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Article

Control of Membrane Biofouling in MBR for Wastewater Treatment byQuorum Quenching Bacteria Encapsulated in Microporous Membrane

Hyun-Suk Oh, Kyung-Min Yeon, Cheon-Seok Yang, Sang-Ryong Kim,Chung-Hak Lee, Son Young Park, Jong Yun Han, and Jung-Kee Lee

Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es204312u • Publication Date (Web): 03 Apr 2012

Downloaded from http://pubs.acs.org on April 6, 2012

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1

Control of Membrane Biofouling in MBR for Wastewater 1

Treatment by Quorum Quenching Bacteria Encapsulated in 2

Microporous Membrane 3

Hyun-Suk Oh,† Kyung-Min Yeon,

† Cheon-Seok Yang,

† Sang-Ryoung Kim,

† 4

Chung-Hak Lee,*,†

Son Young Park,‡ Jong Yun Han,

‡ and Jung-Kee Lee

‡

5

6

†School of Chemical and Biological Engineering, Seoul National University, Seoul 151-744, Republic 7

of Korea 8

‡Department of Life Science and Genetic Engineering, Paichai University, Daejeon 302-735, Republic 9

of Korea 10

11

12

*Corresponding author. Mailing address: School of Chemical and Biological Engineering, 13

Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-744, Republic of Korea. 14

Phone: +82-2-880-7075. Fax: +82-2-874-0896. E-mail: [email protected]

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2

ABSTRACT 1

Recently, enzymatic quorum quenching has proven its potential as an innovative approach for 2

biofouling control in the membrane bioreactor (MBR) for advanced wastewater treatment. 3

However, practical issues on the cost and stability of enzymes are yet to be solved, which 4

requires more effective quorum quenching methods. In this study, a novel quorum quenching 5

strategy, interspecies quorum quenching by bacterial cell, was elaborated and proved to be 6

efficient and economically feasible biofouling control in MBR. A recombinant Escherichia 7

coli which producing N-acyl homoserine lactonase or quorum quenching Rhodococcus sp. 8

isolated from a real MBR plant was encapsulated inside the lumen of microporous hollow 9

fiber membrane, respectively. The porous membrane containing these functional bacteria (i.e., 10

‘Microbial–vessel’) was put into the submerged MBR to alleviate biofouling on the surface 11

of filtration membrane. The effect of bio-fouling inhibition by the Microbial-vessel was 12

evaluated over 80 days of MBR operation. Successful control of biofouling in a laboratory 13

scale MBR suggests that the biofouling control through the interspecies quorum quenching 14

could be expanded to the plant scale of MBR and various environmental engineering systems 15

with economic feasibility. 16

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INTRODUCTION 1

Over the past two decades, a membrane bioreactor (MBR) where a biological activated 2

sludge process is combined with membrane filtration has emerged as an innovative 3

technology for the effective wastewater treatment and reuse because it provides a treated 4

effluent of high quality.1, 2

However, the application of MBR has been hampered, particularly 5

by membrane biofouling, which is closely associated with the naturally attached microbial 6

growth on the membrane surface.3, 4

Especially, biofouling control has been a critical research 7

issue because around 60 % of MBR operating cost is directly related to biofouling.2 8

Therefore, tremendous efforts to inhibit biofouling in membrane processes for water 9

treatment have been made through engineering, material, and chemistry platforms.4, 5

10

However, all these attempts are limited by the fact that they are essentially not able to uproot 11

the natural biofilm formation on the membrane surface. 12

Since the regulation of microbial group behaviors by cell-to-cell communication (i.e., 13

quorum sensing) was reported to be involved in the biofilm formation,6 the quorum 14

quenching has been regarded as a fundamental approach of biofouling inhibition on diverse 15

surfaces.7

Recently, the concept of bacterial quorum sensing was also introduced to an MBR 16

as a new biofouling control paradigm. In detail, it was experimentally observed that quorum 17

sensing is closely associated with the formation of a biofouling layer on the immersed 18

membrane surface in a submerged MBR for wastewater treatment.8 Furthermore, AHL-19

acylase which degrades the N-acyl homoserine lactone (AHL) type quorum sensing signal 20

molecules has proven its potential to inhibit biofouling when the AHL-acylase immobilized 21

magnetic carriers were put into the submerged MBR9 or when the AHL-acylase was 22

immobilized onto the nanofiltration membrane surface in the crossflow nanofiltration of 23

microbial suspension.10

24

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Practical issues of cost and stability of enzymes, however, are yet to be solved, which 1

