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Macroencapsulation of quorum quenching bacteria by polymeric membrane layer and its application to MBR for biofouling control
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Macroencapsulation of quorum quenching bacteria by Polymeric Membrane Layer and Its Application to MBR for Biofouling Control Sang-Ryoung Kim, Ki-Baek Lee, Jeong-Eun Kim, Young-June Won, Kyung-Min Yeon, Chung-Hak Lee, Dong-Joon Lim
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Received date: 9 July 2014 Revised date: 29 August 2014 Accepted date: 8 September 2014 Cite this article as: Sang-Ryoung Kim, Ki-Baek Lee, Jeong-Eun Kim, Young-June Won, Kyung-Min Yeon, Chung-Hak Lee, Dong-Joon Lim, Macroencapsulation of quorum quenching bacteria by Polymeric Membrane Layer and Its Application to MBR for Biofouling Control, Journal of Membrane Science, http: //dx.doi.org/10.1016/j.memsci.2014.09.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Macroencapsulation of Quorum Quenching Bacteria by Polymeric Membrane Layer and Its Application to MBR for Biofouling Control
Sang-Ryoung Kim1, Ki-Baek Lee1, Jeong-Eun Kim1, Young-June Won1, Kyung-Min Yeon1, Chung-Hak Lee*1, Dong-Joon Lim2
1
School of Chemical and Biological Engineering, Seoul National University, Seoul 151-744,
Republic of Korea 2
School of Chemical Engineering, Yeungnam University, Gyeongsan 712-749, Republic of
Korea
*Corresponding author. Phone: +82-2-880-7075. Fax: +82-2-874-0896. E-mail: [email protected] Address: School of Chemical and Biological Engineering, Seoul National University 1 Gwanak-ro, Gwanak-gu, Seoul, 151-744, Korea
Abstract We report the preparation and characterization of macrocapsules coated by a polymeric membrane layer for the stable encapsulation of quorum quenching (QQ) bacteria (Rhodococcus sp. BH4). As a new macrocapsule manufacturing platform, a phase inversion method was adopted to cover an alginate core with a microporous membrane layer of poly(vinylidene) fluoride (PVDF), polyethersulfone (PES) or polysulfone (PSf). Scanning electron microscopy revealed that membrane layer was successfully formed on the outside of the alginate-QQ bacteria matrix. Although contact with the organic solvent during the phase inversion step caused minor cell damage, the QQ activity of macrocapsules could be reactivated through cultivation. The QQ bacteria entrapping macrocapsules showed an excellent anti-biofouling capacity in the continuous membrane bioreactor (MBR) fed even with real wastewater. The macrocapsules were capable of maintaining QQ activity more safely than previously reported alginate beads in harsher environmental conditions such as in real wastewater or in the presence of chelating agent (EDTA) which can disintegrate an alginate matrix. Particularly, the PSf coated membrane layer was pronounced in preventing QQ bacteria from leaking outside the macrocapsules. This study is expected to bring the bacterial QQ closer to being a practical solution to the biofouling problem in MBR for wastewater treatment.
Keywords Cell encapsulation, Alginate bead, Membrane bioreactor (MBR), Quorum quenching, Biofouling.
1. Introduction Immobilized microorganisms have been extensively used in environmental [1-5], pharmaceutical [6] and biological [7] processes for the production of enzymes, organic acids, antibiotics, lipids and amino acids. Immobilization provides target microorganisms nutrients without competing with other microorganisms and protects them from environmental stress [8-10]. The immobilization of microorganisms in spherical calcium alginate has been reported to be useful in the laboratory and in commercial applications due to its excellent biocompatibility and ease of preparation [8]. Bacteria or yeast entrapping alginate beads have been applied in the production of ethanol [11], monoclonal antibodies [10] and insulin [12], as well as in the removal of nitrogen, phosphorous and metal in the wastewater treatment process [13-15]. Recently, Kim et al. [4] encapsulated quorum quenching (QQ) bacteria into an alginate matrix and demonstrated that the QQ bacteria entrapping alginate beads were very efficient for biofouling control in a membrane bioreactor (MBR) for wastewater treatment via both physical washing and quorum quenching. However, they also found it was unavoidable that the calcium alginate matrix gradually decomposed during a long-term MBR operation although the feed to MBR was a relatively mild synthetic wastewater (Figure S1). Alginate has been reported to be susceptible to disintegration in the presence of excess monovalent ions and Ca2+ chelating agents like EDTA (Ethylene Diamine Tetraacetic Acid), which is a main obstacle in its wider application [8, 10]. Some efforts have been made to increase the chemical and physical stability of the alginate matrix in a biological environment by coating with polyelectrolytes such as polylysine [16], poly(ethylene glycol) [17], chitosan [18], polyvinylamine [19]. However, coating of alginate beads with these materials suffered from relatively high costs or complex preparation steps, which are limitations for use in wastewater
treatment from a practical point of view. In this study, we enclosed the QQ bacteria-containing alginate core by a polymeric membrane layer to solve these limitations of the conventional alginate immobilization platform. Using various commercial polymers such as poly(vinylidene) fluoride (PVDF), polyethersulfone (PES) and polysulfone (PSf), a porous membrane layer was formed around the alginate core through a phase inversion process, which is commonly used for the preparation of low pressure membranes [20-23]. The macrocapsules consisting of the membrane coating layer and alginate core with QQ bacteria were characterized and compared with previously reported alginate beads [4] in terms of structure and QQ activity. The feasibility of macrocapsules was evaluated not only under a harsh chemical condition, but also in the continuous operation of MBR fed with real wastewater.
2. Materials and methods 2.1. Materials PVDF pellet (average Mw ~530,000), N-methyl-2-pyrrolidone (NMP), ethylenediamine tetraacetic acid disodium salt (EDTA) and calcium chloride (CaCl2) were purchased from Sigma Aldrich Korea. PSf (Udel®P1800) and PES (Ultrason E6020P) pellets were obtained from Solvay-Korea (Korea) and BASF (Germany), respectively. Sodium alginate powder was purchased from Junsei Chemical (Japan). All chemicals were of analytical grade and used as received. A Millipore Synergy filtration system was used to purify all water. 2.2. Microorganisms and growth conditions N-acyl homoserine lactone (AHL) quorum sensing (QS) autoinducers were detected using a reporter strain of Agrobacterium tumefaciens A136 (Ti-)(pCF218)(pCF372). It was grown at 30°C in Luria-Bertani (Miller, US) medium with streptomycin (0.25 v/v%) and tetracycline
(0.05 v/v%) [24-26]. The quorum quenching Rhodococcus sp. BH4 (KCTC 33122) strain was previously isolated from the a MBR wastewater treatment plant (Ok-cheon, Korea) and was cultured in Luria-Bertani medium at 30°C [4, 5]. 2.3. Preparation of macrocapsules The overall preparation scheme of macrocapsules is graphically depicted in Figure 1. First, QQ bacteria (Rhodococcus sp. BH4) were entrapped in the alginate matrix (alginate beads with QQ bacteria) (Step 1 in Figure 1). Subsequently, cultured Rhodococcus sp. BH4 were centrifuged (12,000 g, 15 min) and resuspended in 10 ml of deionized water. This Rhodococcus sp. BH4 suspension was mixed with 90 ml of the alginate solution. The alginate concentration in the final mixture was 2 w/v%. The mixture was dropped into 500 ml of calcium chloride solution (4 w/v%) using a syringe needle to form spherical beads and stirred for 3 hours. After washing and swelling in deionized water, the alginate beads were used for subsequent polymeric coating. The QQ bacteria content of alginate beads was 2.0 mg QQ bacteria/g alginate solution. Microporous membrane layers were formed on the surface of alginate beads using the phase inversion technique [27, 28]. In detail, PVDF, PSf and PES pellets were dissolved in NMP at 60°C for 12 hours. The concentration of each polymer solution was set to 10 w/v%. Each polymeric solution was stirred overnight for complete mixing and cooling. Alginate beads were immersed in each polymeric solution for 30 seconds (Step 2 in Figure 1). The water contained in the alginate beads contacted the polymeric solution and thus induced phase separation. Consequently, the inner membrane layer was formed at the interface between the alginate bead and the polymer coating layer. And then, the alginate bead surrounded by the polymeric solution was immersed in a water coagulation bath for 1 hour (Step 3 in Figure 1). During Step 3, the polymeric solution still enveloping the outer coating layer made contact
