Fungal Quorum Quenching: A Paradigm Shift for Energy Savings in

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Fungal Quorum Quenching: A Paradigm Shift for Energy Savings in Membrane Bioreactor (MBR) for Wastewater Treatment Kibaek Lee, Seonki Lee, Sang Hyun Lee, Sang-Ryoung Kim, Hyun-Suk Oh, PyungKyu Park, Kwang-Ho Choo, Yea-Won Kim, Jung-Kee Lee, and Chung-Hak Lee Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b00313 • Publication Date (Web): 16 Sep 2016 Downloaded from http://pubs.acs.org on September 17, 2016

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Environmental Science & Technology

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Fungal Quorum Quenching: A Paradigm Shift for Energy Savings in Membrane Bioreactor (MBR) for Wastewater Treatment

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Kibaek Lee1, Seonki Lee1, Sang Hyun Lee1, Sang-Ryoung Kim1, Hyun-Suk Oh1, Pyung-Kyu Park2,

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Kwang-Ho Choo3, Yea-Won Kim4, Jung-Kee Lee4, and Chung-Hak Lee1*

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Department of Environmental Engineering, Kyungpook National University, Daegu, 702-701, Republic of Korea

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*Corresponding author: Chung-Hak Lee, School of Chemical and Biological Engineering, Seoul National University, Seoul, 151-742, Republic of Korea, Tel: +82-2-880-7075, E-mail: [email protected]

School of Chemical and Biological Engineering, Seoul National University, Seoul, 151-742, Republic of Korea Department of Environmental Engineering, Yonsei University, Wonju, 220-710, Republic of Korea

Department of Biomedicinal Science and Biotechnology, Paichai University, Daejeon 302-735, Republic of Korea

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Abstract

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In the last thirty years, the use of membrane bioreactors (MBRs) for advanced wastewater

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treatment and reuse have been expanded continuously, but they still suffer from excessive

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energy consumption resulting from the intrinsic problem of membrane biofouling. One of the

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major causes of biofouling in MBRs is bacterial quorum sensing (QS) via N-acylhomoserine

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lactones

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communications, respectively. In this study, we demonstrate that farnesol can substantially

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mitigate membrane biofouling in a MBR due to its quorum quenching (QQ) activity. When

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Candida albicans (a farnesol producing fungus) entrapping polymer beads (AEBs) were

(AHLs)

and/or

autoinducer-2

(AI-2),

enabling

1 ACS Paragon Plus Environment

intra-

and

inter-species


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placed in the MBR, the rate of transmembrane pressure (TMP) rise-up was substantially

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decreased, even for lower aeration intensities. This finding corresponds to a specific aeration

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energy savings of approximately 40% (25% through the physical washing effect and a further

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15% through the biological QQ effect of AEBs) compared to conventional MBRs without

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AEBs. A real-time RT-qPCR analysis revealed that farnesol secreted from C. albicans

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mitigated the biofilm formation in MBRs via the suppression of AI-2 QS. Successful control

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of biofouling and energy savings through fungal-to-bacterial QQ could be expanded to the

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plant

scale

for

MBRs

in

wastewater

treatment

with

36

37

TOC Art

38

39

2 ACS Paragon Plus Environment

economic

feasibility.

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1. Introduction

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As water shortages are exacerbated due to a fast-growing global population and climate

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change, water reuse has become a more urgent issue. Membrane bioreactors (MBRs) have

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emerged as one of the innovative options for advanced wastewater treatment and reuse. The

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global MBR market is forecast to reach more than $800 million by 2017.1 However,

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biofouling in MBRs, i.e., biofilm formation on the membrane surface causes high energy

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costs, which are the most common and critical hurdle for the expansion of MBRs throughout

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the world. Among the operating and maintenance costs for MBRs, the cost of membrane

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replacement has decreased drastically from 1992 to 2005, whereas the electrical power

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portion (energy cost) has largely increased.2, 3 In particular, the major cause for the overall

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energy consumption is the aeration required for the control of membrane biofouling.4-7 Since