requires more effective quorum quenching methods. The purpose of this study was to devise 2

and investigate the inhibition of quorum sensing in MBR by interspecies interference using 3

quorum quenching bacteria. Application of bacterial quorum quenching can be more 4

economic than enzymatic quorum quenching because the former has longer life span and 5

does not need any enzyme purification process. Two types of quorum quenching bacteria, a 6

recombinant Escherichia coli which producing AHL-lactonase or quorum quenching 7

Rhodococcus sp. isolated from a real MBR plant for wastewater treatment, were encapsulated 8

into microporous membranes, respectively. The microporous membranes containing these 9

functional bacteria were directly put into the MBR systems, respectively, to confirm the 10

feasibility of interspecies quorum quenching which is definitely more economic than the 11

enzymatic quorum quenching reported previously. 12

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EXPERIMENTAL SECTION 1

Preparation of Microbial-vessels. A microbial-vessel was designed to keep quorum 2

quenching bacteria using polyethylene hollow fiber membrane (Econity, Co. Ltd., Korea). 3

The bottom side of the Microbial-vessel was sealed with epoxy resin and the quorum 4

quenching bacteria were packed into the Microbial-vessel from the open top side using a 5

peristaltic pump (Figure 1). The same type of vessel not containing bacteria was named as the 6

Vacant-vessel. Detailed specifications of the Microbial-vessel are shown in Table S1. 7

8

Quorum quenching bacteria. Two types of quorum quenching bacteria were used for the 9

quorum quenching experiment in MBR. One was AHL-lactonase-producing recombinant E. 10

coli. The AHL-lactonase, AiiA, is known to hydrolyze the lactone ring of various AHL 11

molecules.11, 12

The AHL-lactonase gene (aiiA) from Bacillus thuringiensis serovar kurstaki 12

HD263 was introduced into E. coli XL1-Blue using pMBP-His-Parallel1 vector.13

Before the 13

recombinant E. coli was packed into the Microbial-vessel, it was cultured on Luria-Bertani 14

(LB) broth supplemented with ampicillin to maintain plasmids that provide AHL-lactonase 15

producing system. The other one was obtained using an enrichment culture method. Activated 16

sludge and biocake on the used membrane were taken respectively from the real MBR plant 17

for wastewater treatment (Okcheon, Korea). The mixed bacteria from each sample (activated 18

sludge or biocake) were seeded in a minimal medium containing AHL (2.5 mM N-butyryl-L-19

homoserine lactone or N-hexanoyl-L-homoserine lactone or N-(3-oxohexanoyl)-L-20

homoserine lactone) as a sole carbon source. After 3 days of incubation, 1 % (V/V) transfer 21

was made to a new minimal medium containing AHL. For each sample, this transfer 22

procedure was done three times to confirm the isolation of bacteria which can live with only 23

AHL as the carbon source. Then the final culture was spread on the LB agar to form single 24

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colonies. The single colony was incubated separately in the minimal medium containing AHL 1

again, and then 16S rRNA gene sequences of cultured strains were analyzed. 2

3

Strain identification. The 16S rRNA genes of the isolated strains were PCR amplified from 4

the colonies using two universal primers H+ (5‘-GAGTTTGATCCTGGCTCAG-3’) and E- 5

(5‘-AGAAAGGAGGTGATCCAGCC-3’). The PCR conditions were denaturation at 95 °C 6

for 5 min, followed by 30 cycles at 95 °C for 45 s, 50 °C for 45 s, and 72 °C for 60 s. The 7

sequencing of the PCR product was performed by an ABI3700 automatic sequencer (Applied 8

Biosystems, U.S.). The sequence identification was performed using the EzTaxon server 2.1 9

(www.eztaxon.org).14

The 16S rRNA gene sequences of Rhodococcus sp. BH4, Paenibacillus 10

sp. SYP2, Enterobacter sp. SHEB1, and Micrococcus sp. SHMC have been deposited in the 11

GenBank database under accession numbers JN378528, JN378529, JN378530, and 12

JN378531, respectively. 13

14

Inoculums and MBR operation. Activated sludge from a wastewater treatment plant (Sihwa, 15

Korea) was taken and acclimated to the synthetic wastewater before starting MBR. The 16

composition of the synthetic wastewater in the batch experiment was as follows (mg/l)8: 17

glucose, 250; yeast extract, 12.5; bactopeptone, 12.5; (NH4)2SO4, 125; K2HPO4, 300; 18

KH2PO4, 300; MgSO4, 2.25; FeCl3, 0.2; NaCl, 1.75; CaCl2, 0.2; CoCl2, 0.6; and NaHCO3, 19

