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Quorum Quenching Bacteria Isolated from the Sludge of a Wastewater Treatment Plant and Their Application for Controlling Biofilm Formation
Quorum Quenching Bacteria Isolated from the Sludge of a Wastewater Treatment Plant and Their Application for Controlling Biofilm Formation
Journal of Microbiology and Biotechnology. 2014. Nov, 24(11): 1574-1582
Copyright © 2014, The Korean Society For Microbiology And Biotechnology
  • Received : July 07, 2014
  • Accepted : August 10, 2014
  • Published : November 28, 2014
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About the Authors
A-Leum Kim
Department of Biomedicinal Science and Biotechnology, Paichai University, Daejeon 302-735, Republic of Korea
Son-Young Park
Department of Biomedicinal Science and Biotechnology, Paichai University, Daejeon 302-735, Republic of Korea
Chi-Ho Lee
Department of Biomedicinal Science and Biotechnology, Paichai University, Daejeon 302-735, Republic of Korea
Chung-Hak Lee
School of Chemical and Biological Engineering, Seoul National University, Seoul 151-744, Republic of Korea
Jung-Kee Lee
Department of Biomedicinal Science and Biotechnology, Paichai University, Daejeon 302-735, Republic of Korea
leejk@pcu.ac.kr

Abstract
Bacteria recognize changes in their population density by sensing the concentration of signal molecules, N -acyl-homoserine lactones (AHLs). AHL-mediated quorum sensing (QS) plays a key role in biofilm formation, so the interference of QS, referred to as quorum quenching (QQ), has received a great deal of attention. A QQ strategy can be applied to membrane bioreactors (MBRs) for advanced wastewater treatment to control biofouling. To isolate QQ bacteria that can inhibit biofilm formation, we isolated diverse AHL-degrading bacteria from a laboratory-scale MBR and sludge from real wastewater treatment plants. A total of 225 AHL-degrading bacteria were isolated from the sludge sample by enrichment culture. Afipia sp., Acinetobacter sp. and Streptococcus sp. strains produced the intracellular QQ enzyme, whereas Pseudomonas sp., Micrococcus sp. and Staphylococcus sp. produced the extracellular QQ enzyme. In case of Microbacterium sp. and Rhodococcus sp., AHL-degrading activities were detected in the whole-cell assay and Rhodococcus sp. showed AHL-degrading activity in cell-free lysate as well. There has been no report for AHL-degrading capability in the case of Streptococcus sp. and Afipia sp. strains. Finally, inhibition of biofilm formation by isolated QQ bacteria or enzymes was observed on glass slides and 96-well microtiter plates using crystal violet staining. QQ strains or enzymes not only inhibited initial biofilm development but also reduced established biofilms.
Keywords
Introduction
Many bacteria can monitor their population density via a quorum sensing (QS) signaling system, which controls the expression of diverse genes in response to cell density [10 , 25] . N -Acyl-homoserine lactones (AHLs) are common signaling molecules used by mainly gram-negative bacteria [8 , 23 , 34] . Quorum sensing plays a major role in regulating biofilm formation, which shows typical group behavior, and the close correlation between QS and biofilm formation on membranes has been studied in detail [5 , 6] . Biofilm is an extracellular polymeric substance (EPS) produced by microbial cells that grow on a solid surface [6 , 9] . Because the biofilm is one of the major virulence factors in many pathogenic bacteria, QS can be an attractive target for antibiofilms in developing novel anti-infective agents [24 , 27] . Biofilms cause numerous problems not only from the medical viewpoint but also in industrial fields such as biofouling in membrane bioreactors (MBRs) [11 , 28 , 36] . There are several known strategies for disrupting a bacterial quorum sensing system, which is referred to as quorum quenching (QQ), and inhibiting biofilm formation [35] . One of the intervention strategies for AHL-based QS is the enzymatic degradation or modification of AHL signaling molecules for quorum quenching [7 , 32] . Several QQ enzymes that can degrade or modify the signal molecule AHL have been studied. There are two types of AHL-degrading enzymes; AHL-lactonase and AHL-acylase. AHL-lactonase cleaves the ester bond of the lactone ring [7 , 16] , resulting in N -acyl homoserine, and AHL-acylase cleaves the amide bond between the acyl chain and the homoserine lactone ring [12 , 17] . On the other hand, oxidoreductase modifies the AHL to 3-hydroxy AHL [3 , 30] , and the resulting hydrolyzed or modified chemicals do not function as signal molecules for biofilm formation [32] .