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2009, quorum quenching (QQ), i.e., the inhibition of quorum sensing (QS) between

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microorganisms, has demonstrated its potential as a novel biofouling control technique in

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MBRs for wastewater treatment.8,

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surface in MBRs have been based on the decomposition of N-acylhomoserine lactones

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(AHLs), which are signal molecules used for QS between Gram-negative bacteria.10-13 Until

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now, the decomposition of signal molecules in MBRs, both enzymatic QQ 8, 9, 14 and bacterial

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QQ

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quorum sensing by fungus) has not been reported to date for biofouling control in MBRs used

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for wastewater treatment. Candida albicans, a ubiquitous fungus, living in activated sludge in

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MBRs16 has been reported to produce farnesol, which is the first known fungal QS signal

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molecule.17 Farnesol is used by the dimorphic fungus, C. albicans to inhibit filamentation, i.e.,

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the suppression of the conversion of yeast-to-mycelium.18 Farnesol was also reported to

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interfere with biofilm formation of some bacteria and fungi by inhibiting their cell growth or

10, 13, 15

9

QQ strategies for anti-biofouling on the membrane

have been reported. However, fungal-to-bacterial QQ (i.e., inhibition of bacterial

3

ACS Paragon Plus Environment


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affecting a metabolite pathways,19-25 but the mechanism behind this action still remains

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unclear. Hogan’s group

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producing fungi) and Pseudomonas aeruginosa (AHL-producing bacteria), but the direct

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correlation of farnesol with AI-2 quorum sensing has not yet been reported. In this study, we

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investigated the potential of farnesol to inhibit biofilm formation on the membrane surface in

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MBRs for wastewater treatment. Taking into account the practical application of fungal-to-

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bacterial QQ as an effective anti-biofouling strategy, we prepared C. albicans entrapping

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polymer beads and applied them to an MBR fed using real wastewater. To explore in greater

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detail the inhibition of bacterial biofilm formation by farnesol, we isolated three AI-2 QS

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bacteria from the activated sludge in a lab-scale MBR and tested the potential of farnesol to

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inhibit AI-2 QS in those bacteria as well as in two well-known AI-2 QS bacteria (V. harveyi

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BB152 and E. coli K12), respectively. The mechanism to inhibit AI-2 QS by farnesol was

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explored by monitoring the expression level of LuxS protein in the BB152 or K12 cultures

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grown in the presence of farnesol. We also quantitatively estimated the extent of energy

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savings through the fungal-to-bacterial QQ for an MBR operated over an extended period of

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about six months.

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investigated the interaction between C. albicans (farnesol-

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2. Materials and Methods

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2.1 Biofilm formation assay using 96-well plates: V. harveyi BB152, E. coli K12,

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activated sludge and isolated AI-2 QS bacteria (YB-1, YB-2, and YB-3) in the presence

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of farnesol

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The extent of anti-biofilm formation in the presence of farnesol was measured using

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microtiter dish biofilm formation assays.27 Vibrio harveyi BB152 was incubated for 24 hours

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in polystyrene 96-well plates at 30℃ in marine broth with either farnesol (dissolved in

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methanol) or an equivalent volume of methanol as a control. E. coli K12, activated sludge,

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Aeromonas sp. YB-1, Aeromonas sp. YB-2, and Enterobacter sp. YB-3 were incubated,

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respectively, using the same method as above, with the exception of the culture medium

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containing LB broth instead of marine broth. The polystyrene 96-well plate was washed three

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times with DI water. Then, the biofilms formed on the wells were stained with 1% crystal

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violet for 15 minutes, washed three times with DI water and dried completely. Then, the

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plates were destained with 95% ethanol and incubated at room temperature for 10 minutes.

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The absorbance at 595 nm of the solution containing crystal violet was measured using a

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microplate reader (SUNRISE™, TECAN, Switzerland). Each data point was averaged from

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six replicate wells and the standard deviations were calculated.