with the non-solvent (i.e., water) to induce the second phase inversion on the outer surface of the polymeric layer. Finally, the macrocapsules were repeatedly washed and stored in deionized water at 4°C until use. 2.4. Bioassay for AHL autoinducer quantification The AHL level was quantitatively determined using a bioassay with the luminescence substrate Beta-Glo (Promega, USA) [29, 30]. Briefly, 5 l of AHL samples were loaded into a 96-well-plate with 95 l of an overnight culture of A. tumefaciens A136. After incubation for 90 minutes at 30°C, 30 l of Beta-Glo were added into each well. This generated the luminescence of oxyluciferin, whose intensity is proportional to the amount of betagalactosidase released from the A. tumefaciens A136 biosensor. After incubation for 30 minutes at 30°C, the bioluminescence intensity of each sample was recorded using a luminometer (Synergy 2, Bio-Tek, USA). A calibration curve was prepared using a standard AHL solution of N-octanoyl-L-homoserine lactone (C8-HSL, Sigma-Aldrich), which was previously found to be present as a major AHL in the activated sludge used in this study [4, 26]. Each bioassay was conducted in triplicate to assess repeatability. 2.5. Determination of quorum quenching activity The quorum quenching (QQ) activity of alginate beads and macrocapsules was quantitatively determined by the reduction rate of C8-HSL standard solution [4, 5]. Briefly, C8-HSL was dissolved in deionized water to a concentration of 200 nM. Then, 40 beads were added to 40 ml of the standard AHL solution and incubated at 30°C for 0, 30, 60, 120 minutes using an orbital shaker at 200 rpm. The residual concentration of C8-HSL at each reaction time point was determined using the A. tumefaciens A136 bioluminescence assay. The “QQ activity” of beads (i.e., alginate beads or macrocapsules) was defined by the rate of AHL (C8HSL) degradation during initial 60 minutes (nmol C8-HSL/min). On the other hand, the
“Relative QQ activity” was also introduced to monitor the QQ stability of various beads and was defined by the percentage ratio of residual QQ activity to initial QQ activity. 2.6. Measurement of mechanical strength The mechanical resistance of alginate beads and macrocapsules were determined using a texture analyzer (CT3 4500, Brookfield, USA) [31]. The mechanical deformation tests were performed at a mobile probe (TA44) speed of 0.5 mm/s until the bead matrix was observed to burst. The hardness work, a measure of the energy required to crush the container, was calculated as the area under the curve of the compression plot. An average of at least 20 beads was assessed to obtain statistically relevant data. 2.7. Measurement of chemical stability To evaluate the chemical stability of any type of beads under a harsh chemical environment, a buffered EDTA solution was selected to make up a harsh environment. It is because EDTA is well known to be a strong complexing agent with a calcium ion and thus is expected to easily disintegrate any alginate bead matrix containing calcium ions [32]. Beads to be tested were placed in citrate buffer (30 mM EDTA, 55 mM sodium citrate and 0.15 M sodium chloride) and then the mixture was incubated for 60 minutes with gentle agitation [8, 33]. During the incubation, 3 ml were taken out of the suspension every ten minutes and the concentration of leaked cells from disintegrated beads were measured using a spectrophotometer at 600 nm because it correlates with the chemical stability of beads. 2.8. Restoration of quorum quenching activity of disintegrated beads Restoration of beads (i.e., alginate beads or macrocapsules) were conducted as follows: Fresh alginate beads or fresh macrocapsules were deliberately put into a harsh chemical environment by placing them in citrate buffer (30 mM EDTA, 55 mM sodium citrate and 0.15 M sodium chloride) for 10 minutes, respectively. For the restoration of such chemically
treated, i.e., disintegrated beads, they were washed with deionized water three times and then placed in Luria-Bertani (LB) medium at 30°C for 12 hours using a shaking incubator at 200 rpm. The extent of restoration was evaluated by comparing the Relative QQ activities of tested beads at each step, i.e., before and after chemical treatment, and after restoration, respectively. 2.9. MBR operation Two lab-scale MBRs (one control & one QQ MBR) were constructed in a similar way to those described by other researches and were operated in parallel (Figure 2) [4, 5]. To evaluate biofouling control only through the QQ activity of macrocapsules, one control MBR was operated with vacant macrocapsules containing no QQ bacteria (Rhodococcus sp. BH4) as shown in Figure 2. The MBR was fed with real wastewater which was generated in a restaurant after screening collected real wastewater through 1 mm wire mesh. Membrane modules were made of poly(vinylidene) fluoride hollow fiber (ZeeWeed500, GE-Zenon, USA). The permeate flux was set to 30 l/m2/h (LMH) and the transmembrane pressure (TMP) was continuously monitored to determine the degree of membrane fouling. Other detailed operating conditions are given in Table S1. 2.10. Scanning electron microscopy (SEM) The morphology of the macrocapsules was examined by SEM (JEOL JSM-6701F). Macrocapsules were cut in half and the membrane coating layer was detached from the alginate core. This detached membrane layer, after ethanol dehydration, was embedded in epoxy resin for 2 hours at room temperature. This embedded membrane specimen was cut with a razor blade for structural observation. Samples were placed on a conducting stage with a platinum coating, and then analyzed using SEM. Alginate beads were cut by a razor blade for structural observation, then dehydrated in ethanol, transferred to a critical point dryer for
2 hours, coated with platinum, and examined by SEM.
2.11. Confocal laser scanning microscopy (CLSM) The three-dimensional structure of beads and the membrane-biocake was visually monitored with confocal laser scanning microscope (CLSM, C1 plus, Nikon, Japan) [4]. Samples were fluorescently stained with nucleic acid-specific SYTO9 or a BacLight Live/Dead staining kit according to the observational aim (Molecular Probes, Eugene, OR). Z-section image stacks (5 m) of each channel were reconstructed using IMARIS software (Bitplane AG, Switzerland).
3. Results and discussion 3.1. Preparation and characterization of macrocapsules with various polymeric coatings Three kinds of microporous polymeric layers, i.e. PSf, PES and PVDF, were formed on the surface of alginate beads. As shown in Figure 3a, these macrocapsules were globular in shape and their diameters ranged from 3.2 to 3.8 mm (mean 3.5 mm), indicating that the type of polymer material had little influence on the size and shape of the macrocapsules. SEM observations clearly showed the outer surface, the inner surface and the cross-section of a piece of macrocapsule coated with PSf, PES and PVDF (Figure 3b). In all macrocapsules, the polymeric coating consisted of inner and outer membrane layers, of which the inner layer had a denser structure than the outer membrane layer. This was thought to be caused by the relatively slower demixing rate of the polymeric solution in the first phase inversion step than in the second phase inversion step. The cross-sectional image of each polymeric coating layer clearly demonstrated asymmetric finger-like structures. The overall thickness of each
membrane coating layer was found to be 60-100 m. The main reason for the unevenness of the membrane thickness was attributed to the fact that the phase inversion of the polymeric solution took place not on a flat sheet, but on a spherical bead. In the next step, the mechanical strengths of the three types of macromolecules were evaluated by compression tests using a texture analyzer (Figure 4). The mechanical strengths of the PSf-, PES- and PVDF-macrocapsules were 1.43 (±0.24), 1.11 (±0.11) and 1.04 (±0.22) mJ, respectively, whereas that of the alginate bead without any polymeric coating layer was 0.73 (±0.06) mJ. In other words, each macrocapsule showed 1.95 (PSf), 1.51 (PES) and 1.41 (PVDF) times greater mechanical strength than the alginate bead with no polymeric coating layer. As the PSf-macrocapsule had the highest mechanical strength, it was adopted for subsequent tests such as QQ activity and biofouling control efficiency in the continuous MBR operation. 3.2. Quorum quenching activity of PSf-macrocapsules The QQ activity of PSf-macrocapsules was evaluated using the standard C8-HSL as a representative signal molecule [4]. The QQ bacteria content of alginate beads was 2.0 mg QQ bacteria/g alginate solution. Because the alginate beads could remove the C8-HSL by adsorption, the removal of C8-HSL only by quorum quenching should be differentiated from that by adsorption. Consequently, the adsorption of C8-HSL was quantified using vacant alginate beads which contained no QQ bacteria. As shown in Figure 5, however, the adsorption of C8-HSL by vacant alginate beads was not significant, i.e. less than 5%. On the other hand, the decomposition rate of C8-HSL during initial 60 minutes (i.e., QQ activity) by macrocapsules (0.059 nmol C8-HSL/min) decreased to around one half of that by alginate beads (0.114 nmol C8-HSL/min), indicating that the QQ activity of macrocapsules decreased due to the membrane coating layer.