Recently, many QQ bacteria and enzymes have been applied to MBRs for advanced wastewater treatment as a new strategy to control biofouling, and effective reduction of biofouling by quorum quenching enzymes that inactivate the AHLs has been reported [2 , 19 , 37] . The purpose of our study was to isolate indigenous quorum quenching bacteria that can inhibit biofilm formation and eventually be used to reduce biofouling in MBR systems of wastewater treatment plants. We isolated and analyzed diverse AHL-degrading bacteria from the sludge of real wastewater treatment plants. Biofilm formation was inhibited by the isolated QQ bacteria or enzymes.
Materials and Methods
- Bacterial Strains and Culture Media
As reporter strains for the detection of N -acyl homoserine lactone, Chromobacterium violaceum CV026 [14 , 29] and Agrobacterium tumefaciens NT1 (pDCI41E33) [4] were used, and cultured in LB and the defined minimal medium [38] at 30℃, respectively. For the bioassay plate using A. tumefaciens NT1 (pDCI41E33), it was cultivated overnight in the minimal medium, and then added to a minimal agar medium at an optical density of 0.1. Subsequently, 5 ml of the mixture was overlaid on the surface of a minimal agar plate containing 40 µg/ml of 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside. AHL-degrading bacteria were screened on a modified AHL-minimal medium (pH 5.5 or 6.0) containing 2.5 mM N -butyryl-L-homoserine lactone (BHL), N -hexanoyl-L-homoserine lactone (HHL), N -3-oxohexanoyl-L-homoserine lactone (OHHL), or N -octanoyl-L-homoserine lactone (OHL) as the sole carbon source and NH 4 Cl as the nitrogen source [15] . All isolated bacteria, including the biofilm producer Aeromonas sp. T3-4 and Raoultella sp. D1-6, were cultivated in LB medium at 30℃.
- Screening and Isolation of AHL-Producing and AHL-Degrading Bacteria
Activated sludge sampled from wastewater treatment plants (Okcheon and Daejeon, Korea) and a laboratory-scale MBR in which sludge was taken from the same place were used as seed for screening of AHL-producing and AHL-degrading bacteria. A C. violaceum CV026-overlaid plate was used for the detection of BHL and HHL and an A. tumefaciens NT1 (pDCI41E33)-overlaid plate for OHHL and OHL. For screening AHL-degrading bacteria, an enrichment culture method was used as previously described [22] . The activated sludge samples were inoculated in a minimal medium containing AHL (2 mM) as a sole carbon source and incubated for 3 days. Then, the cultured broth was transferred to a new AHL-minimal medium (5% (v/v)) and this transfer procedure was repeated three times. Then the final culture was spread on LB agar to isolate single colonies.
- Strain Identification
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’). The PCR conditions involved denaturation at 95℃ for 5 min, followed by 30 cycles at 95℃ for 30 sec, 58℃ for 30 sec, and 72℃ for 90 sec, using a Taq polymerase (BIOFACT, Korea). The resulting PCR product was sequenced by an ABI3700 automatic sequencer (Applied Biosystems, USA), and the sequence was identified using the Ezbiocloud ( http://www.ezbiocloud.net/eztaxon ).
- Bioassay of AHL-Degrading Activity
The AHL substrates BHL, HHL, and OHL were purchased from Bnpharm (Korea). Each strain’s culture broth was fractionated into three fractions; the supernatant of the cultured broth, the cellfree lysate, and the whole cell. For the whole-cell assay, overnight cultures (5 ml) of the isolates were washed and resuspended with 300 µl of Tris-HCl buffer (10 mM, pH 7.0). The resuspended samples were diluted using the same Tris-HCl buffer to adjust their concentrations to the equivalent of OD 600 1.0. Then, 80 µl of the cell resuspension was mixed with 20 µl of AHL (final 20 µM HHL and 5 µM OHL) and incubated at 37℃ with gentle agitation. For the assay with the culture supernatant and cell-free lysate, after centrifugation of 5 ml of the cultured broth (12,000 × g , 10 min), the cell pellet was resuspended in 300 µl of Tris-HCl buffer (10 mM, pH 7.0). Then, the resuspended cells were disrupted using a bead beater to obtain the cell-free lysate. Finally, 80 µl of the culture supernatant and cell-free lysate were mixed with AHL as described above. After boiling at 95℃ for 5 min to stop the reaction, the reaction mixture (20 µl) was loaded into the hole of the two reporter strain-overlaid agar plates. The residual AHL amounts were calculated using a regression equation based on the color zone size and known amounts of AHLs.