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2.2 Preparation of C. albicans entrapping beads (AEBs)

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A farnesol over-producing C. albicans strain (approximately 20 times more than the wild

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type

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Sabouraud broth at 37℃. Cultured C. albicans was centrifuged (7,000 g, 20 minutes), and

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resuspended in the sterilised DI water. AEBs were prepared using the dripping method.13

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However, the material and solidification method were modified to reinforce their stability by

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mixing polyvinyl alcohol and sodium alginate solutions because AEBs made of an alginate

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matrix are easily decomposed in real wastewater. The mixture of polyvinyl alcohol, alginate

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and C. albicans were dripped into the solution of CaCl2-Boric acid to form beads. Then, the

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beads were immersed in a sodium sulphate solution and stored in water. The content of C.

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albicans in the AEBs was approximately 17 ~ 18 mg cell/g bead. Non-cell entrapping beads

28, 29

) was used for the preparation of bead. C. albicans was incubated for 24 hours in a

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(NEBs), i.e., vacant beads without C. albicans, were also prepared using the same method as

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for AEBs.

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2.3 MBR operations

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Three lab-scale MBRs were operated in parallel, using wastewater discharged from a kitchen

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located in the Seoul National University (Figure 1). The conventional-MBR was operated

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without any beads, the vacant-MBR was operated with vacant beads (NEBs, dose-vol. 0.5%

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v/v), and the QQ-MBR was operated with C. albicans entrapping beads (AEBs, dose-vol. 0.5%

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v/v). The filtration membrane modules were hydrophilic polyvinylidene fluoride (PVDF)

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hollow fibres (Zeeweed 10, GE-Zenon, USA) and a nominal pore size of 0.04 µm and an

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effective surface area of 150 cm2. The MBRs were operated in two phases, Phase 1 under the

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same aeration intensity and Phase 2 under the different aeration intensities, as shown in Table

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1. The transmembrane pressure (TMP) was continuously monitored because the TMP is

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directly related to membrane biofouling, which is the most important factor in the evaluation

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of the MBR performance. For comparison of anti-biofouling performance of AEBs in an

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MBR, the average number of days for one TMP-Jump to occur (TTMP), was computed by

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dividing the operation period in days by the number of TMP-Jumps to 30 kPa during the

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operation period (see Table S4 of the supporting information).

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2.4 Confirmation of the AI-2 QS activity

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The AI-2 QS activity was evaluated using the luminescence of the reporter strain V. harveyi

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BB170.30 Briefly, BB170 was cultured overnight in an orbital shaker using an AB medium at

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30℃. BB170 was diluted to 1:5000 with a fresh AB medium. Next, 10 µl of each sample 6


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(cell-free supernatant) and 90 µl of the diluted BB170 were placed in 96-well plates.31 The

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luminescence of each well was measured every 30 minutes for 5 to 7 hours with a

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luminometer (Synergy2, BioTek, USA).

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2.5 Inhibition of AI-2 QS via farnesol in V. harveyi BB152, E. coli K12 and isolated AI-2

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QS bacteria (YB-2 and YB-3)

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When each culture reached the early exponential phase (OD600 of 0.2 ~ 0.3) in the AB

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medium, the culture was distributed equally in the conical tubes, and either farnesol

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(dissolved in methanol) or an equivalent volume of methanol as a control was added,

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respectively. The cell-free supernatants from each culture of BB152, K12, YB-2, or YB-3

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were collected every 30 minutes for 90 minutes and were stored at -20℃ for future

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experiments. The inhibition of AI-2 QS was evaluated by the degree of AI-2 QS activity upon

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the addition of farnesol. Data from three replicate wells were averaged and the standard

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deviations were calculated.