In order to elucidate the negative effect of the polymeric coating layer on the QQ activity, macrocapsules were fluorescently stained with a live/dead kit and observed by CLSM to check the active state of encapsulated whole cells. It was because the quorum quenching efficiency of various beads, including alginate beads and macrocapsules, could be assessed in close association with the microbial activity of QQ bacteria inside beads. As shown in Figure 6, a substantial amount of dead cells (red) were observed along the interface between the alginate core (green) and the polymer coating layer (green and red). During the first phase inversion process in which the demixing of water and organic solvent (NMP) took place, the organic solvent could come into direct contact the QQ bacteria in the alginate matrix and thus damage the QQ bacteria located in the vicinity of the polymer solution. Such partial damage of QQ bacteria could result in the relatively lower QQ activity of macrocapsules compared to that of the alginate beads shown in Figure 5. It is worth noting that, in this CLSM image, the PSf membrane layer was stained both green and red because the fluorescent dyes were adsorbed onto the PSf membrane layer during the staining step. Therefore, we tried to reactivate the macrocapsules with damaged QQ bacteria. In detail, macrocapsules were incubated for 12 hours in Luria-Bertani growth medium in order to stimulate the growth of active cells in macrocapsules. We observed the proliferation of QQ bacteria on the merged CLSM image of the restored macrocapsule, although dead cells were still found along the interface between the membrane layer and alginate matrix (Figure S2). As a consequence of the proliferation of live QQ bacteria, the QQ activity which is defined by the C8-HSL degradation rate of macrocapsules during initial 60 minutes, after reactivation was increased to 0.084 nmol C8-HSL/min, which is higher by about 40% than the initial level (0.059 nmol C8-HSL/min). 3.3. Stability of macrocapsules in a harsh environment
Taking into account macrocapsule's potential practical applications under harsh environmental conditions such as in a sewage treatment plant fed with real wastewater or shock loadings, the chemical stabilities of both beads (alginate beads and macrocapsules) were tested and compared to each other in terms of the QQ activity and leakage of QQ bacteria. Firstly, the extent of cell leakage from each QQ bead was evaluated quantitatively by measuring the optical density (OD600) in the buffered EDTA solution (Figure 7a). Vacant alginate beads and vacant macrocapsules also went through the same test to check whether any other material than the QQ bacteria leaked into the suspension from the bead and interfered with the measurement of the cell concentration in the suspension. Fortunately, the contributions from both vacant beads to the OD600 were too small to induce significant interference with the measurement of QQ bacteria. On the contrary, the suspension with alginate beads resulted in a great increase in OD600, suggesting that the alginate matrix was severely disrupted under such chemically harsh conditions, leading to leakage of QQ bacteria from the alginate beads into the suspension. On the other hand, the suspension with macrocapsules showed a negligible change in the OD600, suggesting that the membrane layer coating the alginate matrix successfully prevented the QQ bacteria from leaking out of the macrocapsules. Secondly, we monitored the change of Relative QQ activity, defined by the percentage ratio of residual QQ activity to initial QQ activity, for both alginate beads and macrocapsules at each step through a cycle of chemical treatment and restoration. As clearly shown in Figure 7b, the Relative QQ activity of alginate beads continuously decreased to 81% after chemical treatment and further to 56% despite the restoration step, suggesting the continuous leakage of QQ bacteria from the damaged alginate matrix. This represents a definite limitation of
alginate beads in terms of practical applications to a MBR plant fed with real wastewater. On the contrary, the macrocapsules displayed a continuous increase in Relative QQ activity up to 115% even after chemical treatment and further up to 190% after restoration. This result indicates that the membrane layer enveloping the alginate matrix was able to completely encapsulate the QQ bacteria, but allowed them to proliferate inside the macrocapsules during the restoration step. The slight increase in QQ activity (15%) after chemical treatment might be attributed to the fact that the polymeric coating layer was too resistant to be destructed under the chemically harsh condition, but the alginate matrix located inside the polymeric layer was less resistant against the attack of EDTA, which may have loosened the alginate network and thus facilitated the mass transfer of signal molecules (C8-HSL) to QQ bacteria through the macrocapsule [34]. 3.4. Biofouling inhibition by macrocapsules in a continuous MBR fed with real wastewater The feasibility of QQ macrocapsules was tested through the parallel operation of two labscale MBRs fed with real wastewater generated from a kitchen (Figure 2). 500 pieces of vacant macrocapsules containing no QQ bacteria (Rhodococcus sp. BH4) were put into one MBR (i.e., the control MBR), while 500 pieces of QQ macrocapsules were put into the other MBR (i.e., the QQ MBR). The COD removal efficiencies were maintained more than 93% based on permeate for both the control and QQ MBRs over the operation period of 34 days. The TMP variations were monitored for both MBRs as shown in Figure 8. In the first cycle, it took 3-10 days to reach TMP of 40 kPa for the control MBR in which only the physical cleaning effect would be expected through collisions between moving vacant macrocapsules and hollow fiber membrane surfaces [4]. On the other hand, it took 23 days to reach the same TMP for the QQ MBR in which both physical and biological (i.e., quorum quenching) effects
would be expected. In summary, the QQ effect of macrocapsules was pronounced even in the MBR fed with real wastewater, such that the rate of TMP rise-up was delayed by about threefold overall in Figure 8. At the end of the first cycle, the used filtration membranes from both MBRs were cleaned with 1000 ppm NaOCl solution and then reinserted into each MBR for the second cycle in Figure 8. Just before the second cycle started, activated sludges were taken out from both the control and QQ MBRs and remixed together and then redistributed equally into each MBR. After the same operating period of nine days, the used membrane modules were taken out of both MBRs to measure total attached biomass (TAB) as well as to visualize the biocakes formed on the surface of both membranes. The CLSM images clearly show that the amount of biocakes formed on the used membrane in the QQ MBR [13.3 (±1.2) mg] was much less than that in the control MBR [25.8 (±1.2) mg] (Figure S3). Comparing the protein and polysaccharides mass (mg) accumulated per unit membrane area (m2) between the control and QQ MBRs, the amounts of protein and polysaccharides in the biocakes in the control MBR were greater than those in the QQ MBR (Figure S4). It is worth noting that when the alginate beads with neither polymeric coating layer nor QQ bacteria was put into one MBR, the delay of TMP rise-up was not observed, but the rate of TMP rise up was nearly the same as that in the control MBR with no bead (Figure S3a). It was because the alginate beads became disintegrated from the early stage of operation in the MBR fed with real wastewater and thus physical cleaning effect of the alginate beads disappeared. As a matter of fact, we observed most of alginate beads were dissolved like porridge at the end of run (Figure S3b). Since 2009, QQ effects in MBR for wastewater treatment have been reported successively [2, 4, 5, 26]. However, they achieved successful QQ stories in MBR fed with synthetic
wastewater rather than with real wastewater. Taking into account that the rate of TMP rise-up is directly linked to the energy consumption of a MBR, the delay of TMP rise-up in MBR with QQ macrocapsules in Figure 8 is very encouraging from the view point of energy saving in the MBR operation. Consequently, macrocapsules might play an important role in the design of a future MBR with energy saving. 3.5. Effect of macrocapsule on MBR performance The water quality of permeate as well as the average microbial floc size in mixed liquor were monitored to check the effects of macrocapsules on the general performance of MBR. The COD removals in two MBRs were calculated on the basis of their feed and permeate concentrations. As shown in Figure S5 both MBRs generated similar COD concentrations in permeates with more than 93% of COD removal efficiencies: 6.712.5 mg/L with vacant macrocapsules and 5.311.4 mg/L with QQ-macrocapsules. In addition, the change of average microfloc size in mixed liquor did not make any significant difference between two MBRs over the entire operating period (Figure S6).
