Membrane layers intensifying quorum quenching alginate cores and its potential for membrane biofouling control

Accepted Manuscript Membrane layers intensifying quorum quenching alginate cores and its potential for membrane biofouling control Jinhui Huang, Ying Yang, Guangming Zeng, Yanling Gu, Yahui Shi, Kaixin Yi, Yichen Ouyang, Jianglin Hu, Lixiu Shi PII: DOI: Reference:

S0960-8524(19)30167-1 https://doi.org/10.1016/j.biortech.2019.01.134 BITE 21011

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

26 November 2018 26 January 2019 28 January 2019

Please cite this article as: Huang, J., Yang, Y., Zeng, G., Gu, Y., Shi, Y., Yi, K., Ouyang, Y., Hu, J., Shi, L., Membrane layers intensifying quorum quenching alginate cores and its potential for membrane biofouling control, Bioresource Technology (2019), doi: https://doi.org/10.1016/j.biortech.2019.01.134

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Membrane layers intensifying quorum quenching alginate cores and its potential for membrane biofouling control Jinhui Huang a, b, *,Ying Yang a, b , Guangming Zeng a, b ,Yanling Gu

a, b

, Yahui Shi a, b,

Kaixin Yi a, b , Yichen Ouyang a, b , Jianglin Hu a, b , Lixiu Shi a, b a

College of Environmental Science and Engineering, Hunan University, Changsha,

Hunan 410082, China. b

Key Laboratory of Environmental Biology and Pollution Control, Hunan University,

Ministry of Education, Changsha, Hunan 410082, China. * Corresponding author. College of Environmental Science and Engineering, Hunan University, Changsha, Hunan 410082, China. Tel./ Fax: + 86 731 88821413. E-mail addresses: [email protected] (J.H. Huang).

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Abstract Quorum quenching (QQ) has been proved to be an efficient method to mitigate biofouling in membrane bioreactors (MBRs). In this paper, in order to enhance practicability of QQ microcapsules, we prepared three types microcapsules with same alginate cores (SAs). The microcapsules with polyacrylonitrile (PAN) layer showed excellent performance in preventing cell leakage from the microcapsules, increasing service life and improving mechanical strength. And confocal laser scanning microscopy images demonstrated that there were very little dead bacteria in the microcapsules with both chitosan and PAN layer than microcapsules with only PAN layer because chitosan layer can protect bacteria entrapped in cores from the hurt caused by poisonous PAN solution. At the same time, the microcapsules with PAN layer presented more efficient anti-biofouling ability in the physical washing test. At last, the bacterial microcapsules coated with both chitosan and PAN layer showed an obvious biofouling mitigation during the MBRs operation.

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Keywords: Polyacrylonitrile; Chitosan; Alginate cores; Microcapsules; Quorum quenching; Biofouling

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1. Introduction

Membrane bioreactors (MBRs) are a kind of highly effective wastewater treatment technique that combines an active sludge process with a membrane filtration and can offer high quality effluent (Maqbool et al., 2015; Meng et al., 2017; Waheed et al., 2018; Shi et al., 2017a; Gu et al., 2018a). However, membrane fouling in MBRs hinders its wide application in actual use (Shi et al., 2017b). Among the all types of membrane fouling (organic adsorption, inorganic precipitation, biological), biofouling, as a dynamic change process, is so complex that the interactions between bacteria during the period of biofilm formation has not been completely figured out for now (Malaeb et al., 2013; Mark Pasmore, 2001). Plentiful efforts such as membrane modification, optimization in MBR operation, aeration scouring, antibiotics, in-situ chemical cleaning and Quorum quenching (QQ) (Anja, 2010; Iorhemen et al., 2017; Tan et al., 2017; Zeng et al., 2018) have been attempted to mitigate membrane biofouling. QQ, as a novel biological fouling inhibition approach that can block N-acyl homoserine lactone (AHL) for cell-to-cell communication stands out for its potential to mitigate membrane biofouling (Weerasekara et al., 2016; Xiong and Liu, 2010). Many study results indicated that QQ has prominent performance in biofouling mitigation in MBRs, especially QQ bacterial immobilization technology. A core- shell structure QQ media was designed to achieve biostimulation in QQ technology and has realized efficient biofouling control successfully (Yu et al., 2018). Kim et al. (2013) immobilized QQ bacteria into cross-linked alginate beads and found that the bacterial beads had excellent effect in biofouling mitigation. Meanwhile, it has been pointed out that the alginate beads had a low stability and tended to collapse in long4