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3. Results and discussion

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3.1 The effect of farnesol on the biofilm formation of activated sludge

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It has been reported that farnesol has inhibited single culture bacterial biofilm formation 19, 21,

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has a similar effect on the biofilm formation of activated sludge taken from an MBR. As

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shown in Figure 2, a clear dose response was observed over a farnesol concentration ranging

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from 600 to 800 µM. Biofilm formation was reduced by approximately 25% and 43% for

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farnesol concentrations of 600 and 800 µM, respectively, compared to the control in which

and increased the susceptibility of biofilms to antibiotics

20, 22

7


. We investigated if farnesol


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only activated sludge was present without any farnesol. It is suggested that the biofilm

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formation on the membrane surface in an MBR was closely related to the concentration of

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farnesol present in solution. In the next stage, the effect of C. albicans on the biofilm

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formation of activated sludge was tested using a Transwell system in which activated sludge

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was co-cultured with farnesol or C. albicans, respectively. After 24 hour of incubation at

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30℃, the extent of biofilm formation in each Transwell system was compared to each

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another. When farnesol (at a final concentration of 800 µM) was added in the upper well of

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the Transwell system, the biofilm of the activated sludge in the bottom well was reduced by

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approximately 30% (Figure 3b) compared to the control well in which only activated sludge

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was present (Figure 3a). In the presence of C. albicans in the upper well (Figure 3c), the

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biofilm of activated sludge in the bottom well also decreased by approximately 45%

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compared with the control (Figure 3a). C. albicans was reported to produce and accumulate

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farnesol in the extracellular medium.17, 28 Consequently, the reduction of biofilm formation

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for the activated sludge with C. albicans co-culture was attributed to the presence of farnesol

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secreted from C. albicans. To further confirm that the reduction of the biofilm was caused by

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the farnesol secreted from C. albicans in Figure 3c, the extract from a C. albicans supernatant

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from 12 hr culture was analysed by HPLC (see Figure S1 of the supporting information).

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Based on the chromatogram in Figure S1a of the supporting information, the amount of

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farnesol extracted from C. albicans supernatant was calculated to be approximately 670 µg of

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farnesol per g of C. albicans cells.

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3.2 Preparation and application of C. albicans entrapping beads (AEBs)

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To demonstrate the practical application of farnesol to the mitigation of membrane biofouling

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in a real MBR for wastewater treatment, C. albicans was entrapped in spherical beads to 8


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protect them against other microorganisms in the activated sludge of an MBR. C. albicans

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entrapping beads (AEBs) were considered more appropriate than the direct injection of C.

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albicans into MBR. The morphologies of the vacant beads (NEBs, i.e., beads without C.

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albicans) and C. albicans entrapping beads (AEBs) were determined by SEM (Figure 4). As

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shown in Figure S2 of the supporting information, only metabolically active C. albicans (live

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cells) were marked clearly with an orange-red or yellow-orange fluorescent intravacuolar

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structure, indicating that C. albicans was well adjusted and entrapped in beads. Although C.

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albicans was confirmed to secrete farnesol substantially (see Figure S1a of the supporting

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information), it was required to verify whether AEBs produced sufficient amounts of farnesol.

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The extract from the AEBs and standard farnesol were analysed by HPLC. The peak of the

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extract from the AEBs appeared at the same retention time as those of standard farnesol.

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Based on the results in Figure S1b of the supporting information, the amount of farnesol

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extracted was calculated to be approximately 16.7 µg of farnesol per g of AEB. However,

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considering the result from the previous report that substantial amounts of farnesol could be

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lost in the extraction process

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would be greater than that.

17

, it was thought that the actual amount of farnesol secreted

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3.3 The effect of C. albicans on biofouling control in the continuous MBRs

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To evaluate the effect of C. albicans on biofouling control, three MBRs fed with real

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wastewater were run in parallel for more than 40 days: a conventional-MBR without any

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beads, a vacant-MBR with vacant beads (NEBs, 0.5% v/v), and a QQ-MBR with C. albicans

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entrapping beads (AEBs, 0.5% v/v). The conventional- and vacant-MBRs were also run to

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differentiate the physical effect of that using the beads might have from the biological effect

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of beads. Operating conditions for the three MBRs are given in greater detail in Table 1

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(Phase 1). As shown in Figure 5a, the biofouling was greatly mitigated in the QQ-MBR

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compared to the vacant-MBR as well as the conventional-MBR. The values of average

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number of days for one TMP-Jump to occur, TTMP, calculated from Figure 5a were 1.7, 11.7

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and 41.5 days for conventional-MBR, vacant-MBR, and QQ-MBR, respectively (see Table