4. Conclusions We prepared and characterized “macrocapsule” which is coated by a polymeric membrane layer for the stable encapsulation of quorum quenching (QQ) bacteria (Rhodococcus sp. BH4 strain) and then investigated its stability under a harsh chemical condition as well as its potential for the control of biofouling in the continuous MBR fed with real wastewater. The following conclusions were made: 1) We successfully coated alginate beads encapsulating quorum quenching (QQ) bacteria by a polymeric membrane layer using a phase inversion method, which is well-known in the conventional preparation of asymmetric membrane.
2) During the phase inversion process for the preparation of macrocapsule, bacterial deaths were unavoidable to some extent. However, the decreased QQ activity of macrocapsules could be recovered by a reactivation process. 3) The membrane coated layer prevented QQ bacteria from leaking out of the macrocapsule in a harsh chemical environment. Moreover, the QQ bacteria within a macrocapsule were able to restore their QQ activities by intensive culturing in LB broth. 4) When the macrocapsules were applied to continuous operating MBR fed with real wastewater, it substantially alleviated membrane biofouling. 5) This study would bring bacterial QQ closer to being a practical solution to the current biofouling problem in MBR for wastewater treatment.
Acknowledgements This research was supported by the Korea Ministry of the Environment “Converging Technology Project” (2012001440001).
Supplementary data Operating conditions of MBR are described in detail, and SEM images of decomposed alginate bead in MBR are given in supporting information.
References (1)
M. B. Cassidy, H. Lee, J. T. Trevors, Environmental applications of immobilized
microbial cells: a review. J Ind Microbiol. 16 (1996) 79-101. (2)
W. S. Cheong, C. H. Lee, Y. H. Moon, H. S. Oh, S. R. Kim, S. H. Lee, C. H. Lee, J.
K. Lee, Isolation and identification of indigenous quorum quenching bacteria, Pseudomonas
sp 1A1, for biofouling control in MBR. Ind Eng Chem Res. 252 (2013) 10554-10560. (3)
L. E. de-Bashan, Y. Bashan, Immobilized microalgae for removing pollutants:
Review of practical aspects. Bioresource Technol. 101 (2010) 1611-1627. (4)
S. R. Kim, H. S. Oh, S. J. Jo, K. M. Yeon, C. H. Lee, D. J. Lim, C. H. Lee, J. K. Lee,
Biofouling control with bead-entrapped quorum quenching bacteria in membrane bioreactors: Physical and biological effects. Environ Sci Technol. 47 (2013) 836-842. (5)
H. S. Oh, K. M. Yeon, C. S. Yang, S. R. Kim, C. H. Lee, S. Y. Park, J. Y. Han, J. K.
Lee, Control of membrane biofouling in MBR for wastewater treatment by quorum quenching bacteria encapsulated in microporous membrane. Environ Sci Technol. 46 (2012) 4877-4884. (6)
A. I. Kallenberg, F. van Rantwijk, R. A. Sheldon, Immobilization of penicillin G
acylase: the key to optimum performance. Adv Synth Catal. 347 (2005) 905-926. (7)
J. Szebeni, E. E. Diiorio, H. Hauser, K. H. Winterhalter, Encapsulation of
hemoglobin in phospholipid liposomes: characterization and stability. Biochemistry 24 (1985) 2827-2832. (8)
O. Smidsrod, G. Skjakbraek, Alginate as immobilization matrix for cells. Trends
Biotechnol. 8 (1990) 71-78. (9)
Y. Tal, J. van Rijn, A. Nussinovitch, Improvement of mechanical and biological
properties of freeze-dried denitrifying alginate beads by using starch as a filler and carbon source. Appl Microbiol Biot. 51 (1999) 773-779. (10) F. Thorsen, T. A. Read, M. Lund-Johansen, B. B. Tysnes, R. Bjerkvig, Alginateencapsulated producer cells: a potential new approach for the treatment of malignant brain tumors. Cell Transplant. 9 (2000) 773-783. (11) D. Serp, U. von Stockar, I. W. Marison, Enhancement of 2-phenylethanol
productivity by Saccharomyces cerevisiae in two-phase fed-batch fermentations using solvent immobilization. Biotechnol Bioeng. 82 (2003) 103-110. (12) N. E. Simpson, S. C. Grant, L. Gustavsson, V. M. Peltonen, S. J. Blackband, I. Constantinidis, Biochemical consequences of alginate encapsulation: a NMR study of insulin-secreting cells. Biomaterials 27 (2006) 2577-2586. (13) L. E. de-Bashan, J. P. Hernandez, T. Morey, Y. Bashan, Microalgae growthpromoting bacteria as "helpers" for microalgae: a novel approach for removing ammonium and phosphorus from municipal wastewater. Water Res. 38 (2004) 466-474. (14) S. X. Liu, S. W. Hermanowicz, M. Peng, Nitrate removal from drinking water through the use of encapsulated microorganisms in alginate beads. Environ Technol. 24 (2003) 1129-1134. (15) N. Mallick, Biotechnological potential of immobilized algae for wastewater N, P and metal removal: a review. Biometals 15 (2002) 377-390. (16) G. Orive, S. K. Tam, J. L. Pedraz, J-P. Halle, Biocompatibility of alginate-poly-Llysine microcapsules for cell therapy. Biomaterials 27 (2006) 3691-3700. (17) T. Chandy, D. L. Mooradian, G. H. Rao, Chitosan/polyethylene glycol-alginate microcapsules for oral delivery of hirudin. J Appl Polymer Sci. 70 (1998) 2143-2153. (18) Y. Y. Wong,; S. J. Yuan, C. Choong, Degradation of PEG and non-PEG alginatechitosan microcapsules in different pH environments. Polym Degrad Stabil. 96 (2011) 21892197. (19) J. M. Guisan, Immobilization of enzymes and cells, second ed., Humana press, New Jersey, 2006. (20) T. H. Young, L. P. Cheng, D. J. Lin, L. Fane, W. Y. Chuang, Mechanisms of PVDF membrane formation by immersion-precipitation in soft (1-octanol) and harsh (water)
nonsolvents. Polymer 40 (1999) 5315-5323. (21) Y. J. Won, J. Lee, D. C. Choi, H. R. Chae, I. Kim, C. H. Lee, I. C. Kim, Preparation and application of patterned membranes for wastewater treatment. Environ Sci Technol. 46 (2012) 11021-11027. (22) I. M. Wienk, R. M. Boom, M. A. M. Beerlage, A. M. W. Bulte, C. A. Smolders, H. Strathmann, Recent advances in the formation of phase inversion membranes made from amorphous or semi-crystalline polymers. J Membrane Sci. 113 (1996) 361-371. (23) J. Lee, H. R. Chae, Y. J. Won, K. Lee, C. H. Lee, H. H. Lee, I. C. Kim, J. M. Lee, Graphene oxide nanoplatelets composite membrane with hydrophilic and antifouling properties for wastewater treatment. J Membrane Sci. 448 (2013) 223-230. (24) W. C. Fuqua, S. C. Winans, A Luxr-Luxi type regulatory system activates Agrobacterium Ti plasmid conjugal transfer in the presence of a plant tumor metabolite. J Bacteriol. 176 (1994) 2796-2806. (25) C. Fuqua, S. C. Winans, Conserved cis-acting promoter elements are required for density-dependent transcription of Agrobacterium tumefaciens conjugal transfer genes. J Bacteriol. 178 (1996) 435-440. (26) K. M. Yeon, W. S. Cheong, H. S. Oh, W. N. Lee, B. K. Hwang, C. H. Lee, H. Beyenal, Z. Lewandowski, Quorum sensing: a new biofouling control paradigm in a membrane bioreactor for advanced wastewater treatment. Environ Sci Technol. 43 (2009) 380-385. (27) P. van de Witte, P. J. Dijkstra, J. W. A. vandenBerg, J. Feijen, Phase separation processes in polymer solutions in relation to membrane formation. J Membrane Sci. 117 (1996) 1-31. (28) M. R. M. Abed, S. C. Kumbharkar, A. M. Groth, K. Li, Ultrafiltration PVDF hollow
fibre membranes with interconnected bicontinuous structures produced via a single-step phase inversion technique. J Membrane Sci. 407 (2012) 145-154. (29) T. Kawaguchi, Y. P. Chen, R. S. Norman, A. W. Decho, Rapid screening of quorumsensing signal N-acyl homoserine lactones by an in vitro cell-free assay. Appl Environ Microb. 74 (2008) 3667-3671. (30) R. Geiger, E. Schneider, K. Wallenfels, W. Miska, A New ultrasensitive bioluminogenic enzyme substrate for beta-galactosidase. Biol Chem. H-S 373 (1992) 11871191. (31) F. Edwards-Levy, M. C. Levy, Serum albumin-alginate coated beads: Mechanical properties and stability. Biomaterials 21 (2000) 645-646. (32) D. C. Harris, Quantitative Chemical Analysis, eighth ed., W. H. Freeman and Company, New York, 2010. (33) H. J. Hauselmann, R. J. Fernandes, S. S. Mok, T. M. Schmid, J. A. Block, M. B. Aydelotte, K. E. Kuettner, E. J. M. A. Thonar, Phenotypic stability of bovine articular chondrocytes after long-term culture in alginate beads. J Cell Sci. 107 (1994) 17-27. (34) C. Yi, M. Le-He, W. Guo-Liang, L. Dong-Qiang, Y. Shan-Jing, Gelation conditions and transport properties of hollow calcium alginate capsules. Biotechnol Bioeng. 87 (2004) 228-233.