37.5. 20

Two batch types of the MBR (Figure 2a), i.e., the Control reactor with Vacant-vessel and 21

the Microbial-vessel reactor with a Microbial-vessel, were designed and operated under total 22

recycle modes in which all permeates from the filtration membranes were recycled to the 23

reactors. The working volume and the mixed liquor suspended solid (MLSS) concentration of 24

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each reactor were 800 ml and 200 mg/l respectively. Polyvinylidene fluoride (PVDF) hollow 1

fiber modules (ZeeWeed 500, GE-Zenon, U.S.) with an effective area of 19 cm2 were inserted 2

in both reactors together with the Vacant- or the Microbial-vessel. 3

In addition, two continuous MBRs with 1.2 liter working volume (Figure 2b) were 4

operated in a way similar to that described by other MBR researchers.3, 9

The composition of 5

the synthetic wastewater in the continuous experiment was as follows (mg/l)3: glucose, 400; 6

yeast extract, 14; bactopeptone, 115; (NH4)2SO4, 104.8; KH2PO4, 21.75; MgSO4, 15.63; 7

FeCl3, 0.075; CaCl2, 2.45; MnSO4, 1.8; and NaHCO3, 255.5. The effective area of the hollow 8

fiber membrane module (ZeeWeed 500) was 86 cm2. Hydraulic retention time (HRT) and 9

sludge retention time (SRT) were set to 12 h and 40 d, respectively. MLSS in both reactors 10

were maintained within the range of 4,500 ~ 5,000 mg/l. MLSS and chemical oxygen 11

demand (COD) were determined according to standard methods.15 12

The biocake (or biofilm) on a membrane specimen was stained with 100 µl SYTO 9 13

(Molecular Probes, Eugene, US; ex = 488 nm; em = 515/30 nm) for 20 minutes in the dark to 14

visualize bacterial cells. After careful washing with distilled water, the stained biocake was 15

observed using confocal scanning laser microscopy (CLSM, C1 plus, Nikon, Japan). 16

17

Measurement of quorum quenching activity. The quorum quenching activity was 18

measured by the degradation of AHLs in aqueous Tris-HCl buffer (pH 7, 50 mM). To 19

determine the quorum quenching activity of the Microbial-vessel, N-octanoyl-L-homoserine 20

lactone (C8-HSL) was added at a final concentration of 0.2 µM to the buffer. C8-HSL was 21

chosen because it was identified as one of the major signal molecules in our previous study.8 22

The Microbial-vessel was then inserted into the buffer and the mixture was incubated at 30°C 23

with orbital shaking (200 rpm) for different lengths of time, as indicated. The remaining 24

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concentrations of C8-HSL were measured using bioassay. In the activity test of the whole cell, 1

C8-HSL was added at a final concentration of 0.2 µM to the overnight bacterial culture, 2

which was diluted to an optical density at 600 nm (OD600) of 1.0. 3

4

Bioassay for detecting AHL molecules. All the AHLs were purchased from Sigma-Aldrich 5

(U.S.). AHLs were detected using the indicating agar plate, which was made by mixing an 6

overnight culture of Agrobacterium tumefaciens A136 (AHL biosensor),16

LB agar and X-gal. 7

The samples were loaded into the well of the indicating agar plate and the amounts of AHLs 8

were calculated using relationship equations based on the color zone size and known amounts 9

of AHLs.8, 12, 17

10

11

AHL extraction and detection. AHLs were extracted from the MBR broth as follows: 20 ml 12

of sludge was centrifuged and the supernatant was mixed with an equal volume of acidified 13

ethyl acetate (0.1% acetic acid).18

After shaking the mixture for 2 hours, the upper layer was 14

separated and dried in a rotary evaporator at 30 °C. The residues of each sample were 15

suspended in 250 µl of methanol. 10 µl of each extracts was loaded on the indicating agar 16

plate and they were incubated at 30 °C for 10 hours. Samples were also analyzed by TLC-17

bioassay, as previously described.8 18

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RESULTS AND DISCUSSION 1

Preparation of Microbial-vessel with recombinant E. coli. To decompose the AHL type 2

quorum sensing signal molecules (i.e., quorum quenching) in the MBR, we applied 3

recombinant E. coli expressing the heterologous AHL-lactonase gene (aiiA) for quorum 4

quenching. Although the recombinant cell was constructed in such a way that isopropyl β-D-5

