measured by a luminometer (Synergy 2, Biotek, U.S.). The amounts of AHLs were calculated using relationship equations based on the calibration curve derived from standard samples of AHLs. 2.7. Preparation of Microbial Vessel. For the application of Pseudomonas sp. 1A1 to the continuous MBR, the bacterium was encapsulated in the microbial vessel.5,15 A polyethylene hollow fiber membrane (Econity Co. Ltd., Korea) was used for the microbial vessel (Figure 1). The bottom side of the microbial vessel was sealed with epoxy resin, and the QQ

at 95 °C for 5 min, followed by 30 cycles at 95 °C for 45 s, 54 °C for 45 s, and 72 °C for 90 s. The resulting PCR product was sequenced by an ABI3700 automatic sequencer (Applied Biosystems, U.S.), and the sequence was identified by using the EzTaxon server 2.1 (www.eztaxon.org).9 Finally, in order to screen out the isolated strains showing quorum sensing activity, each strain was examined by a crossfeeding assay for the detection of AHL production.10 2.2. Comparison of QQ Activity among the Isolated Strains. In order to select appropriate QQ bacteria in MBR, the QQ activity of the supernatant as well as the whole cell was tested for each strain’s culture medium. Each strain was incubated in 20 mL of Luria−Bertani (LB) medium for 1 day (30 °C, 200 rpm), and then the optical density at 600 nm (OD600) of each culture was measured by spectrophotometry. Each strain’s culture medium was fractionated into the supernatant and the cells by centrifugation (12,000 g, 10 min), and then the cell pellet was resuspended in 10 mL of Tris-HCl buffer (50 mM, pH 7.0). Finally, the supernatant and whole cell suspension were diluted by using Tris-HCl buffer to adjust their concentrations to the equivalent of OD600 1.0, and both QQ activities of the supernatant and whole cell suspension for each strain’s culture medium were determined by measuring the degradation of N-(octanoyl)-DL-homoserine lactone (C8HSL) (200 nM) in 1 h. 2.3. Comparison of QQ Enzyme Location between Strain 1A1 and BH4. Among the isolated strains, Pseudomonas sp. 1A1 was selected and compared to another QQ bacterium, Rhodococcus sp. BH45 with respect to the location of QQ enzyme. The indicating agar plate was prepared by mixing an overnight culture of Agrobacterium tumefaciens A136 (AHL biosensor),11 LB agar, and X-gal. A sterilized 0.45 μm filter (Super 450, Pall Corporation, U.S.) was soaked in 100 mg/L of N-(decanoyl)-DL-homoserine lactone (C10-HSL) and then put on the agar plate. On the filter, two strains were loaded separately. After overnight incubation, we checked whether or not a blue color develops around each colony in order to identify the location of QQ activity for each strain. 2.4. Identification of QQ Enzyme of Pseudomonas sp. 1A1 . Pseudomonas sp. 1A1 was investigated for whether or not it has an AHL-acylase homologue gene. The primer sequences for PCR, which were based on the nucleotide sequence of AHL-acylase homologue genes from the P. putida GB-1 genome sequence annotated as penicillin amidase, were designed as following: pvdQ homologue (PF - GGC TAT GCC TAT GCC CAG, PR - TGC AAT ACA CGT GTG TTC TC), quiP homologue (QF − TAT CGC GAC AAG CTG CCG, QR - TAA CGC CCA TCC CAG CC), hacB homologue (HF - TAC CGT GCG CTG GGC TA, HR - TCG TAC CAC ACG GCT GG).12 The PCR protocol used is given as denaturation at 95 °C for 5 min, followed by 30 cycles at 95 °C for 45 s, 58 °C for 40 s, and 72 °C for 60 s. Molecular weight of the enzyme that Pseudomonas sp. 1A1 produces was also estimated. Supernatant of strain 1A1 culture medium was fractionated into three portions using centrifugal filters with molecular weight cut off of 3 kDa, 50 kDa, and 100 kDa. The QQ activity of each fraction was examined by measuring the degradation rate of C8-HSL (200 nM) during 1 h. 2.5. Characterization of QQ Bacteria. The growth rate of Pseudomonas sp. 1A1 was compared with that of Rhodococcus sp. BH4. The two stains were inoculated separately in a sterilized volumetric flask with 100 mL of LB medium and incubated at