Figure 1. Preparation n scheme off a macrocappsule coatedd with a mem mbrane layeer through thhe phase inv version meth hod.
Figure 2.. Schematic diagram of the lab-scalee MBR in continuous opeeration.
(a)
(b) Figure 3.. (a) Photogrraphs of an alginate beadd and PSf, PES, PVDF coated macroocapsules. (bb)
SEM im mages of thee outer surfaace, inner ssurface and cross-sectioon of each macrocapsule coated with w PSf, PES S, and PVDF F, respectivelly.
Figure 4. Comparisoon of mechaanical strenggth between an alginatee bead and three t types of o coated macrocapsule m es. Error bar:: standard deeviation (n=220).
Figure 5.. Comparison n of the AHL L (C8-HSL) removal ratte (i.e., QQ activity) betw ween variouss types of beads. b Errorr bar: standarrd deviation (n=3).
Figure 6.. CLSM imaage of the liive/dead celll distribution n in a macroocapsule. No ote that greeen and red colors appeear in the PSf-membraane layer because b the fluorescencce dyes werre adsorbedd onto the meembrane layer during thee staining steep.
(a)
(b) Figure 7. Chemical stability andd relative quuorum quennching activiity of alginaate beads annd macrocap psules. (a) Leakage L of quorum quuenching baacteria in booth beads after chemical treatmentt using citraate buffer (3 30 mM EDT TA, 55 mM M sodium cittrate and 0.115 M sodium m chloride)). (b) Relativve QQ activ vity of bothh beads befo ore and afterr chemical treatment t annd
after resttoration. Rellative QQ acctivity: the percentage p of o residual Q QQ activity to initial QQ activity. Error E bar: standard deviaation (n=3).
Figure 8.. TMP profilles during th he operation of continuoous MBR fedd with real wastewater. w I In the 1st cyycle, the vaccant-macroccapsule and macrocapsu ule with QQ bacteria weere inserted in i the Contrrol and QQ MBRs, resppectively. Att the end of the 2nd cycle, used mem mbranes werre taken ou ut of both MBRs M for analyzing a bioocakes withh CLSM and EPS conccentrations in i biocakes.
Highlights z The first report on bacterial quorum quenching in MBR fed with real wastewater. z Macrocapsule mitigated biofouling in MBR through bacterial quorum quenching. z Microbial alginate bead was coated by membrane layer via phase inversion method. z Macrocapsule showed greater physicochemical stability than microbial alginate bead.
Alginate bead
Alginate matrix
Quorum quenching bacteria
Graphical Abstract (for review)
Coating step
Polymeric solution
Macrocapsule
Membrane coating layer
Macroencapsulation of quorum quenching bacteria by Polymeric Membrane Layer and Its Application to MBR for Biofouling Control Sang-Ryoung Kim, Ki-Baek Lee, Jeong-Eun Kim, Young-June Won, Kyung-Min Yeon, Chung-Hak Lee, Dong-Joon Lim
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PII: DOI: Reference:
S0376-7388(14)00702-9 http://dx.doi.org/10.1016/j.memsci.2014.09.009 MEMSCI13180
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Journal of Membrane Science
Received date: 9 July 2014 Revised date: 29 August 2014 Accepted date: 8 September 2014 Cite this article as: Sang-Ryoung Kim, Ki-Baek Lee, Jeong-Eun Kim, Young-June Won, Kyung-Min Yeon, Chung-Hak Lee, Dong-Joon Lim, Macroencapsulation of quorum quenching bacteria by Polymeric Membrane Layer and Its Application to MBR for Biofouling Control, Journal of Membrane Science, http: //dx.doi.org/10.1016/j.memsci.2014.09.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Macroencapsulation of Quorum Quenching Bacteria by Polymeric Membrane Layer and Its Application to MBR for Biofouling Control
Sang-Ryoung Kim1, Ki-Baek Lee1, Jeong-Eun Kim1, Young-June Won1, Kyung-Min Yeon1, Chung-Hak Lee*1, Dong-Joon Lim2
1
School of Chemical and Biological Engineering, Seoul National University, Seoul 151-744,
Republic of Korea 2
School of Chemical Engineering, Yeungnam University, Gyeongsan 712-749, Republic of
Korea
*Corresponding author. Phone: +82-2-880-7075. Fax: +82-2-874-0896. E-mail: [email protected] Address: School of Chemical and Biological Engineering, Seoul National University 1 Gwanak-ro, Gwanak-gu, Seoul, 151-744, Korea
Abstract We report the preparation and characterization of macrocapsules coated by a polymeric membrane layer for the stable encapsulation of quorum quenching (QQ) bacteria (Rhodococcus sp. BH4). As a new macrocapsule manufacturing platform, a phase inversion method was adopted to cover an alginate core with a microporous membrane layer of poly(vinylidene) fluoride (PVDF), polyethersulfone (PES) or polysulfone (PSf). Scanning electron microscopy revealed that membrane layer was successfully formed on the outside of the alginate-QQ bacteria matrix. Although contact with the organic solvent during the phase inversion step caused minor cell damage, the QQ activity of macrocapsules could be reactivated through cultivation. The QQ bacteria entrapping macrocapsules showed an excellent anti-biofouling capacity in the continuous membrane bioreactor (MBR) fed even with real wastewater. The macrocapsules were capable of maintaining QQ activity more safely than previously reported alginate beads in harsher environmental conditions such as in real wastewater or in the presence of chelating agent (EDTA) which can disintegrate an alginate matrix. Particularly, the PSf coated membrane layer was pronounced in preventing QQ bacteria from leaking outside the macrocapsules. This study is expected to bring the bacterial QQ closer to being a practical solution to the biofouling problem in MBR for wastewater treatment.
Keywords Cell encapsulation, Alginate bead, Membrane bioreactor (MBR), Quorum quenching, Biofouling.
1. Introduction Immobilized microorganisms have been extensively used in environmental [1-5], pharmaceutical [6] and biological [7] processes for the production of enzymes, organic acids, antibiotics, lipids and amino acids. Immobilization provides target microorganisms nutrients without competing with other microorganisms and protects them from environmental stress [8-10]. The immobilization of microorganisms in spherical calcium alginate has been reported to be useful in the laboratory and in commercial applications due to its excellent biocompatibility and ease of preparation [8]. Bacteria or yeast entrapping alginate beads have been applied in the production of ethanol [11], monoclonal antibodies [10] and insulin [12], as well as in the removal of nitrogen, phosphorous and metal in the wastewater treatment process [13-15]. Recently, Kim et al. [4] encapsulated quorum quenching (QQ) bacteria into an alginate matrix and demonstrated that the QQ bacteria entrapping alginate beads were very efficient for biofouling control in a membrane bioreactor (MBR) for wastewater treatment via both physical washing and quorum quenching. However, they also found it was unavoidable that the calcium alginate matrix gradually decomposed during a long-term MBR operation although the feed to MBR was a relatively mild synthetic wastewater (Figure S1). Alginate has been reported to be susceptible to disintegration in the presence of excess monovalent ions and Ca2+ chelating agents like EDTA (Ethylene Diamine Tetraacetic Acid), which is a main obstacle in its wider application [8, 10]. Some efforts have been made to increase the chemical and physical stability of the alginate matrix in a biological environment by coating with polyelectrolytes such as polylysine [16], poly(ethylene glycol) [17], chitosan [18], polyvinylamine [19]. However, coating of alginate beads with these materials suffered from relatively high costs or complex preparation steps, which are limitations for use in wastewater
treatment from a practical point of view. In this study, we enclosed the QQ bacteria-containing alginate core by a polymeric membrane layer to solve these limitations of the conventional alginate immobilization platform. Using various commercial polymers such as poly(vinylidene) fluoride (PVDF), polyethersulfone (PES) and polysulfone (PSf), a porous membrane layer was formed around the alginate core through a phase inversion process, which is commonly used for the preparation of low pressure membranes [20-23]. The macrocapsules consisting of the membrane coating layer and alginate core with QQ bacteria were characterized and compared with previously reported alginate beads [4] in terms of structure and QQ activity. The feasibility of macrocapsules was evaluated not only under a harsh chemical condition, but also in the continuous operation of MBR fed with real wastewater.