1-thiogalactopyranoside (IPTG) is needed for the overexpression of the AHL-lactonase, the 6

bacterial cells showed quorum quenching activity which is needed to degrade AHLs even 7

without IPTG induction (Figure S1). It is because pMal-His-Parallel1 vector has the tac 8

promoter, a derivative of lac promoter, but the regulation of tac promoter is not tight enough. 9

Hence the recombinant E. coli could produce AHL-lactonase without induction of IPTG. In 10

this study, no IPTG was added to the recombinant E. coli for the application. 11

To continuously keep quorum quenching bacteria in the MBR, it was necessary to select an 12

appropriate technique for the whole cell immobilization. Various polymer matrices such as 13

alginate,19

agarose,20

and polyacrylamide21

have been widely used for the entrapment of 14

bacteria, but the unstability of polymer matrices and the leakage of cells have always been the 15

limitations to the application. Therefore, we designed a Microbial-vessel using hollow fiber 16

(HF) membranes which can encapsulate quorum quenching bacteria inside the fibers (Figure 17

1). Because the nominal pore size of a Microbial-vessel is 0.4 µm, smaller than the size of 18

one bacterial cell, quorum quenching bacteria cannot escape from the Microbial-vessel. On 19

the contrary, the signal molecules can freely pass through the pores of Microbial-vessel so as 20

to be degraded by quorum quenching bacteria inside the Microbial-vessel. Also, the nutrients 21

can come into the Microbial-vessel and be used to maintain the activity of quorum quenching 22

bacteria. Furthermore, the population of quorum quenching bacteria can be maintained in the 23

reactor regardless of the periodic withdrawal of excess sludge from the MBR. 24

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As shown in Figure 3, the degradation efficiency of C8-HSL with the Microbial-vessel was 1

measured to be 58 % in the reaction time of 90 minutes when the Microbial-vessel contains 2

360 mg of the recombinant E. coli. As the adsorption of C8-HSL on the Vacant-vessel was 3

negligible, the decrease in the concentration of C8-HSL was attributed mainly to its 4

decomposition by the Microbial-vessel. 5

6

Application of the Microbial-vessel with recombinant E. coli to the batch MBR. The 7

biofouling control in MBR by the Microbial-vessel containing the recombinant E. coli was 8

tested in the batch and the continuous modes, respectively (Figure 2). The extent of 9

biofouling in each MBR was quantitatively evaluated by monitoring the increase of 10

transmembrane pressure (TMP) under constant flux operation. In the batch MBR, it took 11

about 28 hours in the Control reactor with a Vacant-vessel to reach the TMP of 25 kPa, 12

whereas it took 39 hours in the Microbial-vessel reactor to reach the same TMP (Figure 4). 13

This result indicated the biofouling inhibition of the Microbial-vessel encapsulating quorum 14

quenching bacteria in the batch MBR. 15

To further confirm the relationship between quorum quenching and biofouling, AHL 16

molecules were extracted by ethyl acetate from the broths of both reactors and their levels 17

were compared with each other using A. tumefaciens A136. As shown in Figure 5a, the fade-18

out of blue color developments for the extract from the Microbial-vessel reactor, unlike those 19

from the Control reactor was observed, indicating that the AHL concentration was decreased 20

in the Microbial-vessel reactor. Then the extracts from both reactors were also analyzed by 21

thin layer chromatography (TLC) and an AHL biosensor (A. tumefaciens A136) to identify 22

the types of AHLs present in the reactors. Three different types of AHLs were detected in the 23

extracts from the Control reactor, and following comparison with the Rf values of standard 24

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AHLs, they were confirmed to be C6-HSL, C8-HSL, and C10-HSL, respectively (Figure 5b). 1

Yeon et al.8 also reported the presence of C6-HSL, and C8-HSL in the MBR. On the contrary, 2

extracts from the Microbial-vessel reactor did not show any AHLs in TLC analysis, which 3

means that the concentrations of AHLs might be reduced to a level below the detection limit 4

in the TLC-bioassay. From these results, we can conclude that the Microbial-vessel 5

encapsulating the quorum quenching bacteria degraded the AHL molecules, which resulted in 6

the inhibition of biofouling, i.e., the delay of TMP rise-up by the quorum quenching 7

mechanism in the batch type MBR (Figure 4). 8

9

Application of the Microbial-vessel with recombinant E. coli to the continuous MBR. To 10

verify the long term effect of quorum quenching, two MBRs (reactor A & B) were run in 11

parallel1 with continuous feeding (Figure 2b). Two Vacant-vessels were inserted in Reactor 12

A, while two Microbial-vessels containing the recombinant E. coli were inserted in Reactor 13