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S4 of the supporting information). For better comparison of anti-fouling performance

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between the three MBRs, the ratio of the TTMP values were used. The ratio of the TTMP value

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for the vacant-MBR to that for the conventional-MBR is 6.9. This indicates that the physical

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washing effect, the detachment of biocakes from the membrane surfaces via collisions with

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membrane surface, of vacant beads was capable of alleviating biofouling by 6.9-fold. On the

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other hand, the ratio of TTMP value for QQ-MBR to that for the conventional MBR was 24.4,

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which is significantly larger than the ratio between the vacant- and conventional-MBRs. This

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demonstrates that the QQ activity, combined with the physical washing effect, of AEBs could

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more effectively reduce biofouling in an MBR.

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3.4 The comparison of energy consumption between conventional- and QQ-MBRs

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From a practical point of view, the alleviation of biofouling in an MBR by AEBs, as

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evidenced in Figure 5a and Table S4 (supporting information), is very important because it is

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closely associated with energy consumption during MBR operations. It is worth to note that

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among the MBR maintenance and operation costs, the aeration for the membrane scouring

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accounts for the highest energy consumption in MBR operations.4-7 This is why tremendous

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efforts have been undertaken to reduce the aeration energy in both academic and industrial

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sectors. However, the high aeration energy consumption is still an issue that needs to be

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resolved. In this context, it was meaningful to estimate how much energy would be saved

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through the application of QQ-MBR with C. albicans entrapping beads. The assessment of

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energy savings is conducted by operating the vacant- and QQ-MBRs at lowered aeration

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intensities and by calculating the specific energy consumption for membrane filtration, as

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well as aeration during 140 days of operation. The different aeration intensities were applied

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were 2.0 L/min for the conventional-MBR, 1.5 L/min for the vacant-MBR and 1.2 L/min for

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the QQ-MBR as shown in Table 1 (Phase 2). Generally, TMP rises more quickly when

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aeration intensity is lower. However, although the aeration intensity was the lowest in the

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QQ-MBR, the TTMP value for the QQ-MBR (42.8 days) was larger than that for conventional-

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MBR (19.0 days) and similar to that for vacant-MBR (40.2 days) in spite of 40% and 20%

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reduced aeration intensity, respectively, as shown in Figure 5b and Table S4 (supporting

237

information). Based on Figure 5b, the specific energy (i.e., energy consumed per unit volume

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of permeate) for membrane filtration and the specific energy for aeration were evaluated

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using equations (supporting information – Materials & Methods 1.5 [1]) and (supporting

240

information – Materials & Methods 1.5 [2]), respectively. In the conventional-, vacant-, and

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QQ-MBRs, the average specific membrane filtration energies were 3.8 Wh/m3, 2.8 Wh/m3,

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and 4.0 Wh/m3, respectively, whereas the specific aeration energies were 192 Wh/m3, 144

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Wh/m3, and 115 Wh/m3, respectively (Figure 6). The specific energy consumptions for

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membrane filtration were relatively small and did not result in any significant differences

245

between the three MBRs. However, the specific energy consumption for aeration in the

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vacant-MBR decreased by approximately 25% (Figure 6b) compared to the conventional-

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MBR (Figure 6a). Furthermore, the specific energy in the QQ-MBR decreased by

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approximately 40% (Figure 6c) compared to the conventional-MBR (Figure 6a). In other

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words, the specific aeration energy saved was approximately 25% via the physical washing

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effect of the beads and a further 15% through the biological QQ effect of the beads.

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3.5 The effect of farnesol on the production of extracellular polymeric substances (EPS)

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EPS, which is excreted by microorganisms in MBRs, consists of a variety of organic

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substances. Because an increase in EPS is one of the factors that cause flux decline in MBRs

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32

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bound-EPS in the microbial flocs in the aerobic membrane tank were measured and

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compared in terms of protein and polysaccharide between three MBRs (see Figure S3 of the

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supporting information). The protein concentration in the bound-EPS in the QQ-MBR was

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substantially reduced compared to the other two MBRs (conventional- and vacant-MBRs). It

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was thought that the bound-EPS could be reduced by farnesol secreted from the AEBs.