2. Materials and methods 2.1. Materials PVDF pellet (average Mw ~530,000), N-methyl-2-pyrrolidone (NMP), ethylenediamine tetraacetic acid disodium salt (EDTA) and calcium chloride (CaCl2) were purchased from Sigma Aldrich Korea. PSf (Udel®P1800) and PES (Ultrason E6020P) pellets were obtained from Solvay-Korea (Korea) and BASF (Germany), respectively. Sodium alginate powder was purchased from Junsei Chemical (Japan). All chemicals were of analytical grade and used as received. A Millipore Synergy filtration system was used to purify all water. 2.2. Microorganisms and growth conditions N-acyl homoserine lactone (AHL) quorum sensing (QS) autoinducers were detected using a reporter strain of Agrobacterium tumefaciens A136 (Ti-)(pCF218)(pCF372). It was grown at 30°C in Luria-Bertani (Miller, US) medium with streptomycin (0.25 v/v%) and tetracycline
(0.05 v/v%) [24-26]. The quorum quenching Rhodococcus sp. BH4 (KCTC 33122) strain was previously isolated from the a MBR wastewater treatment plant (Ok-cheon, Korea) and was cultured in Luria-Bertani medium at 30°C [4, 5]. 2.3. Preparation of macrocapsules The overall preparation scheme of macrocapsules is graphically depicted in Figure 1. First, QQ bacteria (Rhodococcus sp. BH4) were entrapped in the alginate matrix (alginate beads with QQ bacteria) (Step 1 in Figure 1). Subsequently, cultured Rhodococcus sp. BH4 were centrifuged (12,000 g, 15 min) and resuspended in 10 ml of deionized water. This Rhodococcus sp. BH4 suspension was mixed with 90 ml of the alginate solution. The alginate concentration in the final mixture was 2 w/v%. The mixture was dropped into 500 ml of calcium chloride solution (4 w/v%) using a syringe needle to form spherical beads and stirred for 3 hours. After washing and swelling in deionized water, the alginate beads were used for subsequent polymeric coating. The QQ bacteria content of alginate beads was 2.0 mg QQ bacteria/g alginate solution. Microporous membrane layers were formed on the surface of alginate beads using the phase inversion technique [27, 28]. In detail, PVDF, PSf and PES pellets were dissolved in NMP at 60°C for 12 hours. The concentration of each polymer solution was set to 10 w/v%. Each polymeric solution was stirred overnight for complete mixing and cooling. Alginate beads were immersed in each polymeric solution for 30 seconds (Step 2 in Figure 1). The water contained in the alginate beads contacted the polymeric solution and thus induced phase separation. Consequently, the inner membrane layer was formed at the interface between the alginate bead and the polymer coating layer. And then, the alginate bead surrounded by the polymeric solution was immersed in a water coagulation bath for 1 hour (Step 3 in Figure 1). During Step 3, the polymeric solution still enveloping the outer coating layer made contact
with the non-solvent (i.e., water) to induce the second phase inversion on the outer surface of the polymeric layer. Finally, the macrocapsules were repeatedly washed and stored in deionized water at 4°C until use. 2.4. Bioassay for AHL autoinducer quantification The AHL level was quantitatively determined using a bioassay with the luminescence substrate Beta-Glo (Promega, USA) [29, 30]. Briefly, 5 l of AHL samples were loaded into a 96-well-plate with 95 l of an overnight culture of A. tumefaciens A136. After incubation for 90 minutes at 30°C, 30 l of Beta-Glo were added into each well. This generated the luminescence of oxyluciferin, whose intensity is proportional to the amount of betagalactosidase released from the A. tumefaciens A136 biosensor. After incubation for 30 minutes at 30°C, the bioluminescence intensity of each sample was recorded using a luminometer (Synergy 2, Bio-Tek, USA). A calibration curve was prepared using a standard AHL solution of N-octanoyl-L-homoserine lactone (C8-HSL, Sigma-Aldrich), which was previously found to be present as a major AHL in the activated sludge used in this study [4, 26]. Each bioassay was conducted in triplicate to assess repeatability. 2.5. Determination of quorum quenching activity The quorum quenching (QQ) activity of alginate beads and macrocapsules was quantitatively determined by the reduction rate of C8-HSL standard solution [4, 5]. Briefly, C8-HSL was dissolved in deionized water to a concentration of 200 nM. Then, 40 beads were added to 40 ml of the standard AHL solution and incubated at 30°C for 0, 30, 60, 120 minutes using an orbital shaker at 200 rpm. The residual concentration of C8-HSL at each reaction time point was determined using the A. tumefaciens A136 bioluminescence assay. The “QQ activity” of beads (i.e., alginate beads or macrocapsules) was defined by the rate of AHL (C8HSL) degradation during initial 60 minutes (nmol C8-HSL/min). On the other hand, the
“Relative QQ activity” was also introduced to monitor the QQ stability of various beads and was defined by the percentage ratio of residual QQ activity to initial QQ activity. 2.6. Measurement of mechanical strength The mechanical resistance of alginate beads and macrocapsules were determined using a texture analyzer (CT3 4500, Brookfield, USA) [31]. The mechanical deformation tests were performed at a mobile probe (TA44) speed of 0.5 mm/s until the bead matrix was observed to burst. The hardness work, a measure of the energy required to crush the container, was calculated as the area under the curve of the compression plot. An average of at least 20 beads was assessed to obtain statistically relevant data. 2.7. Measurement of chemical stability To evaluate the chemical stability of any type of beads under a harsh chemical environment, a buffered EDTA solution was selected to make up a harsh environment. It is because EDTA is well known to be a strong complexing agent with a calcium ion and thus is expected to easily disintegrate any alginate bead matrix containing calcium ions [32]. Beads to be tested were placed in citrate buffer (30 mM EDTA, 55 mM sodium citrate and 0.15 M sodium chloride) and then the mixture was incubated for 60 minutes with gentle agitation [8, 33]. During the incubation, 3 ml were taken out of the suspension every ten minutes and the concentration of leaked cells from disintegrated beads were measured using a spectrophotometer at 600 nm because it correlates with the chemical stability of beads. 2.8. Restoration of quorum quenching activity of disintegrated beads Restoration of beads (i.e., alginate beads or macrocapsules) were conducted as follows: Fresh alginate beads or fresh macrocapsules were deliberately put into a harsh chemical environment by placing them in citrate buffer (30 mM EDTA, 55 mM sodium citrate and 0.15 M sodium chloride) for 10 minutes, respectively. For the restoration of such chemically
treated, i.e., disintegrated beads, they were washed with deionized water three times and then placed in Luria-Bertani (LB) medium at 30°C for 12 hours using a shaking incubator at 200 rpm. The extent of restoration was evaluated by comparing the Relative QQ activities of tested beads at each step, i.e., before and after chemical treatment, and after restoration, respectively. 2.9. MBR operation Two lab-scale MBRs (one control & one QQ MBR) were constructed in a similar way to those described by other researches and were operated in parallel (Figure 2) [4, 5]. To evaluate biofouling control only through the QQ activity of macrocapsules, one control MBR was operated with vacant macrocapsules containing no QQ bacteria (Rhodococcus sp. BH4) as shown in Figure 2. The MBR was fed with real wastewater which was generated in a restaurant after screening collected real wastewater through 1 mm wire mesh. Membrane modules were made of poly(vinylidene) fluoride hollow fiber (ZeeWeed500, GE-Zenon, USA). The permeate flux was set to 30 l/m2/h (LMH) and the transmembrane pressure (TMP) was continuously monitored to determine the degree of membrane fouling. Other detailed operating conditions are given in Table S1. 2.10. Scanning electron microscopy (SEM) The morphology of the macrocapsules was examined by SEM (JEOL JSM-6701F). Macrocapsules were cut in half and the membrane coating layer was detached from the alginate core. This detached membrane layer, after ethanol dehydration, was embedded in epoxy resin for 2 hours at room temperature. This embedded membrane specimen was cut with a razor blade for structural observation. Samples were placed on a conducting stage with a platinum coating, and then analyzed using SEM. Alginate beads were cut by a razor blade for structural observation, then dehydrated in ethanol, transferred to a critical point dryer for
2 hours, coated with platinum, and examined by SEM.