term MBRs operation (Kim et al., 2015). It is because alginate matrices are sensitive to chelating compounds and anti-gelling ions which can sequester Ca2+ and result in structure disintegrating ultimately (Skjåk-Braek, 1990). So many efforts were made to overcome this limitation. Li et al. (2017) prepared photo-cross-linking reinforced alginate-methacrylate (MA) beads and found that reinforced alginate-MA beads had the higher stability and the lower swelling ratio. Another research discovered that the combination of ionic cross-linking and dehydration could enhance durability and lifespan of QQ media (Lee et al., 2017). In addition, bacterial alginate cores were enclosed to polymeric membrane layer by Kim et al. (2015) and the result showed that macroencapsulation had the higher practicability in biofouling mitigation than common alginate beads. However, what cannot be ignored is that there are always some irreversible damage (most are the death of bacteria caused by poisonous matters or harmful processes) to the bacteria immobilized in QQ beads during these beads reinforcement process. Alginate is a kind of commonly used bacterial immobilization material because it has well biocompatibility and gentle gelation process, meanwhile, alginate is easy to get and has low cost (Skjåk-Braek, 1990; Arruda and Vitolo, 1999; Rathore et al., 2013), so alginate was chosen as the core of microcapsules in this study. And chitosan with excellent biodegradability was chosen as a coating layer because chitosan is a rare cationic polymer in nature (Qi et al., 2006; Kamalian et al., 2014) and its amino in molecular chain can trigger polyelectrolyte reaction with carboxyl in alginate molecular chain to form a semipermeable membrane on the surface of alginate cores (Rathore et al., 5

2013; Yu et al., 2011). And PAN membrane was chosen for its low cost and high chemical stability and mechanical strength (Braun et al., 2005). In this study, we selected two covering membrane materials and manufactures three microcapsules with same alginate cores but different outer layers which has never been used in QQ technology in MBRs: only a layer of chitosan (SA-Cs), only a layer of PAN membrane (SA-Ps) and a layer-by-layer (LBL) structure composed of a chitosan layer and a PAN membrane layer (SA-C-Ps). And the properties of these prepared microcapsules were characterized and were compared with cross-linked SAs from different aspects in a series of experiments (physics, chemistry, physical washing and QQ activity) to prove the effect of membrane layers in intensifying QQ media and to evaluate their feasibility in biofouling mitigation in MBRs.

2. Material and methods

2.1. Preparation of alginate microcapsules

Rhodococcus sp. BH4 (Oh et al., 2013) were inoculated in Luria-Bertani (LB) culture at 30℃. After 18 h cultured, the LB broth were centrifuged, washed with sterile water, and centrifuged again to obtain bacteria. The wet weight of bacteria was recorded, and the bacteria were resuspended in 5 ml sterile water. The BH4 suspension were mixed with 3% (w/v) sodium alginate solution (1 g bacteria /100 g sodium alginate solution) and the mixed solution were dripped into 5% CaCl2 solution through a sterile injection syringe to form beads for 1 h (Zeng et al., 2018; Huang et al., 2018a). The SA beads were washed with deionized water for three times. Then the SAs were immersed in 0.4% (w/v) chitosan 6

solution (chitosan dissolved in acetic acid aqueous solution, pH= 5.7- 6.0) and stirred for 40 mins to obtain alginate-chitosan（SA-Cs）microcapsules. Phase separation process (Witte et al., 1996) were used to form polymeric membrane layers on the surface of SAs and SA-Cs, the steps were as follows: firstly, PAN powders dried for 24 h in a vacuum dryer, then dissolved in N,N-Dimethylacetamide (DMAC) and stirred for 12 h at 60℃ to acquire 10% (w/v) PAN casting solution. Secondly, the SAs and the SA-Cs were immersed in PAN solution completely for 30 s to form polymeric membrane layer on the outer surface of the SAs and the SA-Cs. Finally, the beads with polymeric membrane layer were immediately immersed in water bath for 1 h, washed with deionized water for three times to remove excessive PAN solution and named SAPs/ SA-C-Ps microcapsules. Vacant microcapsules (no bacteria enclosed in the alginate cores) were manufactured following the process of bacterial microcapsules except that the BH4 suspension was replaced with sterile water. Four types vacant/bacterial QQ microcapsules (SA-Cs, SA-Ps and SA-C-Ps) were stored at 4℃ until use.

2.2. Chemical stability test of prepared microcapsules

To evaluate the capacity of membrane layers in resisting chemical impact, calcium chelating-EDTA buffer solution (30 mM EDTA, 55 mM sodium citrate and 0.15 M NaCl) was adopted for its ability to destroy the three-dimensional network of alginate cores through capturing Ca2+ fixed in cross-linked alginate cores. 30 of bacterial microcapsules were put in 100 ml EDTA buffer solution for 60 mins and 4 ml liquid samples were taken at same time intervals (every 10 mins). The absorption value was measured using an 7

ultraviolet-visible spectrophotometer at OD600 to evaluate the cell leakage. Moreover, the chemical stability experiment of vacant microcapsules were also carried out to eliminate interference of other compounds (those materials used for manufacturing microcapsules such as sodium alginate, chitosan and PAN) to the change of absorption value.