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Previous studies also reported that EPS was reduced by farnesol.19, 37

, EPS is usually regarded as a control parameter for membrane biofouling in MBRs.33-36 The

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3.6 The effects of farnesol on the characteristics of the activated sludge and the effluent

264

quality

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As mentioned previously, QQ-MBR inhibited biofouling via physical and biological effects

266

and thus substantially reduced aeration energy consumption. However, we cannot proceed

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further without verifying the stability of effluent quality in the QQ-MBR, as the effluent

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quality is one of the primary factors in the evaluation of MBR performance. Thus, COD

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removal and mixed liquor suspended solids (MLSS) in each MBR were continuously

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monitored during the extended operations (140 days) for the three MBRs. As shown in Figure

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S4a of the supporting information, the differences in COD removal efficiency were shown to 12


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be negligible between the three MBRs, suggesting that the addition of NEBs or AEBs did not

273

affect the ability of the activated sludge to decompose organic pollutants in the feed

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wastewater. Moreover, the addition of farnesol did not make any significant difference in the

275

mixed liquor suspended solids in three MBRs over the entire running period (see Figure S4b

276

of the supporting information). The addition of beads (NEBs or AEBs) resulted in a reduction

277

in the microbial floc size in the aerobic tank (see Figure S4c of the supporting information).

278

However, the floc sizes were similar between the vacant- and QQ-MBRs. This may be

279

attributed to the continuous collisions between the beads containing the microbial flocs.

280

Consequently, we conclude that the QQ-MBR (with AEBs) exhibits better performance in

281

terms of energy savings than the conventional-MBR without compromising the

282

biodegradable activity of the activated sludge.

283 284

3.7 The mechanism behind the biofilm formation inhibition via farnesol

285

Determining the inhibitory mechanism associated with biofilm formation in the presence of

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farnesol in MBRs is a worthwhile activity. Biofilm formation is one of various phenotypes

287

regulated by bacterial quorum sensing (QS) via N-acylhomoserine lactones (AHLs) and/or an

288

autoinducer-2 (AI-2).38-42 AHLs are intra-species QS signal molecules for Gram-negative

289

bacteria, whereas AI-2 is a universal QS signal molecule for inter-species communications

290

between Gram-negative and Gram-positive bacteria.38 Taking into account the fact that

291

farnesol has been reported mostly to inhibit biofilm formation in gram-positive bacteria 19, 21,

292

23

293

because AHLs QS are not related to Gram-positive bacteria but, rather, Gram-negative

294

bacteria. First, we examined if AI-2 QS bacteria really exist in the activated sludge of an

, we suspected and investigated step by step the potential inhibition of AI-2 QS via farnesol

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MBR. As shown in Table S1 and Figure S5 of the supporting information, three species of

296

AI-2 QS bacteria were isolated from the lab-scale MBR operated in this study (i.e.,

297

Aeromonas sp. YB-1, Aeromonas sp. YB-2, and Enterobacter sp. YB-3). Second, we

298

examined if farnesol has anti-biofilm formation effects on two well-known AI-2 QS bacteria

299

(BB152 and K12) as well as three isolated AI-2 QS bacteria (YB-1, YB-2, and YB-3). For all

300

five species of bacteria, a clear dose response to biofilm formation was observed over

301

farnesol concentrations ranging from 600 to 800 µM (Figure 7). However, it is worth noting

302

that farnesol over a range of 600 to 800 µM had no effect on the cell growth of six different

303

AI-2 producing microorganisms (see Figure S7 of the supporting information). Third, C.

304

albicans or farnesol was co-cultured with YB-3 in a Transwell system. After a 24 incubation

305

period at 30℃, the extent of biofilm formation was measured (see Figure S6 of the

306

supporting information). When farnesol (final conc. 800 µM) was added in the upper well of

307

a Transwell system, the biofilm of the YB-3 in the bottom well was reduced by

308

approximately 33% (see Figure S6b of the supporting information) compared to the sample

309

without farnesol in the bottom well (see Figure S6a of the supporting information). In the

310

presence of C. albicans in the upper well, the biofilm of YB-3 in the bottom well also

311

decreased by approximately 40% (see Figure S6c of the supporting information) compared to

312

that without farnesol in the bottom well (see Figure S6a of the supporting information).