2.11. Confocal laser scanning microscopy (CLSM) The three-dimensional structure of beads and the membrane-biocake was visually monitored with confocal laser scanning microscope (CLSM, C1 plus, Nikon, Japan) [4]. Samples were fluorescently stained with nucleic acid-specific SYTO9 or a BacLight Live/Dead staining kit according to the observational aim (Molecular Probes, Eugene, OR). Z-section image stacks (5 m) of each channel were reconstructed using IMARIS software (Bitplane AG, Switzerland).
3. Results and discussion 3.1. Preparation and characterization of macrocapsules with various polymeric coatings Three kinds of microporous polymeric layers, i.e. PSf, PES and PVDF, were formed on the surface of alginate beads. As shown in Figure 3a, these macrocapsules were globular in shape and their diameters ranged from 3.2 to 3.8 mm (mean 3.5 mm), indicating that the type of polymer material had little influence on the size and shape of the macrocapsules. SEM observations clearly showed the outer surface, the inner surface and the cross-section of a piece of macrocapsule coated with PSf, PES and PVDF (Figure 3b). In all macrocapsules, the polymeric coating consisted of inner and outer membrane layers, of which the inner layer had a denser structure than the outer membrane layer. This was thought to be caused by the relatively slower demixing rate of the polymeric solution in the first phase inversion step than in the second phase inversion step. The cross-sectional image of each polymeric coating layer clearly demonstrated asymmetric finger-like structures. The overall thickness of each
membrane coating layer was found to be 60-100 m. The main reason for the unevenness of the membrane thickness was attributed to the fact that the phase inversion of the polymeric solution took place not on a flat sheet, but on a spherical bead. In the next step, the mechanical strengths of the three types of macromolecules were evaluated by compression tests using a texture analyzer (Figure 4). The mechanical strengths of the PSf-, PES- and PVDF-macrocapsules were 1.43 (±0.24), 1.11 (±0.11) and 1.04 (±0.22) mJ, respectively, whereas that of the alginate bead without any polymeric coating layer was 0.73 (±0.06) mJ. In other words, each macrocapsule showed 1.95 (PSf), 1.51 (PES) and 1.41 (PVDF) times greater mechanical strength than the alginate bead with no polymeric coating layer. As the PSf-macrocapsule had the highest mechanical strength, it was adopted for subsequent tests such as QQ activity and biofouling control efficiency in the continuous MBR operation. 3.2. Quorum quenching activity of PSf-macrocapsules The QQ activity of PSf-macrocapsules was evaluated using the standard C8-HSL as a representative signal molecule [4]. The QQ bacteria content of alginate beads was 2.0 mg QQ bacteria/g alginate solution. Because the alginate beads could remove the C8-HSL by adsorption, the removal of C8-HSL only by quorum quenching should be differentiated from that by adsorption. Consequently, the adsorption of C8-HSL was quantified using vacant alginate beads which contained no QQ bacteria. As shown in Figure 5, however, the adsorption of C8-HSL by vacant alginate beads was not significant, i.e. less than 5%. On the other hand, the decomposition rate of C8-HSL during initial 60 minutes (i.e., QQ activity) by macrocapsules (0.059 nmol C8-HSL/min) decreased to around one half of that by alginate beads (0.114 nmol C8-HSL/min), indicating that the QQ activity of macrocapsules decreased due to the membrane coating layer.
In order to elucidate the negative effect of the polymeric coating layer on the QQ activity, macrocapsules were fluorescently stained with a live/dead kit and observed by CLSM to check the active state of encapsulated whole cells. It was because the quorum quenching efficiency of various beads, including alginate beads and macrocapsules, could be assessed in close association with the microbial activity of QQ bacteria inside beads. As shown in Figure 6, a substantial amount of dead cells (red) were observed along the interface between the alginate core (green) and the polymer coating layer (green and red). During the first phase inversion process in which the demixing of water and organic solvent (NMP) took place, the organic solvent could come into direct contact the QQ bacteria in the alginate matrix and thus damage the QQ bacteria located in the vicinity of the polymer solution. Such partial damage of QQ bacteria could result in the relatively lower QQ activity of macrocapsules compared to that of the alginate beads shown in Figure 5. It is worth noting that, in this CLSM image, the PSf membrane layer was stained both green and red because the fluorescent dyes were adsorbed onto the PSf membrane layer during the staining step. Therefore, we tried to reactivate the macrocapsules with damaged QQ bacteria. In detail, macrocapsules were incubated for 12 hours in Luria-Bertani growth medium in order to stimulate the growth of active cells in macrocapsules. We observed the proliferation of QQ bacteria on the merged CLSM image of the restored macrocapsule, although dead cells were still found along the interface between the membrane layer and alginate matrix (Figure S2). As a consequence of the proliferation of live QQ bacteria, the QQ activity which is defined by the C8-HSL degradation rate of macrocapsules during initial 60 minutes, after reactivation was increased to 0.084 nmol C8-HSL/min, which is higher by about 40% than the initial level (0.059 nmol C8-HSL/min). 3.3. Stability of macrocapsules in a harsh environment
Taking into account macrocapsule's potential practical applications under harsh environmental conditions such as in a sewage treatment plant fed with real wastewater or shock loadings, the chemical stabilities of both beads (alginate beads and macrocapsules) were tested and compared to each other in terms of the QQ activity and leakage of QQ bacteria. Firstly, the extent of cell leakage from each QQ bead was evaluated quantitatively by measuring the optical density (OD600) in the buffered EDTA solution (Figure 7a). Vacant alginate beads and vacant macrocapsules also went through the same test to check whether any other material than the QQ bacteria leaked into the suspension from the bead and interfered with the measurement of the cell concentration in the suspension. Fortunately, the contributions from both vacant beads to the OD600 were too small to induce significant interference with the measurement of QQ bacteria. On the contrary, the suspension with alginate beads resulted in a great increase in OD600, suggesting that the alginate matrix was severely disrupted under such chemically harsh conditions, leading to leakage of QQ bacteria from the alginate beads into the suspension. On the other hand, the suspension with macrocapsules showed a negligible change in the OD600, suggesting that the membrane layer coating the alginate matrix successfully prevented the QQ bacteria from leaking out of the macrocapsules. Secondly, we monitored the change of Relative QQ activity, defined by the percentage ratio of residual QQ activity to initial QQ activity, for both alginate beads and macrocapsules at each step through a cycle of chemical treatment and restoration. As clearly shown in Figure 7b, the Relative QQ activity of alginate beads continuously decreased to 81% after chemical treatment and further to 56% despite the restoration step, suggesting the continuous leakage of QQ bacteria from the damaged alginate matrix. This represents a definite limitation of