2.3. Physical stability test of prepared microcapsules

To evaluate the physical performance of these microcapsules and figure out the effect of membrane layers in physical performance, SR test reflecting change in volume and weight change test reflecting loss in weight were carried out. 30 of vacant microcapsules were immersed in deionized water for 25 d with gentle vibration (Yu et al., 2011). During the period, the diameter of microcapsules was measured using an electronic digital indicator at different time intervals. The volume of alginate microcapsules were defined by the following Eq. (1). Swelling ratio (SR) was calculated by the following Eq. (2). V = (4⁄3) 𝜋(𝐷⁄2)

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Eq. (1)

where V represents the volume of alginate microcapsules, cm3; D represents the diameter of alginate microcapsules, cm3. SR = (𝑉𝑡 ⁄𝑉0 − 1) ∗ 100%

Eq. (2)

where V0 represents the volume of alginate microcapsules at time 0, cm3; Vt represents the volume of alginate microcapsules at time t, cm3. As for weight change test, before the total mass of 50 microcapsules was weighed, 50 of vacant alginate microcapsules were first dried in ovens at 37℃ for 30 mins (Li et al., 2017). Then these dried microcapsules were immersed in 400 ml water with continual 8

aeration. At different time intervals, the microcapsules were taken out and the water on it were removed using filter paper, then the microcapsules were dried in ovens at 37℃ for 30 mins again and weighed. Weight change rate was calculated by the following Eq. (3). Weight change = 𝑊𝑡 ⁄𝑊0 ∗ 100%

Eq. (3)

where W0 represents the total weight of 50 alginate microcapsules at time 0, g; Wt represents the total weight of 50 alginate microcapsules at time t, g.

2.4. Quorum quenching activity test of prepared microcapsules

To evaluate the quorum quenching activity of prepared bacterial microcapsules with membrane covering and compared with SAs, C8-HSL degradation activity of QQ microcapsules was tested (Gu et al., 2018b). The bacterial microcapsules were cultured in LB broth for 12 h. After washed with deionized water, 80 of these cultured microcapsules were put in 50 ml Tris-buffer solution with 200 ng/ml C8-HSL. At the set time intervals, 500 μl samples were taken for QQ activity detection. The concentration of C8-HSL in the sample was determined using the A. tumefaciens A136 bioluminescence assay (Oh et al., 2012; Fuqua C, 1996). In brief, every 10ml of the A136 broth were mixed with every 90ml of LB agar contained spectinomycin, tetracycline and 5-Bromo-4chloro-3-indolyl β-D-galactopyranoside (X-gal). After LB agar coagulated, 6 μl samples were spotted on it carefully and these medium were cultured for 12 h in biochemical incubator for chromogenic reaction. At last, the residual concentration of C8-HSL was calculated according to the diameter of color zone. In addition, the QQ activity of vacant

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microcapsules were also carried out as a negative control. And the QQ activity was defined by the following Eq. (4). QQ activity = (1 − 𝑛𝑡 ⁄𝑛0 ) ∗ 100%

Eq. (4)

where n0 represents the concentration of C8-HSL at time 0, mg/L; nt represents the residual concentration of C8-HSL at time t, mg/L.

2.5. Physical washing test of prepared microcapsules

Considering there are two main aspects (QQ activity and physical washing effect) (S.H. Lee et al., 2016) for QQ microcapsules to mitigate biofouling formed on the membrane, so the physical washing ability of the alginate microcapsules in biofilm formation was carried out and compared with SAs as well. 20 of every type vacant alginate microcapsules were placed in four batch reactors respectively. In each batch reactor, there were three PVDF coupons fixed on the internal surface of the container. All batch reactors were at same aeration intensity, working volume (100 ml) and sludge concentration. After 24 h aeration, the PVDF coupons were taken out to measure the biofilm formed on it. The PVDF coupons were first stained with 10 ml 0.1% (w/v) crystal violet (CV) for 30 mins, and the floating color on the coupons was washed carefully with deionized water. Next, the coupons were discarded after the coupons were immersed in 10 mL ethanol solution for 30 mins. At last, the concentration of CV in ethanol was measured using an ultravioletvisible spectrophotometer at OD590. The formed biofilm mass was defined as the absorption value.

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2.6. Membrane bioreactors operation

Two parallel MBRs (pH=6.9-7.2) with a working volume of 4.5 L were operated with same aeration intensity of 1.5 mg/L. The MLSS and flux of MBRs was 5000±500 mg/L and 12 L/ m2/h. The HRT was 18 h and there was no drained activated sludge during the experiment. And these two MBRs were feed with artificial waste water which composed as follows (every 50 L): glucose, 25 g; yeast extract powder, 1.25 g; peptone, 1.25 g; (NH4)2SO4, 12.5 g; KH2PO4, 7.5 g; K2HPO4, 7.5 g; MgSO4, 0.225g; CaCl2, 0.099 g; NaCl3, 1.75 g; FeCl3, a little; CoCl2, a little; and NaHCO3, 28g. One MBR was set as control with vacant microcapsules, the other was set as QQ with bacterial microcapsules. The biofouling mitigation of microcapsules was shown by the value of the transmembrane pressure (TMP) during the MBRs operation.