- Thin-Layer Chromatography
The AHL profile of the AHL-producing strain, Aeromonas sp., was analyzed by C18 reversed-phase thin-layer chromatography (TLC) (aluminum sheets; Merck) using C. violaceum CV026 as the AHL indicator strain [26] . Cell-free supernatants from each strain were mixed with an equal volume of acidified ethyl acetate (0.1% acetic acid). The extracted samples were separated using methanol (60% (v/v)) in water as the solvent. Then the TLC plates were overlaid with a thin film of CV026-seeded LB soft agar (0.7% (w/v)). After overnight incubation at 30℃, the AHLs were located as purple spots. Synthetic AHL standards (BHL, HHL, and OHHL) were used as references.
- Biofilm Inhibition Test Using Biofilm-Producing Strains and QQ Bacteria
For visualization of the inhibition of biofilm formation by QQ bacteria, the biofilm-producing Aeromonas sp. T 3-4 strain and AHL-degrading Rhodococcus sp. or Microbacterium sp. were cultivated together on a glass slide (2.6 × 7.6 cm) that was submerged in a 10 ml LB broth contained in a 50 ml conical tube. For other strains that showed AHL-degrading activity in the cell-free lysate or culture supernatant, the cell-free lysate or culture supernatant were added to the LB broth (1-3% (v/v)) when inoculating the biofilm-producing Aeromonas sp. T3-4 strain. After overnight cultivation or 20 h of incubation, the glass slides were withdrawn and stained with 0.1% crystal violet for 30 min. Finally, the glass slides were washed with distilled water three times to compare the biofilm formation. To test the inhibition of mature biofilm formation by QQ strains, the biofilm was allowed to grow on a glass slide placed in LB by overnight cultivation with only the biofilm producer Aeromonas sp. T3-4. After biofilm formation, the QQ strain, cell-free lysate, or culture supernatant was added to this Aeromonas sp. T3-4 culture. Then, the mixtures were incubated 15 and 24 h more, and the glass slide was taken out to compare the extent of biofilm formation. To check the inhibition of biofilm formation by QQ strains, a static microtiter plate assay was used as well, and the biofilm formation was determined as described previously [20] . Overnight culture of the biofilm-producing Raoultella sp. D1-6 strain and QQ strains were seeded (5%) together in LB in a 96-well polystyrene microtiter plate and incubated at 30℃ for 16 h without agitation. For strains that showed AHL-degrading activity in the cell-free lysate or culture supernatant, each sample containing QQ enzyme was dispensed into a 96-well microtiter plate containing the inoculum of the biofilm-producing Raoultella sp. D1-6 strain in LB. The planktonic cells were removed and washed with distilled water. Biofilms were detected by staining with 0.1% crystal violet for 30 min at room temperature and then washing thoroughly with sterile distilled water. For the quantitative analysis of biofilm formation, 200 µl of ethanol (95%) was used to destain the wells, and the absorbance was determined at 550 nm using a microplate reader (VersaMax; Molecular Devices Inc., USA). The glass slides or wells without addition of QQ samples were used as controls.
- Nucleotide Accession Numbers
The 16S rRNA gene sequences of Rhodococcus sp. B5, Microbacterium sp. HSY-2, Micrococcus sp. SY-1, Staphylococcus sp. ML-1, Pseudomonas sp. SS-1, Streptococcus sp. SL-2, Afipia sp. MS-1, and Acinetobacter sp. HMS-1 were deposited in the GenBank database under the accession numbers KJ028074, KJ028677, KJ145900, KJ541504, KJ816640, KJ541506, KJ541505, and KJ816639, respectively.