B. Both reactors were operated under the same constant flux of 18-20 l/m2/h (LMH) and TMP 14

variations were monitored for three cycles (Figure 6). In the 1st cycle, Reactor B with the 15

Microbial-vessels showed the mitigation of biofouling compared with Reactor A having the 16

Vacant-vessels in the continuous system. In the 2nd

cycle both reactors were operated at a 17

constant flux of 18 LMH using the cleaned membranes with NaOCl right after the 1st cycle. 18

We could observe a more dramatic difference of TMP rise-up between two reactors. In order 19

to verify possible doubt that the slow rate of TMP rise-up in the Microbial-vessel reactor 20

might be caused by the differences in other environmental factors (e.g., hydrodynamic regime, 21

etc.) rather than quorum quenching, in the 3rd

cycle the Vacant- and Microbial-vessels were 22

taken out of Reactor A and B, respectively and then they were interchanged. After two days 23

of stabilization period, two reactors were run again using chemically cleaned membranes. 24

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Less biofouling, (i.e., slower TMP rise-up) was also observed in the reactor with the 1

Microbial-vessel (Reactor A) (Figure 6). There were variations in the rate of TMP rise-up in 2

all cycles for both rectors. It was attributed to the continuous dynamic changes in the 3

microbial community in the MBR.22

Despite those variations in the rate of TMP rise-up, the 4

Microbial-vessel always delayed the TMP rise-up substantially during all three cycles. 5

In a final step to ensure whether the recombinant E. coli inhibited biofouling by the 6

quorum quenching mechanism or by other effects, we prepared a ‘Control-vessel’ containing 7

E. coli harboring only an empty vector (pMBP-His-parallel1) without the aiiA gene which is 8

unable to produce the AHL-lactonase. The TMP in the Control-vessel reactor rose up much 9

faster than that in the Microbial-vessel reactor (Figure S2), indicates that the biofouling 10

inhibition by quorum quenching occurred only in the MBR with the Microbial-vessel 11

containing the AHL-lactonase-producing E. coli. In view of these results, it can be concluded 12

that the Microbial-vessel inhibited the biofouling on the membrane surface, and thus delayed 13

the TMP rise-up through the quorum quenching effect. 14

15

Stability of quorum quenching activity in the long term operation of MBR. In the long 16

term operation of MBRs (80 days), the same trend of TMP variations in each reactor were 17

reproduced (data not shown). During that period, the Microbial–vessel was intermittently 18

taken out of the reactor to monitor its quorum quenching activity (Figure 7). There was a 19

substantial variation in the quorum quenching activity of the Microbial-vessel, which can be 20

represented by the slope of each curve in a plot of the concentration of a signal molecule vs. 21

reaction time. It might be attributed to the continuous change in the population of live 22

quorum quenching bacteria inside the Microbial-vessel during the operation. Comparing the 23

slope for the control (Vacant-vessel) with those of the Microbial-vessel, it has been concluded 24

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that quorum quenching activity of the Microbial-vessel had been maintained for at least 80 1

days. 2

3

Effect of quorum quenching on the biodegradation of organics in MBR. Considering that 4

quorum sensing regulates various microbial physiologies, possible side effects of the 5

Microbial-vessel should be checked. Consequently, the treatment efficiency of organics for 6

both reactors which may be represented by the soluble CODs in the broth and permeate were 7

monitored during the continuous reactor operations. As shown in Figure S3, the differences in 8

COD profiles were negligible, suggesting that the insertion of the Microbial-vessel did not 9

affect the microbial activity for the biodegradation of the organics. 10

11

Isolation of indigenous quorum quenching bacterium from a real MBR plant and its 12

application to the continuous MBR. Even though the recombinant E. coli which producing 13

AHL-lactonase was proved to be effective for the biofouling inhibition in MBR, two 14

problems still remain which need to be overcome: one is that the recombinant E. coli can lose 15

their activity without the antibiotics, while the other is that it may not survive well outside the 16

laboratory, i.e., in a real MBR. Therefore, we tried to find out indigenous quorum quenching 17

bacteria which may inhabit a real MBR plant for wastewater treatment. In order to isolate 18

indigenous quorum quenching bacteria, activated sludge and biocake were sampled from a 19

real MBR plant for wastewater treatment being operated at Okcheon in Korea. AHL 20

degrading bacteria were then isolated using the enrichment culture. 16S rRNA gene 21

sequences of four quorum quenching isolates were analyzed and identified using the EzTaxon 22

server (Table 1). Strain BH4 and SYP2 shared 100% and 98.7 % sequence identity with the 23