2.7. Analytical Methods

The mechanical strength of bacterial microcapsules was measured using a texture analyzer (TA.XT Plus, SMS) equipped with a P/0.5 probe. The hardness work was defined as the mechanical strength (the area under the curve between the trigger plot and the first turning plot). Total 30 of alginate microcapsules were tested to obtain an average value. In order to confirm that alginate and chitosan has formed a PEC membrane layer because of polyelectrolyte reaction, fourier transform infrared spectroscopy (FTIR) characterization was made. The vacant SAs, SA-Cs and chitosan powders were dried in oven at 60℃, and then grinded to powders, pressed into thin slices and analyzed using 11

FTIR from 4000 cm-1 to 400 cm-1. Meantime, to observe the morphology of PAN membrane layer, the SA-C-Ps were cut into half and the PAN membrane layer were separated from the core. The detached PAN membrane layer was pre-frozen in refrigerator at -20℃ for 48 h before drying in freeze drier at -55℃ for 6 h and examined by scanning electron microscopy (SEM, JEOL JSMIT300LA). The live/dead cells immobilized in alginate cores of bacterial SA-Ps and SA-C-Ps microcapsules were observed by confocal laser scanning microscope (CLSM) in order to provide visual evidence that the presence of chitosan layer can protect bacteria from harm during the coating process of PAN membrane layer. The SA-Ps and SA-C-Ps beads were cut into slices to make sure that the microcapsules can be fully stained and the cells immobilized in alginate slices were stained with Live/Dead BacLight Viability Kit (Molecular Probes) under dark conditions for 15 mins according to the operating manual, then the live/dead cells distribution was observed using CLSM (TI-E+A1 SI, Nikon).

3. Results and discussion

3.1. Characterization of alginate microcapsules

Appearance of microcapsules was shown in Supplementary Information. Both SAs and SA-Cs appeared translucent morphology but with different diaphaneity. Moreover, White spherical appearance of SA-Ps and SA-C-Ps proved that a core-shell structure has formed. The diameters of these beads were about: SAs for 3.57 (±0.24 mm), SA-Cs for 4.03 (± 0.26 mm), SA-Ps for 4.72 (±0.32 mm) and SA-C-Ps for 5.18 (±0.36 mm). Compared 12

to SAs, the increased diameter demonstrated the existence of chitosan layer and PAN layer as well. These microcapsules may have different mechanical strength because it coated with different membrane layers, and cause distinction of actual working life time at last. So to figure out mechanical strength of these QQ microcapsules, prepared bacterial microcapsules were tested by a texture analyzer (test speed: 0.5 mm/s, trigger force: 5.0 g, distance: 2.00 mm). As presented in Fig. 1, the hardness work of the alginate microcapsules were SAs for 3.81 (±0.50 mJ), SA-Cs for 3.21 (±0.77 mJ), SA-Ps for 4.87 (±0.80 mJ) and SA-C-Ps for 4.65 (±0.80 mJ). The result indicated that the hardness work of SA-Ps and SA-C-Ps was obviously higher than SAs (approximately 22%-28%), which meant that polymeric membrane layer can efficiently enhance mechanical strength of QQ microcapsules. Noteworthiness, the result showed an interesting phenomenon that the mechanical strength of SA-Cs was lower than cross-linked SAs (approximately 19%) even if there was an additional chitosan layer outside the SAs. It may be because that chitosan layer is a type of cationic polymer and has strong ability to absorb water. The mechanical strength would become lower after absorbing water (Liu and Rempel, 2015). And that SA-C-Ps was a slight inferior than SA-Ps also declared the same consequence. The cross-linked alginate, SA-Cs and pure chitosan powder FTIR spectra were captured to prove that the chitosan has formed a polyelectrolyte complex (PEC) membrane layer with alginate through electrostatic adsorption (Wang et al., 2001) (the FTIR spectra were presented in Supplementary Information). Alginate displayed characteristic peaks of C=O at 1618 cm-1 (Li et al., 2005; Baysal et al., 2013), 13

symmetrical stretching vibration of –COO- at 1421 cm-1, and a typical band of –OH from about 3600 cm-1 to 3000 cm-1 (Baysal et al., 2013).The chitosan spectrum showed vibrations of amide I at 1663 cm-1, amide II at 1598 cm-1 (Li et al., 2005), a characteristic absorption peak of the amino group at 1152 cm-1, and a stretching vibration peak of the C-H at 2873 cm-1 (Ho et al., 2009). In addition, a broad band located from 3600 cm-1 to 3000 cm-1 because –OH bond overlapped N-H bond (Shi et al., 2018; Huang et al., 2018b). And there were peaks at 1092 cm-1 and 1039 cm-1 in both SA and Chitosan corresponding to the stretching of C-O-C and C-O (Espinosa-Andrews et al., 2010). However, the amide I became weaken and the amide II was disappeared, the –OH band became wider and more pointed in SA-Cs spectrum, in the meantime, the C=O bond shifted from 1618 cm-1 to 1635 cm-1, and the peak of the amino group was ceased to be seen. All these changes demonstrated the chitosan has successfully coated on the alginate cores through ionic interaction and has formed a covering layer. In addition, the cross section, outer surface and inner surface SEM photographs of PAN membrane layer were taken (the SEM image were presented in Supplementary Information). We can see that the membrane layer construction was internal porous with surface compact. This may be because that the moment PAN casting solution contacted with coagulating bath (deionized water), the reaction occurred right away on the twophase interface during the process of phase inversion. However, there was no immediate reaction for the component under the lamina, it was after some degree of delay (about a few seconds), the exchange of inner substance accomplished, therefore the cellular structure of the PAN membrane layer has formed. 14