Results and Discussion
- Analysis of AHL- and Biofilm-Producing Strains from Sludge of Wastewater Treatment Plant
To analyze biofilm-producing strains among AHL-producing strains in a real wastewater treatment plant, we first screened strains that produced AHL signals using two reporter strains, C. violaceum CV026 and A. tumefaciens NT1 (pDCI41E33). As AHL-producing strains, Aeromonas sp., Raoultella sp., Enterobacter sp., Enterococcus sp., Haemophilus sp., Klebsiella sp., Lelliottia sp., and Serratia sp., were identified on the basis of 16S rDNA sequences analysis ( Table 1 ). Aeromonas sp. and Serratia sp. strains produced AHLs detected by both reporter strains. Biofilm-producing strains among the screened AHL producers were also analyzed on a 96-well microtiter plate containing LB-glucose (0.2% (w/v)) broth after overnight culturing. Most of the AHL producers, including Aeromonas sp., Raoultella sp., and Enterobacter sp., produced biofilm as well (data not shown). Representative AHL-producing biofilm producers, Aeromonas sp. SL-8 and Aeromonas sp. T3-4 strains, were used to analyze the AHL profile using a C. violaceum CV026-overlaid TLC plate. Compared with standard synthetic AHL spots on TLC, HHL, OHHL, and BHL were detected in the culture supernatant of two strains ( Fig. 1 ).
AHL- and biofilm-producing strains isolated from sludge of a wastewater treatment plant.
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AHL- and biofilm-producing strains isolated from sludge of a wastewater treatment plant.
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Thin-layer chromatogram of the AHLs produced by the representative isolates, Aeromonas sp. SL-8 and Aeromonas sp. T3-4. Lane 1, synthetic AHL mix (BHL, HHL, OHHL, OHL); lane 2-5, BHL, HHL, OHL, and OHHL standard, respectively; lane 6, extract of Aeromonas sp. SL-8 culture supernatant; lane 7, extract of Aeromonas sp. T3-4 culture supernatant. O marks the position of the origin.
- Isolation and Identification of AHL-Degrading Bacteria
To isolate indigenous AHL-degrading bacteria from a wastewater treatment plant using enrichment culture, sludge samples from a laboratory-scale MBR and a real wastewater treatment plant (Daejeon, Korea) were cultivated in minimal medium containing BHL, HHL, or OHHL as the sole carbon source. After three rounds of enrichment culture, the cell suspension was spread on LB agar, and different types of colony were isolated. A total of 225 strains were selected, and then the AHL-degrading activity of the isolates was analyzed. Many kinds of AHL-lactonases and AHL-acylases are reported as intracellular and extracellular enzymes, respectively [1 , 12 , 31] . To identify the location of QQ enzymes for AHL degradation from the isolates, the culture supernatant, cell-free lysate, and whole cells were prepared after overnight cultivation of the isolates. Finally, eight representative strains were selected on the basis of AHL-degrading activity on the reporter plates and the location of the QQ enzymes. These strains were identified based on the analysis of the 16S rDNA sequences ( Table 2 ). And to identify the enzyme responsible for AHL degradation in QQ bacteria, AHL-degrading activities were analyzed using cell-free lysate, culture supernatant and whole cells ( Table 2 ). Staphylococcus sp. ML-1, Micrococcus sp. SY-1 , and Pseudomonas sp. SS-1 strains showed the AHL-degrading activity only on the culture supernatant ( Fig. 2 A). On the other hand, Afipia sp. MS-1, Acinetobacter sp. HMS-1 , and Streptococcus sp. SL-2 strains showed the activity only in cell-free lysate after cell disruption ( Fig. 2 C). In case of Microbacterium sp. HSY-2 and Rhodococcus sp. B5, AHL-degrading activities were detected in the whole-cell assay ( Fig. 2 B). Rhodococcus sp. B5 showed AHL-degrading activity in cell-free lysate as well. Strains belonging to the genera Pseudomonas, Rhodococcus, Micrococcus, Acinetobacter, Staphylococcus , and Microbacterium have been reported to degrade AHL, and the QQ enzymes responsible for the AHL degradation from these bacteria have been identified [13 , 18 , 19 , 21 , 33] . In particular, several Rhodococcus sp. and Pseudomonas sp. have already been reported to produce AHL-lactonase and AHL-acylase, respectively [1 , 12 , 31] . Rhodococcus erythropolis W2 was reported to produce three QQ enzymes: AHL-acylase, AHL-lactonase, and AHL-oxidoreductase [21 , 30 , 31] . However, to date there has been no report for AHL-degrading capability in the case of Streptococcus sp. and Afipia sp. strains. Thus, more studies are needed to identify the QQ enzymes involved in AHL degradation of these isolates.