16S rRNA gene sequences of Rhodococcus qingshengii djl-6T and Paenibacillus turicensis 24

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722T, respectively. As these two isolates degraded AHL molecules more efficiently than other 1

strains, they were compared with the recombinant E. coli cell in terms of quorum quenching 2

activity (Figure 8a). Because Rhodococcus sp. BH4 showed the highest degrading activity 3

against C8-HSL, further study was done to test its quorum quenching ability in the 4

continuous MBR. When the Microbial-vessel containing Rhodococcus sp. BH4 inside was 5

inserted in the MBR, TMP rise-up was substantially delayed as expected (Figure 8b) in two 6

consecutive runs of the continuous MBR. 7

8

Effect of indigenous quorum quenching bacteria on the biocake formation on the 9

membrane surface in MBR. In order to confirm the quorum quenching effect on the 10

inhibition of biofouling in more detail, the used membranes were taken out of both MBRs 11

after 41 hours of operation to measure total attached biomass (TAB) and to visualize biocake 12

layers using CLSM, respectively (Figure S4). The amount of TAB in the control reactor was 13

17.7 mg, whereas that in the Microbial-vessel reactor was only 9.1 mg. Moreover, from the 14

CLSM image, it was clearly seen that the biocake formed on the used membrane in the 15

Microbial-vessel reactor (Figure 9b) was thinner and sparser than that in the Control reactor 16

(Figure 9a). These data support the evidences of quorum quenching effect on the inhibition of 17

biofouling. 18

In summary, we prepared the Microbial-vessel containing recombinant quorum quenching 19

bacteria (AHL-lactonase-producing recombinant E. coli) and used it successfully for the 20

biofouling control by interspecies interference in MBR. In the continuous MBR operation, 21

inserting the Microbial-vessel into the MBR substantially delayed the TMP rise-up (i.e., 22

membrane biofouling) without any deterioration of wastewater treatment performance. 23

Furthermore, an indigenous quorum quenching bacterium, Rhodococcus sp. BH4, screened 24

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and isolated from a real MBR plant proved effective in biofouling inhibition by quorum 1

quenching. 2

This new process for biofouling inhibition with the Microbial-vessel is worth noticing 3

because this technique could open new horizons, in that interspecies quorum quenching could 4

be a novel technique for the control of biofouling in MBR for wastewater with mixed culture 5

where a variety of microorganisms cohabit. 6

7

ACKNOWLEDGEMENTS 8

This work was supported by the National Research Foundation of Korea (NRF) grants 9

funded by the government (MEST) (No. 2010-0018903 and No. 2009-0089695). 10

11

Supporting Information 12

Additional figures and table. This material is available free of charge via the Internet at 13

http://pubs.acs.org. 14

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REFERENCES 1

(1) Meng, F. G.; Chae, S. R.; Drews, A.; Kraume, M.; Shin, H. S.; Yang, F. L., Recent 2

advances in membrane bioreactors (MBRs): Membrane fouling and membrane material. 3

Water Res. 2009, 43 (6), 1489-1512. 4

(2) Judd, S.; Judd, C., The MBR book : principles and applications of membrane 5

bioreactors in water and wastewater treatment. 1st ed.; Elsevier: Amsterdam ; Boston, 2006. 6

(3) Hwang, B. K.; Lee, W. N.; Yeon, K. M.; Park, P. K.; Lee, C. H.; Chang, I. S.; Drews, 7

A.; Kraume, M., Correlating TMP increases with microbial characteristics in the bio-cake on 8

the membrane surface in a membrane bioreactor. Environ. Sci. Technol. 2008, 42 (11), 3963-9

3968. 10

(4) Drews, A., Membrane fouling in membrane bioreactors-Characterisation, 11

contradictions, cause and cures. J. Membr. Sci. 2010, 363 (1-2), 1-28. 12

(5) Xiong, Y. H.; Liu, Y., Biological control of microbial attachment: a promising 13

alternative for mitigating membrane biofouling. Appl. Microbiol. Biot. 2010, 86 (3), 825-837. 14

(6) Davies, D. G.; Parsek, M. R.; Pearson, J. P.; Iglewski, B. H.; Costerton, J. W.; 15

Greenberg, E. P., The involvement of cell-to-cell signals in the development of a bacterial 16

biofilm. Science 1998, 280 (5361), 295-298. 17

(7) Choudhary, S.; Schmidt-Dannert, C., Applications of quorum sensing in 18

biotechnology. Appl. Microbiol. Biot. 2010, 86 (5), 1267-1279. 19

(8) Yeon, K. M.; Cheong, W. S.; Oh, H. S.; Lee, W. N.; Hwang, B. K.; Lee, C. H.; 20