313

Consequently, the reduction of biofilm formation in the co-cultured well (YB-3 and C.

314

albicans) in Figure S6c was attributed to the presence of farnesol secreted from C. albicans.

315

Fourth, to investigate if such a dose response is related to AI-2 QS, we analysed the inhibition

316

of AI-2 QS via farnesol using a luminescence bioassay with BB170 as a reporter strain. As

317

shown in Figure S5 of the supporting information, because YB-1 (colony No. 16) showed

318

relatively smaller AI-2 QS signals than the other two isolated AI-2 QS bacteria, YB-2 14


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(colony No. 6) and YB-3 (colony No. 22), we excluded YB-1 from these analyses. Just as the

320

inhibition of the biofilm formation using farnesol (Figure 7), AI-2 luminescence of the

321

reporter strain (BB170) was also reduced as farnesol concentration increased in each cell-free

322

supernatant from BB152, K12 and two isolated AI-2 QS bacteria (YB-2 and YB-3) (Figure 8).

323

Fifth, finally, we investigated the AI-2 mechanism in depth, which gave rise to the decrease

324

of luminescence levels of the reporter strain (BB170) in BB152 and K12 cultures in the

325

presence of farnesol. LuxS protein is known to be involved in the signal transduction of AI-2

326

QS.30 The LuxS is reported to be directly related to biofilm development in many bacteria

327

species.43-46 The enzyme LuxS produces 4,5-dihydroxy-2,3-pentanedione (DPD) from S-

328

ribosylhomocysteine (SRH).30,

329

spontaneous rearrangement of DPD. As a result, the amount of AI-2 QS signal molecules,

330

which govern the extent of biofilm development, is proportional to the expression level of the

331

gene luxS. Consequently, we extracted RNA from each culture of BB152 or K12 with and

332

without farnesol and then measured the expression level of luxS in each culture by real-time

333

RT-qPCR (real-time reverse transcription quantitative PCR) analysis. As shown in Figure 9,

334

the expression levels of luxS in the BB152 and K12 cultures with farnesol were lower than

335

those without farnesol, respectively. Therefore, we conclude that farnesol secreted from C.

336

albicans is able to mitigate biofilm formation via the suppression of AI-2 QS.

47

The AI-2 QS signal molecule is formed from the

337 338

Notes

339

The authors declare no competing financial interest.

340

Acknowledgements 15



341

We would like to thank Professor Deborah A. Hogan (Geisel School of Medicine at

342

Dartmouth, USA) for providing the fungal strain (Candida albicans) and Professor

343

Kenneth Nickerson (University of Nebraska-Lincoln, USA) for helpful comments.

344

This work was supported through the Korea Ministry of Environment (MOE) as a

345

"Convergence Technology Program (2015001640001)" and Commercializations

346

Promotion Agency for R&D Outcomes (COMPA) (2014K000240). The authors

347

declare no competing financial interest.

348 349

Supporting Information

350

The Supporting Information is available free of charge on ACS Publications website as DOI:

351

List of strains used in this study (Table S1 and S2), List of primers used in this study (Table

352

S3), Average number of days for one TMP-Jump (TTMP) in three MBRs (Table S4),

353

Chromatogram of farnesol extracted from (a) C. albicans and (b) AEBs (Figure S1),

354

Fluorescence microscopy image of AEBs (Figure S2), Comparisons of bound-EPS in the

355

MBRs (Figure S3), Comparison of (a) COD, (b) MLSS, and (c) floc size in the three MBRs

356

(Figure S4), Measurement of AI-2 QS activity (Figure S5), Biofilm formation for YB-3 in

357

various co-culture systems (Figure S6), and Growth of (a) BB152, (b) K12, (c) Activated