Representative AHL-degrading bacteria isolated from sludge of a wastewater treatment plant.
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Representative AHL-degrading bacteria isolated from sludge of a wastewater treatment plant.
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AHL-degrading assay of culture supernatant, cell lysates, and whole cells of QQ strains. To identify the location of QQ enzymes for AHL degradation from the isolates, overnight culture broth of each strain was fractionated into three samples: supernatant of cultured broth (S), cell-free lysate (L), and whole cell (W). (A) Microbacterium sp. HSY-2 and Rhodococcus sp. B5 showed AHL-degrading activities in a whole-cell assay. C. violaceum CV026 was used as a reporter strain for HHL. (B) Micrococcus sp. SY-1, Staphylococcus sp. ML-1, and Pseudomonas sp. SS-1 strains showed the AHL-degrading activity only on culture supernatant. A. tumefaciens NT1 (pDCI41E33) was used as a reporter strain for OHL. (C) Acinetobacter sp. HMS-1, Streptococcus sp. SL-2, and Afipia sp. MS-1 strains showed the activity in cell-free lysate after cell disruption.
- Inhibition of Initial Biofilm Development by AHL-Degrading QQ Strains
To investigate whether isolated AHL-degrading strains could inhibit biofilm formation, we conducted a coculture experiment of the biofilm-forming Aeromonas sp. T3-4 strain and isolated QQ strain; in other words, an AHL producer and an AHL degrader. The biofilm producer Aeromonas sp. T3-4 strain isolated from a wastewater treatment plant produced AHL as well ( Table 1 ). First, we analyzed the extent of biofilm formation on a glass slide submerged in the culture tube using the biofilm producer Aeromonas sp. T3-4 and AHL-degrading strains. In the case of Rhodococcus sp. B5 and Microbacterium sp. HSY-2, which showed whole-cell AHL-degrading activity, each strain was cocultured with the biofilm producer Aeromonas sp. T3-4 strain for 20 h. In the case of the other AHL-degrading strains, a culture supernatant ( Micrococcus sp. SY-1 and Staphylococcus sp. ML-1) or cell-free lysate ( Acinetobacter sp. HMS-1 and Afipia sp.MS-1) was added to the inoculum of the biofilm producer Aeromonas sp. T3-4 at the beginning of cultivation. After 20 h cultivation, the biofilm formation on the glass slide was analyzed using crystal violet staining. As shown in Fig. 3 A, all glass slides submerged in the culture broth containing the AHL-degrading strain or enzyme sample showed much less biofilm formation than the control sample, in which only the biofilm producer Aeromonas sp. T3-4 was cultivated without addition of the QQ sample. In contrast, when the inactivated QQ samples (boiled sample) were added to the biofilm producer Aeromonas sp. T3-4, the extent of biofilm formation was similar to the control. In a similar experiment, a static 96-well microtiter plate assay was used to show the effect of the QQ sample on biofilm formation, in which both the biofilm producer Raoultella sp. D1 -6 and the QQ sample, AHL-degrading strains or enzyme fractions, were incubated together overnight in LB broth. We also measured the adherent biofilm with crystal violet staining. As shown in Fig. 3 B, the sample with AHL-degrading bacteria or enzymes showed a decreased biofilm formation, as in the experiments with glass slides, when compared with the control. These results suggest that QQ bacteria or enzymes are able to control the initial biofilm development by Aeromonas sp. T3-4 or Raoultella sp. D1-6 via AHL degradation.