Beyenal, H.; Lewandowski, Z., Quorum sensing: A new biofouling control paradigm in a 21

membrane bioreactor for advanced wastewater treatment. Environ. Sci. Technol. 2009, 43 (2), 22

380-385. 23

(9) Yeon, K. M.; Lee, C. H.; Kim, J., Magnetic enzyme carrier for effective biofouling 24

Page 16 of 29



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control in the membrane bioreactor based on enzymatic quorum quenching. Environ. Sci. 1

Technol. 2009, 43 (19), 7403-7409. 2

(10) Kim, J. H.; Choi, D. C.; Yeon, K. M.; Kim, S. R.; Lee, C. H., Enzyme-immobilized 3

nanofiltration membrane to mitigate biofouling based on quorum quenching. Environ. Sci. 4

Technol. 2011, 45 (4), 1601-1607. 5

(11) Kim, M. H.; Choi, W. C.; Kang, H. O.; Lee, J. S.; Kang, B. S.; Kim, K. J.; 6

Derewenda, Z. S.; Oh, T. K.; Lee, C. H.; Lee, J. K., The molecular structure and catalytic 7

mechanism of a quorum-quenching N-acyl-L-homoserine lactone hydrolase. Proc. Natl. Acad. 8

Sci. U. S. A. 2005, 102 (49), 17606-17611. 9

(12) Dong, Y. H.; Xu, J. L.; Li, X. Z.; Zhang, L. H., AiiA, an enzyme that inactivates the 10

acylhomoserine lactone quorum-sensing signal and attenuates the virulence of Erwinia 11

carotovora. Proc. Natl. Acad. Sci. U. S. A. 2000, 97 (7), 3526-3531. 12

(13) Kim, M. H.; Kang, H. O.; Kang, B. S.; Kim, K. J.; Choi, W. C.; Oh, T. K.; Lee, C. H.; 13

Lee, J. K., Crystallization and preliminary crystallographic analysis of Bacillus thuringiensis 14

AHL-lactonase. BBA-Proteins Proteom. 2005, 1750 (1), 5-8. 15

(14) Chun, J.; Lee, J. H.; Jung, Y.; Kim, M.; Kim, S.; Kim, B. K.; Lim, Y. W., EzTaxon: a 16

web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene 17

sequences. Int. J. Syst. Evol. Microbiol. 2007, 57, 2259-2261. 18

(15) Standard Methods for the Examination of Water and Wastewater. 20th ed.; American 19

Public Health Association: Washington, DC, 1998. 20

(16) Fuqua, C.; Winans, S. C., Conserved cis-acting promoter elements are required for 21

density-dependent transcription of Agrobacterium tumefaciens conjugal transfer genes. J. 22

Bacteriol. 1996, 178 (2), 435-440. 23

(17) Park, S. Y.; Lee, S. J.; Oh, T. K.; Oh, J. W.; Koo, B. T.; Yum, D. Y.; Lee, J. K., AhlD, 24

Page 17 of 29



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an N-acylhomoserine lactonase in Arthrobacter sp., and predicted homologues in other 1

bacteria. Microbiology-SGM 2003, 149, 1541-1550. 2

(18) Bertani, I.; Venturi, V., Regulation of the N-acyl homoserine lactone-dependent 3

quorum-sensing system in rhizosphere Pseudomonas putida WCS358 and cross-talk with the 4

stationary-phase RpoS sigma factor and the global regulator GacA. Appl. Environ. Microb. 5

2004, 70 (9), 5493-5502. 6

(19) Kierstan, M.; Bucke, C., Immobilization of microbial-cells, subcellular organelles, 7

and enzymes in calcium alginate gels. Biotechnol. Bioeng. 1977, 19 (3), 387-397. 8

(20) Khare, S. K.; Jha, K.; Gandhi, A. P., Use of agarose-entrapped Aspergillus niger cells 9

for the production of citric-acid from soy whey. Appl. Microbiol. Biot. 1994, 41 (5), 571-573. 10

(21) Mittal, Y.; Mishra, I. M.; Varshney, B. S., Characterization of metabolically active 11

developmental stage of Aspergillus niger cells immobilized in polyacrylamide-gel. 12