3.2. Quorum quenching activity of alginate microcapsules

The QQ activity of bacterial microcapsules contained 10mg QQ bacteria/ g alginate was measured. C8-HSL was used as the standard degradation substance for the reason that C8-HSL is a representative quorum sensing signal molecule and usually used in QQ activity test by other researchers (Zeng et al., 2018; Gu et al., 2018b; Huang et al., 2016). As shown in Fig. 2, the C8-HSL degradation rate of SA-Cs was up to 72% at 60 min, inferior to SAs which was about 82% at 60 min. At the same time, the C8-HSL degradation rate of SA-Ps and SA-C-Ps was 46% and 54%, much lower than SAs too, and there may be two reasons for this phenomenon. One is that the chitosan layer and PAN layer of a certain thickness would affect material transfer process, caused decreasing supply of C8-HSL to the alginate cores compared to SAs, and resulted in the reduction of reaction region at last. The other is that PAN casting solution was poisonous, therefore the process of coating PAN layer would cause irreversible damage to QQ bacteria enclosed in the outer edge of alginate cores (Xiong and Liu, 2010), so part of QQ bacteria were dead and lost their QQ ability. But, compared with SA-Ps, chitosan layer in SA-CPs may be able to protect bacteria entrapped in the alginate cores for the reason that it could hinder bacteria from contacting with poisonous PAN solution directly, so the C8HSL degradation rate of SA-C-Ps was slightly higher than SA-Ps.

3.3. Physical stability of alginate microcapsules

To investigate the performance of microcapsules coated with different membrane layers in physical stability, SR test was carried out. Vacant microcapsules were immersed 15

in deionized water with vibration for 25 days. As shown in Fig. 3(A), that the SR of SAPs and SA-C-Ps remained mainly unchanged with time reflected high stability of PAN. On the other hand, the SR of SAs reached 22% at day 1, then kept constant in general with slight fluctuations. And the SR of SA-Cs was 68% at day 1, 120% at day 5, then achieved swelling equilibrium. The reason for this phenomenon may be that both alginate and chitosan is highly hydrophilic, therefore make SAs and SA-Cs possess high waterabsorbing capacity. Meantime, the structure of chitosan layer was loose, made that H2O could easily infiltrate into pores in the microcapsules and stored in these pores. And that would cause the water-holding capacity of SA-Cs greatly heightening. Physical stability was reflected by weight change test as well. The experiment was carried out in aeration conditions to simulate practical environment. As shown in Fig. 3(B), SAs first collapsed gradually in several days at a certain rate and then completely disappeared at day 15. For SA-Cs, it had a sharply weight increase within 2 days, then collapsed rapidly in the next 7 days. However, weight change of SA-Ps and SA-C-Ps was no more than 22% and 13% during the whole experiment. Before three-dimensional structure collapse, the microcapsules would absorb water and those water would be stored in the pore of hydrogel spheres. At the same time, the membrane layer enveloped SAs making that even if SAs collapsed, the collapsed material would be stored in the microcapsules until the membrane layer was destroyed. This was the reason for a drastically rising in the first 2 days for SA-Cs. After the three-dimensional network of SA-Cs absorbed enough water, the structure became very fragile, the chitosan layer fractured and microcapsules collapsed rapidly at last. However, the high mechanical 16

strength of PAN membrane made it not easy to broken. As a result, the weight change of SA-Ps and SA-C-Ps was insignificant. In summary, because of the high physical stability of PAN membrane layer, so the working life of microcapsules can be enhanced and this would make QQ technology closer to practical use in MBRs.

3.4. The capacity of alginate microcapsules to resist chemical impact

The alginate core is easy to collapse because cross-linked Ca2+ can be captured from the formed hydrogel three-dimensional network by chelating agent. And because alginate microcapsules were about to use in an actual wastewater treatment, so the resistance to chemical impact was also taken into account. Bacterial microcapsules were put in EDTA buffer solution which simulated terrible conditions to evaluate chemical stability and cell leakage was measured at OD600. And control experiment of vacant microcapsules proved that there was only bacteria contributed to the absorption value at OD600 (data showed in Supplementary Data). As shown in Fig. 4, the OD600 of SAs increased rapidly at the first 10 min, the bacteria enclosed in alginate cores were released due to the collapse of alginate cores and the absorption value reached to the maximum which may mean that the bacteria have completely released while cell leakage of SA-Cs increased in a relatively slow trend compared to SAs and reached maximum at 30 min. This phenomenon declared that the chitosan layer was resistance to chemical impact, could postpone the disintegration of alginate cores to some extent, and delayed cell leakage from alginate cores at last. Nevertheless, it cannot prevent cell from leakage entirely. However, the microcapsules coating with a PAN membrane layer (SA-Ps and SA-C-Ps) protected 17

bacteria from leakage caused by terrible chemical conditions, so the cell leakage can be ignored basically. And PAN membrane had high resistance to chemical impact, so it could keep porous structure in a long period.

3.5. Effect of membrane layers to bacteria entrapped in alginate microcapsules

Many studies (Kim et al., 2015; Rathore et al., 2013) demonstrated that the process of cross-linking alginate and polyelectrolyte reaction between alginate and chitosan was very gentle and had no damage to bacteria. However, as mentioned before, the process of coating PAN layers could cause damage to QQ bacteria entrapped in alginate cores because PAN casting solution is poisonous to bacteria. So, we adopted a type pf LBL structure (SA-C-Ps) to alleviate this kind of damage. And to intuitively observe the effect of coating PAN layers and the role of chitosan layer in protecting bacteria, CLSM images were taken. The process of coating PAN layers did go against cell survival, and caused a ring band death of bacteria (CLSM images showed in Supplementary Data). But, we can find that there was very little dead bacteria in the peripheral of microcapsules which confirmed the chitosan layer in SA-C-Ps is highly effective in protecting bacteria from the damage caused by PAN casting solution. Besides, the QQ activity of SA-C-Ps is higher than SA-Ps demonstrated the same result as well.