Figure 1. Photograph and enlarged diagram of a microbial vessel. 10555

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bacteria were packed into the microbial vessel from the open top side using a peristaltic pump. The same type of vessel without encapsulated bacteria was named as the “vacant vessel”. The other specifications of microbial vessel are listed in Table 1.

Table 2. Conditions of MBR Operation working volume (L) SRT (d) HRT (h) pH membrane type nominal pore size (μm) membrane area (cm2) flux (L/m2/h) MLSS (mg/L) feed COD (mg/L) COD removal efficiency (%)

Table 1. Specifications of Microbial Vessel Encapsulating Pseudomonas sp. 1A1 membrane material

polyethylene

pore size of a fiber (μm) number of fibers/vessel number of vessels/reactor length (cm) outer surface area (cm2) inner volume (mL) encapsulated biomass of Pseudomonas sp. 1A1 (mg/vessel)

0.4 24 4 10 39.2 0.32 120

F/M ratio

2.5 30 8 6.8−7.0 PVDF, hollow fiber 0.04 155.2 25 7600−8000 575 (±25) broth: 96−97 permeate: 97−98 0.20−0.22

3. RESULTS AND DISCUSSION 3.1. A novel QQ bacterium, Pseudomonas sp. 1A1. Six strains of QQ bacteria were isolated from the lab-scale MBRs in which sludge taken from real wastewater treatment plants were inoculated. Their 16S rRNA gene sequences were identified using the EzTaxon server (Table 3). Among the six isolated

2.7. MBR Operation. Activated sludge taken from a wastewater treatment plant (Sihwa, Korea) was acclimated to synthetic wastewater prior to the MBR operation. The composition of the synthetic wastewater was as follows:16 glucose, 400 mg/L; yeast extract, 14 mg/L; bactopeptone, 115 mg/L; (NH4)2SO4, 104.8 mg/L; KH2PO4, 21.75 mg/L; MgSO4, 15.63 mg/L; FeCl3, 0.075 mg/L; CaCl2, 2.45 mg/L; MnSO4, 1.8 mg/L; and NaHCO3, 255.5 mg/L. Two continuous MBRs, the control MBR with four vacant vessels and the MBR with four microbial vessels, were designed and operated in a way similar to that described by other MBR researchers (Figure 2).16,17 The effective area of the hollow

Table 3. List of Quorum Quenching Isolates from Lab-Scale MBRs strain

16S rDNA

1A1

Pseudomonas monteilii CIP 104883(T) Pseudomonas koreensis Ps 9-14(T) Pseudomonas extremorientalis KMM 3447(T) Bulkholderia multivorans LMG 13010(T) Rhodococcus qingshengii djl-6(T) Stenotrophomonas rhizophila e-p10(T)

1A311 1A2 N2221 1C131 3A121

16S rDNA gene sequence homology (%)

source

97.4

Sihwab

(99.1)a

Sihwa

(99.6)

Sihwa

(97.7)

Tancheonc

(98.7)

Sihwa

(98.1)

Shiwa

a

Values in parentheses indicate sequence homologies using partially sequenced data obtained with only primer A-(5′-GWA TTA CCG CGG CKG CTG-3′) for sequencing. bLab-scale MBR in which was inoculated activated sludge from a real wastewater treatment plant (Sihwa, Korea). cLab-scale MBR in which was inoculated activated sludge from a real wastewater treatment plant (Tancheon, Korea).