Biotechnol. Lett. 1993, 15 (1), 41-46. 13

(22) Lim, S.; Kim, S.; Yeon, K. M.; Sang, B. I.; Chun, J.; Lee, C. H., Correlation between 14

microbial community structure and biofouling in a laboratory scale membrane bioreactor 15

with synthetic wastewater. Desalination 2012, 287, 209-215. 16

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1

Figure 1. Photograph and enlarged diagram of a Microbial-vessel. Specifications of 2

Microbial-vessels are shown in Table S1. 3

4

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http://pubs.acs.org/action/showImage?doi=10.1021/es204312u&iName=master.img-000.jpg&w=305&h=257

2

1

Figure 2. Schematic diagrams of (a) the batch MBRs and (b) the continuous MBRs 2

3

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1

Figure 3. Quorum quenching activity of the Microbial-vessel. The Vacant-vessel was also 2

tested to check the adsorption of C8-HSL on the vessel. The recombinant E. coli was packed 3

into the Microbial-vessel. Error bar: standard deviation (n=4). 4

5

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1

Figure 4. TMP profile in the batch MBR. A Vacant-vessel and a Microbial-vessel with 2

recombinant E. coli were inserted in the Control and the Microbial-vessel reactors, 3

respectively. 4

5

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1

Figure 5. Analysis of AHLs in the batch MBR. (a) Bioassay for the detection of AHL in the 2

batch MBR. 10 μl of extracts from the Control and the Microbial-vessel reactors were spotted 3

on the indicating agar plate together with a standard AHL of C8-HSL (1 ng). (b) TLC-4

bioassay image of the extracted AHLs from both reactors. 20 μl of extracts from both reactors 5

were loaded on TLC plate and the detected spots were compared with the TLC-bioassay 6

result of standard AHLs. 7

8

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1

Figure 6. TMP profiles during the operation of continuous MBR in three cycles. At the end of 2

1st and 2nd cycles, the used filtration membranes were cleaned with 1,000 ppm of NaOCl 3

solution and then reinserted into the reactors for the next cycle, respectively. During the 1st 4

and the 2nd cycles, the Vacant-vessels and the Microbial-vessels with recombinant E. coli 5

were inserted in the Reactor A (Control) and Reactor B (Microbial-vessel), respectively. Then 6

in the 3rd cycle, they were taken out of the reactors and were interchanged, i.e., the Microbial-7

vessels in the Reactor A and the Vacant-vessels in the Reactor B, respectively. 8

9

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1

Figure 7. Stability test for the quorum quenching activity of the Microbial-vessel with 2

recombinant E. coli. The Microbial-vessel was taken out from the continuous MBR at the 3

operating days of 0, 12, 25, and 80 to measure their quorum quenching activities. Error bar: 4

standard deviation (n=2). 5

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1

2

Figure 8. Application of the indigenous quorum quenching bacteria isolated from a real MBR 3

plant for wastewater treatment. (a) Comparison of the quorum quenching activity between 4

quorum quenching bacteria; the recombinant E. coli (pMBP-His-aiiA), Paenibacillus sp. 5

SYP2, and Rhodococcus sp. BH4. Control represents E. coli cells harboring the empty vector 6

pMBP-His-parallel1. Error bar: standard deviation (n=4). (b) TMP profile in the continuous 7

MBRs. The Vacant-vessels and the Microbial-vessels with Rhodococcus sp. BH4 were 8

inserted in the Control and Microbial-vessel reactors, respectively. 9

10

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1

Figure 9. The CLSM images of the biocake formed on the membrane surfaces in (a) the 2

Control reactor and (b) the Microbial-vessel reactor after 41 hours of operation. The 3

microbial cells were stained with SYTO9. Magnification 100 x. Image size 1213 μm x 1213 4

μm. 5

6

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Table 1. List of quorum quenching isolates from biocake and activated sludge of the real MBR plant operated at Okcheon in Korea. 1

Strain Colony morphology Identification (16S rRNA gene sequence homology) Carbon source Sample

BH4 Ivory (viscous) Rhodococcus qingshengii djl-6T (100%) C6-HSL Biocakea

SYP2 White Paenibacillus turicensis MOL 722T (98.7%) C6-HSL Biocake

SHEB1 White Enterobacter ludwigii DSM 16688T (99.8%) 3-oxo-C6-HSL Activated sludgeb

SHMC Yellow Micrococcus luteus NCTC 2665T (99.7%) C6-HSL Activated sludge

2

a Biocake: a mixture of microorganisms on the submerged membrane surface 3

b Activated sludge: a mixture of microorganisms in the broth of a real MBR 4

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455x196mm (300 x 300 DPI)

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