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Inhibition of initial biofilm development by QQ stains on a glass slide (A) and a 96-well microtiter plate (B). Whole cells (Microbacterium sp. HSY-2 and Rhodococcus sp. B5), culture supernatant (Micrococcus sp. SY-1, Staphylococcus sp. ML-1, Pseudomonas sp. SS-1), and cell-free lysate (Acinetobacter sp. HMS-1, Streptococcus sp. SL-2, and Afipia sp. MS-1) were used as QQ samples. (A) The biofilmproducing Aeromonas sp. T3-4 strain and QQ sample were added together, and cultivated for 20 h with a glass slide. (B) The biofilm-producing Raoultella sp. D1-6 strain and QQ sample were mixed together in a 96-well microtiter plate. 1, Control (only Raoultella sp. D1-6); 2, Rhodococcus sp. B5; 3, Microbacterium sp. HSY-2; 4, Pseudomonas sp. 1A1; 5, Micrococcus sp. SY-1; 6, Staphylococcus sp. ML-1; 7, Rhodococcus sp. B5; 8, Acinetobacter sp. HMS-1; and 9, Afipia sp. MS-1.
- Reduction of Established Biofilm by AHL-Degrading QQ Strains
In the previous experiment, the inhibition of initial biofilm development was shown because QQ samples were added at the initial stage of biofilm formation. In addition, we checked the effect of QQ strains and enzyme samples on the established biofilm. For this experiment, mature biofilm on the glass slide was needed before the addition of the QQ sample. After biofilm formation on the glass slide by overnight cultivation using the biofilm producer Aeromonas sp. T3-4, QQ strains or QQ enzymes were added to the culture sample, which was then incubated 15 to 24 h more. As shown in Fig. 4 A, QQ strains or QQ samples also significantly reduced the biofilm formation when comparing with control. In a similar experiment, a static 96-well microtiter plate assay was used to see the effect of the QQ samples on the established biofilm. After biofilm formation using Aeromonas sp. T3-4 strain on a 96-well microtiter plate, the QQ samples were added to this Aeromonas sp. T3-4 culture. Then, the mixtures were incubated 15 and 24 h more, and the adherent biofilm with crystal violet staining was measured. As shown in Fig. 4 B, the sample with AHL-degrading bacteria or QQ samples showed a decreased biofilm formation, as in the experiments with glass slides, when compared with the control. These results suggest that AHL-degrading strains or QQ samples not only inhibited initial biofilm development but also reduced the established biofilm. It remains necessary to clarify the mechanism of reduction of the established biofilm in more detail.
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Effect of quorum quenching activity on established biofilm of Aeromonas sp. T3-4. (A) The biofilm was allowed to grow on a glass slide placed in LB by overnight cultivation with only biofilm producer Aeromonas sp. T3-4. After biofilm formation, the QQ samples were added to this Aeromonas sp. T3-4 culture. Then, the mixtures were incubated 15 and 24 h more and taken out of the glass slide to compare the extent of biofilm formation. Whole cell (Microbacterium sp. HSY-2 and Rhodococcus sp. B5), culture supernatant (Micrococcus sp. SY-1, Staphylococcus sp. ML-1, and Pseudomonas sp. SS-1), and cell-free lysate (Acinetobacter sp. HMS-1, Streptococcus sp. SL-2, and Afipia sp. MS-1) were used as QQ samples. (B) The biofilm-producing Aeromonas sp. T3-4 was cultured in a 96-well microtiter plate for 20 h. After biofilm formation, the QQ samples were added to this Aeromonas sp. T3-4 culture. Then, the mixtures were incubated 15 and 24 h more and the extent of biofilm formation was compared. 1, Control (only Aeromonas sp. T3-4); 2, Rhodococcus sp. B5; 3, Microbacterium sp. HSY-2; 4, Micrococcus sp. SY-1; 5, Stapylococcus sp. ML-1; 6, Afipia sp. MS-1; and 7, Streptococcus sp. SL-2.
In this study, diverse AHL-degrading bacteria from a laboratory-scale MBR and sludge from a wastewater treatment plant were isolated. Afipia sp. and Acinetobacter sp. strains produced the intracellular QQ enzyme, whereas Pseudomonas sp. and Micrococcus sp. produced the extracellular QQ enzyme. Microbacterium sp. and Rhodococcus sp. strains showed AHL-degrading activity in a whole-cell assay. Finally, biofilm formation by AHL-producing bacteria was reduced with isolated QQ bacteria. This study shows the potential application of indigenous QQ bacteria isolated from a wastewater treatment plant for the inhibition of biofilm formation.
Acknowledgements
This research is supported by the Korea Ministry of Environment as Converging Technology Project (2012001440002).
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