3.6. Membrane biofouling control of alginate microcapsules

There is a physical washing role of QQ microcapsules in membrane biofouling except QQ activity because QQ microcapsules always keep moving under aeration condition,

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therefore would cause impact between QQ microcapsules with membrane module placed in the MBRs (S. Lee et al., 2016; S.H. Lee et al., 2016). To compare the physical washing effect of prepared vacant microcapsules, batch experiment was carried out under aeration condition in a square container (length for 4 cm; width for 2 cm and height for 15 cm). The result was shown in Fig. 5. We can see that the formed biofilm mass was SAs > SACs > SA-C-Ps > SA-Ps. In detail, SA-Cs, SA-Ps, SA-C-Ps was about 6%, 20%, 9% lower than SAs, and there was a slight difference between SA-Cs and SA-C-Ps. It was worth mentioning that the weight of single bead were also SAs < SA-Cs < SA-Ps < SA-C-Ps (data not shown), totally corresponding to the change of diameter. General speaking, impact intensity and impact frequency between QQ medium and membrane module are the most two important factors that influence physical washing ability. SA-Ps with a medium weight had more frequent impact frequency and stronger impact intensity than others at same aeration intensity because too light (such as SAs and SA-Cs) would lower impact intensity and too heavy (such as SA-C-Ps) would lower impact frequency. According to the results presented above, for the reason that SA-C-Ps had more superior physical stability, chemical stability and physical washing test than SAs and SACs, at the same time, SA-C-Ps were more effective than SA-Ps in terms of QQ activity and more living QQ bacteria than SA-Ps. So SA-C-Ps were chosen as the bacterial QQ microcapsules to have a further test about the anti-biofouling ability in MBRs. Total 5% (v/v) (volume of media/volume of reactor) bacterial SA-C-Ps or vacant SA-C-Ps were put into two MBRs respectively. The COD removal efficiencies were maintained more than 90% during the whole MBR operation. The TMP results of both MBRs were shown 19

in Fig. 6. The vacant MBRs took about 15 h to reach 60 kPa, however, QQ MBR took about 45 h to reach the same TMP value, which was nearly the 3 times of vacant MBRs. That confirmed the QQ activity of microcapsules in anti-biofouling in MBRs. 4. Conclusion PAN layer realize absolutely remain of bacteria entrapped in microcapsules, improve microcapsules resistance to chemical impact and lengthen microcapsules service life. CLSM proved that chitosan layer in LBL microcapsules could protect bacteria from the

damage caused by poisonous PAN solution. Microcapsules with PAN layer showed better anti-biofouling capacity. Alginate cores coated with chitosan and PAN layer showed obvious membrane biofouling control during MBR operation and proved that it could be a potential bacterial QQ microcapsules. This study came up with a new thought of LBL in QQ media and would bring QQ technology closer to real wastewater treatment in MBRs.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (51578222, 51178172, 51521006 and 51378190), the Project of Chinese Ministry of Education (113049A), the Research Fund for the Program for Changjiang Scholars and Innovative Research Team in University (IRT-13R17). Special thanks to the school of Chemical and Biological Engineering (Seoul National University, Republic of Korea) for the supplies of Rhodococcus sp. BH4 and A. tumefaciens A136.

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References

1. Anja, 2010. Membrane fouling in membrane bioreactors—Characterisation, contradictions, cause and cures. J. Membr. Sci. 363, 1-28. 2. Arruda, Vitolo, 1999. Characterization of invertase entrapped into calcium alginate beads. Appl. Biochem. Biotechnol. 81, 23-33. 3. Braun, Cherdron, Ritter, 2005. Polymer Synthesis: Theory and Practice, fifth ed. Springer Berlin Heidelberg. 4. Espinosa-Andrews, Sandoval-Castilla, Vázquez-Torres, Vernon-Carter, LobatoCalleros, 2010. Determination of the gum Arabic–chitosan interactions by Fourier Transform Infrared Spectroscopy and characterization of the microstructure and rheological features of their coacervates. Carbohydr. Polym. 79, 541-546. 5. Fuqua C, 1996. Conserved cis-acting promoter elements are required for densitydependent transcription of Agrobacterium tumefaciens conjugal transfer genes. J. Bacteriol. 178, 435-440. 6. Gu, Huang, Zeng, L.X. Shi, Y.H. Shi, Yi, 2018a. Fate of pharmaceuticals during membrane bioreactor treatment: Status and perspectives. Bioresour. Technol. 268, 733-748. 7. Gu, Huang, Zeng, Y.H. Shi, Hu, Tang, Zhou, Xu, L.X. Shi, 2018b. Quorum quenching activity of indigenous quorum quenching bacteria and its potential application in mitigation of membrane biofouling. J. Chem. Technol. Biotechnol. 93, 1394-1400. 8. Ho, Mi, Sung, Kuo, 2009. Heparin-functionalized chitosan-alginate scaffolds for controlled release of growth factor. Int. J. Pharm. 376, 69-75. 22