strains, however, strain 1A311 and N-2221 were identified to produce quorum sensing signal molecules (AHLs) (Figure 3b,d). It is well-known that some bacteria could be related to quorum sensing and quorum quenching simultaneously.18 For this reason, strain 1A311 and strain N-2221 were screened out from the selection of QQ bacteria for the control of biofouling in MBR. The QQ activities of the remaining four strains were compared with each other (Figure 4). The culture medium of each strain was separated into the whole cell and supernatant to differentiate the extracellular QQ from the intracellular QQ. The QQ activity of the whole cell represents the intracellular QQ activity of a given strain, while that of the supernatant does the extracellular one. For three strains (1A1, 1A2, and 3A121), their external QQ activities were measured to be higher than their intracellular one, whereas the strain 1C131 showed higher intracellular activity. Taking into account the total QQ activity,

Figure 2. Schematic diagram of a lab-scale MBR system.

fiber membrane module (ZeeWeed 500, GE-Zenon, U.S.) for water filtration was 155.2 cm2. The hydraulic retention time (HRT) and sludge retention time (SRT) were set to 8 h and 30 d, respectively. The mixed liquor suspended solids (MLSS) in both reactors were maintained within the range of 7600−8000 mg/L. The other operating parameters for the continuous MBR are listed in Table 2. 10556

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Figure 5. Bio agar assay for the locations of quorum quenching enzymes produced by Pseudomonas sp. 1A1 and Rhodococcus sp. BH4.

Figure 3. Bio agar assay of AHLs for each strain (a) strain 1A1, (b) strain 1A311, (c) strain 1A2, (d) strain N-2221, (e) strain 1C131, and (f) strain 3A121.

only around the colony of strain 1A1. This implies that Pseudomonas sp. 1A1 produces QQ enzymes and excretes them out of the cell, whereas Rhodococcus sp. BH4 produces QQ enzymes and keeps them inside the cell. 3.2. Identification of AHL-acylase of Pseudomonas sp. 1A1. As shown in Table 3, the 16S rRNA gene sequence of strain 1A1 showed the highest sequence identity (98.9%) when compared to that of Pseudomonas monteilii CIP 104883(T), but it also shared a high degree of sequence identity (98%) with that of P. putida GB-1 (data not shown) of which its whole genome sequence is already known (Gene ID 5867866). Three AHL-acylase homologue genes that show a high identity with pvdQ (Gene ID, 882260), quiP (Gene ID, 880272), and hacB (Gene ID, 877593) of P. aeruginosa PAO1 were found in the P. putida GB-1 genome sequence. Three PCR-amplified gene fragments from the strain 1A1 were compared with those three AHL-acylase homologue genes from the P. putida GB-1 genome annotated as penicillin amidase, and all of them showed over 99% of identity (data not shown). They also showed a high identity with the known AHL-acylase genes of P. aeruginosa PAO1. The deduced amino acid sequence of three gene fragments showed 59%, 58%, and 69% identities with that of pvdQ, quiP, and hacB, respectively (data not shown). To further confirm what kind of enzyme the strain 1A1 produces, the size of extracellular enzyme was estimated. The supernatant of the strain 1A1 culture medium was separated into three fractions using MWCO centrifugal filters based on their molecular weight: < 3 kDa, < 50 kDa, and 95%) with no significant difference between the two reactors. The rate of TMP rise-up is an important factor representing the extent of biofouling in the constant flux MBR operation. At the first cycle, it took about 2.2 days to reach the TMP of 50 kPa in the control MBR, whereas 5.3 days to reach the same TMP in the MBR with a microbial vessel (Figure 12). The trend was repeated in the second cycle. In two consecutive operations of the continuous MBR, the TMP in the control MBR increased more rapidly than that of the MBR with microbial vessels. Therefore, the microbial vessel encapsulated with QQ bacteria demonstrated its potential for efficient biofouling control in MBR.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (C.-H.L.), [email protected] (J.-K.L.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research is supported by the Korea Ministry of Environment as “Converging Technology Project” (2012001440001 and 2012001440002). 10559

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