358

sludge, (d) YB-1, (e) YB-2, and (f) YB-3 in presence of farnesol (Figure S7) (PDF)

359

360

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495

Table 1. MBR operating conditions (Phases 1 and 2). Phase 1

Phase 2

Working volume (L)

2.5

2.5

SRT (day)

30

30

HRT (hour)

12

12

150

150

(Zeeweed-10, GE-Zenon)

(Zeeweed-10, GE-Zenon)

17

15

2

Membrane area (cm ) 2

Flux (L/m /h) Operation cycle

Suction (29 min) / Relaxation (1 min)

off Conventional MBR - 2.0

Aeration (L/min)

1.5

Vacant MBR - 1.5 QQ MBR - 1.2

Feed COD (mg/L) COD removal efficiency (%)

200 ~ 300

200 ~ 300

Broth: 91 ~ 92

Broth: 90 ~ 91

Permeate: 94 ~ 95

Permeate: 92 ~ 93

MLSS (mg/L)

4000 ~ 4500

3900 ~ 4300

F/M ratio

0.12 ~ 0.17

0.12 ~ 0.15

Bead (mg C. albicans/g bead)

Dose-volume of beads

17 ~ 18

0.5 % (v/v)

496 497

23


17 ~ 18

0.5 % (v/v)


498

Figure 1. Schematic diagram of the three parallel MBRs. The conventional-MBR was operated without any beads, the vacant-MBR was operated with vacant beads (NEBs, dose-vol. 0.5% v/v) and the QQ-MBR was operated with C. albicans entrapping beads (AEBs, dose-vol. 0.5% v/v). The average size of the beads is 4.3 ~ 4.5 mm. 499 500

24


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501

Figure 2. Biofilm formation from activated sludge as a function of farnesol concentration. Activated sludge in an LB broth for 24 hr using 96-well plates. The error bars represent the standard deviation (n=6 as the technical replicates). 502 503

25



504

Figure 3. Biofilm formation from the activated sludge in various co-culture systems. (a) Upper well; activated sludge (A.S), bottom well; A.S, (b) upper well; Farnesol (800 µM), bottom well; A.S, and (c) upper well; C. albicans (C.A), bottom well; A.S. The error bars represent the standard deviation (n=2 as the technical replicates). 505 506

26


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507

Figure 4. SEM images of (a) a vacant bead (non-cell entrapping bead, NEB) and (b) C. albicans entrapping bead, AEB). Magnifications are 30X and 2000X for the images on the left and right, respectively. 508 509

27



510

Figure 5. Variation of the TMP in the three MBRs. (a) The three MBRs under the same aeration intensity and (b) the three MBRs under different aeration intensities. When the TMP reached 40 kPa, the used membranes were cleaned with 1000 ppm of NaOCl and then reinserted into the MBR for the next cycle in all MBRs. 511 512 28


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513

Figure 6. Comparisons of the specific energy consumption (Wh/m3) in the MBRs at different aeration intensities. (a) In the conventional-MBR, the aeration intensity was maintained at 2.0 L/min. (b) In the vacant- and (c) QQ-MBR, the aeration intensity was maintained at 1.5 and 1.2 L/min, respectively. 514 515

29



Figure 7. Inhibition of biofilm formation in the five strains; (a) BB152, (b) K12, (c) YB-1, (d) YB-2, and (e) YB-3 with respect to farnesol concentration. Quantitative analysis of the biofilm formation was performed after 24 hr of incubation for each species using 96-well plates. BB152 was cultured in a marine broth, whereas K12, YB1, YB-2, and YB-3 were cultured in LB broth. The error bars represent the standard deviation (n=6 as the technical replicates). 516 517 30


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518

Figure 8. Reduction of AI-2 QS in the four strains; (a) BB152, (b) K12, (c) YB-2, and (d) YB-3 with respect to farnesol concentration. The error bars indicate the standard deviations (n=3). 519 520

31



521

Figure 9. Effect of farnesol on the expression level of the luxS gene in (a) BB152 and (b) K12 using real-time RT-qPCR. The error bars represent the standard deviations (n=3). 522

32


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