9. Huang, Y.H. Shi, Zeng, Gu, Chen, L.X. Shi, Hu, Tang, Zhou, 2016. Acyl-homoserine lactone-based quorum sensing and quorum quenching hold promise to determine the performance of biological wastewater treatments: An overview. Chemosphere 157, 137-151. 10. Huang, Zhou, Zeng, Gu, Hu, Tang, Y.H. Shi, L.X. Shi, 2018a. Biofouling control and sludge properties promotion through quorum quenching in membrane bioreactors at two aeration intensities. Biotechnol. Lett. 40, 1067-1075. 11. Huang, Cheng, Y.H. Shi, Zeng, Yu, Gu, L.X. Shi, Yi, 2018b. Honeycomb-like carbon nitride through supramolecular preorganization of monomers for high photocatalytic performance under visible light irradiation. Chemosphere 211, 324-334. 12. Iorhemen, Hamza, Tay, 2017. Membrane fouling control in membrane bioreactors (MBRs) using granular materials. Bioresour. Technol. 240, 9-24. 13. Kamalian, Mirhosseini, Mustafa, Manap, 2014. Effect of alginate and chitosan on viability and release behavior of Bifidobacterium pseudocatenulatum G4 in simulated gastrointestinal fluid. Carbohydr Polym. 111, 700-706. 14. K. Baysal, Aroguz, Adiguzel, B.M. Baysal, 2013. Chitosan/alginate crosslinked hydrogels: preparation, characterization and application for cell growth purposes. Int. J. Biol. Macromol. 59, 342-348. 15. Kim, Oh, Jo, Yeon, Lim, C.H. Lee, J.K. Lee, 2013. Biofouling control with beadentrapped quorum quenching bacteria in membrane bioreactors: physical and biological effects. Environ. Sci. Technol. 47, 836-842. 16. Li, Ramay, Hauch, Xiao, Zhang, 2005. Chitosan-alginate hybrid scaffolds for bone 23

tissue engineering. Biomaterials 26, 3919-3928. 17. Li, Muller, Chang, Frettloh, Schonherr, 2017. Encapsulation of Autoinducer Sensing Reporter Bacteria in Reinforced Alginate-Based Microbeads. ACS Appl. Mater. Interfaces 9, 22321-22331. 18. Liu, Rempel, 2015. Preparation of superabsorbent polymers by crosslinking acrylic acid and acrylamide copolymers. J. Appl. Polym. Sci. 64, 1345-1353. 19. Malaeb, Le-Clech, Vrouwenvelder, Ayoub, Saikaly, 2013. Do biological-based strategies hold promise to biofouling control in MBRs? Water Res. 47, 5447-5463. 20. Maqbool, Khan, Waheed, Lee, Hashmi, Iqbal, 2015. Membrane biofouling retardation and improved sludge characteristics using quorum quenching bacteria in submerged membrane bioreactor. J. Membr. Sci. 483, 75-83. 21. Mark Pasmore, Smith, Baker, Silverstein, Darrell Coons, 2001. Effects of ultrafiltration membrane surface properties on Pseudomonas aeruginosa biofilm initiation for the purpose of reducing biofouling. J. Membr. Sci. 194, 15-32. 22. Meng, Zhang, Oh, Zhou, Shin, Chae, 2017. Fouling in membrane bioreactors: An updated review. Water Res. 114, 151-180. 23. Oh, Yeon, Yang, Kim, C.H. Lee, Park, Han, J.K. Lee, 2012. Control of membrane biofouling in MBR for wastewater treatment by quorum quenching bacteria encapsulated in microporous membrane. Environ. Sci. Technol. 46, 4877-4884. 24. Oh, Kim, Cheong, C.H. Lee, J.K. Lee, 2013. Biofouling inhibition in MBR by Rhodococcus sp. BH4 isolated from real MBR plant. Appl. Microbiol. Biotechnol. 97, 10223-10231. 24

25. Qi, J. Ma, Yu, Xie, Wang, X. Ma, 2006. Behavior of microbial growth and metabolism in alginate–chitosan–alginate (ACA) microcapsules. Enzyme Microb. Technol. 38, 697-704. 26. Rathore, Desai, Liew, Chan, Heng, 2013. Microencapsulation of microbial cells. J. Food Eng. 116, 369-381. 27.S.H. Lee, S. Lee, K. Lee, Nahm, Kwon, Oh, Won, Choo, C.H. Lee, Park, 2016. More Efficient Media Design for Enhanced Biofouling Control in a Membrane Bioreactor: Quorum Quenching Bacteria Entrapping Hollow Cylinder. Environ. Sci. Technol. 50, 8596-8604. 28. S.H. Lee, S. Lee, K. Lee, Nahm, Jo, J. Lee, Choo, J.K. Lee, C.H. Lee, Park, 2017. Enhancing the Physical Properties and Lifespan of Bacterial Quorum Quenching Media through Combination of Ionic Cross-Linking and Dehydration. J. Microbiol. Biotechnol. 27, 552-560. 29. Skjåk-Braek, 1990. Alginate as immobilization matrix for cells. Trends Biotechnol. 8, 71-78. 30. S. Lee, S.H. Lee, K. Lee, Kwon, Nahm, C.H. Lee, Park, Choo, J.K. Lee, Oh, 2016. Effect of the Shape and Size of Quorum-Quenching Media on Biofouling Control in Membrane Bioreactors for Wastewater Treatment. J. Microbiol. Biotechnol. 26, 1746-1754. 31. S.R. Kim, K.B. Lee, J.E. Kim, Won, Yeon, C.H. Lee, Lim, 2015. Macroencapsulation of quorum quenching bacteria by polymeric membrane layer and its application to MBR for biofouling control. J. Membr. Sci. 473, 109-117. 25

32. Tan, S. B. Liu, Y. G. Liu, Gu, Zeng, Hu, Wang, S. H. Liu, Jiang. 2017. Biochar as potential sustainable precursors for activated carbon production: multiple applications

in

environmental

protection

and

energy

storage. Bioresour.

Technol. 227, 359-372. 33. Waheed, Xiao, Hashmi, Stuckey, Zhou, 2017. Insights into quorum quenching mechanisms to control membrane biofouling under changing organic loading rates. Chemosphere 182, 40-47. 34. Wang, Eugene, Lee-Yong, 2001. Chitosan–Alginate–CaCl2 System for Membrane Coat Application. J. Pharm. Sci. 90, 1134-1142. 35. Weerasekara, Choo, Lee, 2016. Biofouling control: Bacterial quorum quenching versus chlorination in membrane bioreactors. Water Res. 103, 293-301. 36. Witte, Dijkstra, Berg, Feijen, 1996. Phase separation processes in polymer solutions in relation to membrane formation. J. Membr. Sci. 117, 1-31. 37. Xiong, Liu, 2010. Biological control of microbial attachment: a promising alternative for mitigating membrane biofouling. Appl. Microbiol. Biotechnol. 86, 825-837. 38. Y.H. Shi, Huang, Zeng, Gu, Hu, Tang, Zhou, Yang, L.X. Shi, 2017a. Evaluation of soluble microbial products (SMP) on membrane fouling in membrane bioreactors (MBRs) at the fractional and overall level: a review. Rev. Environ. Sci. Bio-Technol. 17, 71-85. 39. Y.H. Shi, Huang, Zeng, Gu, Chen, Hu, Tang, Zhou, Yang, L.X. Shi, 2017b. Exploiting extracellular polymeric substances (EPS) controlling strategies for performance enhancement of biological wastewater treatments: An overview. Chemosphere 180, 26

396-411. 40. Y.H. Shi, Huang, Zeng, Cheng, Yu, Gu, L.X. Shi, Yi, 2018. Stable, metal-free, visible-light-driven photocatalyst for efficient removal of pollutants: Mechanism of action. J. Colloid Interface Sci. 53, 433-443. 41. Yu, Song, Zheng, Liu, Zhang, Ma, 2011. Study on membrane characteristics of alginate–chitosan microcapsule with cell growth. J. Membr. Sci. 377, 214-220. 42. Yu, Lee, Zhang, Choo, 2019. Core-shell structured quorum quenching beads for more sustainable anti-biofouling in membrane bioreactors. Water Res. 150, 321-329. 43. Z.T. Zeng, Tang, Xiao, Huang, Gu, Y.H. Shi, Hu, Zhou, Li, L.X. Shi, G.M. Zeng, 2018. Quorum quenching bacteria encapsulated in PAC-PVA beads for enhanced membrane antifouling properties. Enzyme Microb. Technol. 117, 72-78. 44. Z. W. Zeng, Tang , Y. G. Liu, Tian, G. M. Zeng , Jiang, S. B. Liu, Li, N. Liu, Yin, 2018. Comprehensive adsorption studies of doxycycline and ciprofloxacin antibiotics by biochars prepared at different temperatures. Front. Chem. 6, 80-.

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Figure captions

Fig. 1. Comparison of hardness work of alginate microcapsules.

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Fig. 2. Comparison of QQ activity of prepared microcapsules. The QQ activity was considered as the degradation concentration at the setting time to the initial concentration.

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Fig. 3. (A) Comparison of swelling ratio of prepared microcapsules, (B) Comparison of weight change of prepared microcapsules.

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Fig. 4. Comparison of chemical stability of prepared microcapsules (The calcium chelating-EDTA buffer solution was composed of 30 mM EDTA, 55 mM sodium citrate and 0.15 M NaCl.).

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Fig. 5. Comparison of physical washing effect of prepared microcapsules. The experimental period was 24 hours under a continuous aeration condition.

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Fig. 6. TMP profiles of MBRs during a period of 48 h. CK was the TMP of the vacant microcapsules MBR, and QQ was the TMP of the bacterial microcapsules MBR.

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Highlights: 1. Polyacrylonitrile layer can enhance chemical stability 2. Polyacrylonitrile layer can lengthen service life of microcapsules 3. Anti-biofouling ability of microcapsules with polyacrylonitrile layer was superior 4. Chitosan layer can protect bacteria from damage caused by toxic casting solution 5. Biofouling was mitigated by quorum quenching microcapsules during MBR operation

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