alginate beads in terms of practical applications to a MBR plant fed with real wastewater. On the contrary, the macrocapsules displayed a continuous increase in Relative QQ activity up to 115% even after chemical treatment and further up to 190% after restoration. This result indicates that the membrane layer enveloping the alginate matrix was able to completely encapsulate the QQ bacteria, but allowed them to proliferate inside the macrocapsules during the restoration step. The slight increase in QQ activity (15%) after chemical treatment might be attributed to the fact that the polymeric coating layer was too resistant to be destructed under the chemically harsh condition, but the alginate matrix located inside the polymeric layer was less resistant against the attack of EDTA, which may have loosened the alginate network and thus facilitated the mass transfer of signal molecules (C8-HSL) to QQ bacteria through the macrocapsule [34]. 3.4. Biofouling inhibition by macrocapsules in a continuous MBR fed with real wastewater The feasibility of QQ macrocapsules was tested through the parallel operation of two labscale MBRs fed with real wastewater generated from a kitchen (Figure 2). 500 pieces of vacant macrocapsules containing no QQ bacteria (Rhodococcus sp. BH4) were put into one MBR (i.e., the control MBR), while 500 pieces of QQ macrocapsules were put into the other MBR (i.e., the QQ MBR). The COD removal efficiencies were maintained more than 93% based on permeate for both the control and QQ MBRs over the operation period of 34 days. The TMP variations were monitored for both MBRs as shown in Figure 8. In the first cycle, it took 3-10 days to reach TMP of 40 kPa for the control MBR in which only the physical cleaning effect would be expected through collisions between moving vacant macrocapsules and hollow fiber membrane surfaces [4]. On the other hand, it took 23 days to reach the same TMP for the QQ MBR in which both physical and biological (i.e., quorum quenching) effects
would be expected. In summary, the QQ effect of macrocapsules was pronounced even in the MBR fed with real wastewater, such that the rate of TMP rise-up was delayed by about threefold overall in Figure 8. At the end of the first cycle, the used filtration membranes from both MBRs were cleaned with 1000 ppm NaOCl solution and then reinserted into each MBR for the second cycle in Figure 8. Just before the second cycle started, activated sludges were taken out from both the control and QQ MBRs and remixed together and then redistributed equally into each MBR. After the same operating period of nine days, the used membrane modules were taken out of both MBRs to measure total attached biomass (TAB) as well as to visualize the biocakes formed on the surface of both membranes. The CLSM images clearly show that the amount of biocakes formed on the used membrane in the QQ MBR [13.3 (±1.2) mg] was much less than that in the control MBR [25.8 (±1.2) mg] (Figure S3). Comparing the protein and polysaccharides mass (mg) accumulated per unit membrane area (m2) between the control and QQ MBRs, the amounts of protein and polysaccharides in the biocakes in the control MBR were greater than those in the QQ MBR (Figure S4). It is worth noting that when the alginate beads with neither polymeric coating layer nor QQ bacteria was put into one MBR, the delay of TMP rise-up was not observed, but the rate of TMP rise up was nearly the same as that in the control MBR with no bead (Figure S3a). It was because the alginate beads became disintegrated from the early stage of operation in the MBR fed with real wastewater and thus physical cleaning effect of the alginate beads disappeared. As a matter of fact, we observed most of alginate beads were dissolved like porridge at the end of run (Figure S3b). Since 2009, QQ effects in MBR for wastewater treatment have been reported successively [2, 4, 5, 26]. However, they achieved successful QQ stories in MBR fed with synthetic
wastewater rather than with real wastewater. Taking into account that the rate of TMP rise-up is directly linked to the energy consumption of a MBR, the delay of TMP rise-up in MBR with QQ macrocapsules in Figure 8 is very encouraging from the view point of energy saving in the MBR operation. Consequently, macrocapsules might play an important role in the design of a future MBR with energy saving. 3.5. Effect of macrocapsule on MBR performance The water quality of permeate as well as the average microbial floc size in mixed liquor were monitored to check the effects of macrocapsules on the general performance of MBR. The COD removals in two MBRs were calculated on the basis of their feed and permeate concentrations. As shown in Figure S5 both MBRs generated similar COD concentrations in permeates with more than 93% of COD removal efficiencies: 6.712.5 mg/L with vacant macrocapsules and 5.311.4 mg/L with QQ-macrocapsules. In addition, the change of average microfloc size in mixed liquor did not make any significant difference between two MBRs over the entire operating period (Figure S6).
4. Conclusions We prepared and characterized “macrocapsule” which is coated by a polymeric membrane layer for the stable encapsulation of quorum quenching (QQ) bacteria (Rhodococcus sp. BH4 strain) and then investigated its stability under a harsh chemical condition as well as its potential for the control of biofouling in the continuous MBR fed with real wastewater. The following conclusions were made: 1) We successfully coated alginate beads encapsulating quorum quenching (QQ) bacteria by a polymeric membrane layer using a phase inversion method, which is well-known in the conventional preparation of asymmetric membrane.
2) During the phase inversion process for the preparation of macrocapsule, bacterial deaths were unavoidable to some extent. However, the decreased QQ activity of macrocapsules could be recovered by a reactivation process. 3) The membrane coated layer prevented QQ bacteria from leaking out of the macrocapsule in a harsh chemical environment. Moreover, the QQ bacteria within a macrocapsule were able to restore their QQ activities by intensive culturing in LB broth. 4) When the macrocapsules were applied to continuous operating MBR fed with real wastewater, it substantially alleviated membrane biofouling. 5) This study would bring bacterial QQ closer to being a practical solution to the current biofouling problem in MBR for wastewater treatment.
Acknowledgements This research was supported by the Korea Ministry of the Environment “Converging Technology Project” (2012001440001).
Supplementary data Operating conditions of MBR are described in detail, and SEM images of decomposed alginate bead in MBR are given in supporting information.
References (1)
M. B. Cassidy, H. Lee, J. T. Trevors, Environmental applications of immobilized
microbial cells: a review. J Ind Microbiol. 16 (1996) 79-101. (2)
W. S. Cheong, C. H. Lee, Y. H. Moon, H. S. Oh, S. R. Kim, S. H. Lee, C. H. Lee, J.
K. Lee, Isolation and identification of indigenous quorum quenching bacteria, Pseudomonas
sp 1A1, for biofouling control in MBR. Ind Eng Chem Res. 252 (2013) 10554-10560. (3)
L. E. de-Bashan, Y. Bashan, Immobilized microalgae for removing pollutants:
Review of practical aspects. Bioresource Technol. 101 (2010) 1611-1627. (4)
S. R. Kim, H. S. Oh, S. J. Jo, K. M. Yeon, C. H. Lee, D. J. Lim, C. H. Lee, J. K. Lee,
Biofouling control with bead-entrapped quorum quenching bacteria in membrane bioreactors: Physical and biological effects. Environ Sci Technol. 47 (2013) 836-842. (5)
H. S. Oh, K. M. Yeon, C. S. Yang, S. R. Kim, C. H. Lee, S. Y. Park, J. Y. Han, J. K.
Lee, Control of membrane biofouling in MBR for wastewater treatment by quorum quenching bacteria encapsulated in microporous membrane. Environ Sci Technol. 46 (2012) 4877-4884. (6)
A. I. Kallenberg, F. van Rantwijk, R. A. Sheldon, Immobilization of penicillin G
acylase: the key to optimum performance. Adv Synth Catal. 347 (2005) 905-926. (7)
J. Szebeni, E. E. Diiorio, H. Hauser, K. H. Winterhalter, Encapsulation of
hemoglobin in phospholipid liposomes: characterization and stability. Biochemistry 24 (1985) 2827-2832. (8)
O. Smidsrod, G. Skjakbraek, Alginate as immobilization matrix for cells. Trends
Biotechnol. 8 (1990) 71-78. (9)
Y. Tal, J. van Rijn, A. Nussinovitch, Improvement of mechanical and biological
properties of freeze-dried denitrifying alginate beads by using starch as a filler and carbon source. Appl Microbiol Biot. 51 (1999) 773-779. (10) F. Thorsen, T. A. Read, M. Lund-Johansen, B. B. Tysnes, R. Bjerkvig, Alginateencapsulated producer cells: a potential new approach for the treatment of malignant brain tumors. Cell Transplant. 9 (2000) 773-783. (11) D. Serp, U. von Stockar, I. W. Marison, Enhancement of 2-phenylethanol
productivity by Saccharomyces cerevisiae in two-phase fed-batch fermentations using solvent immobilization. Biotechnol Bioeng. 82 (2003) 103-110. (12) N. E. Simpson, S. C. Grant, L. Gustavsson, V. M. Peltonen, S. J. Blackband, I. Constantinidis, Biochemical consequences of alginate encapsulation: a NMR study of insulin-secreting cells. Biomaterials 27 (2006) 2577-2586. (13) L. E. de-Bashan, J. P. Hernandez, T. Morey, Y. Bashan, Microalgae growthpromoting bacteria as "helpers" for microalgae: a novel approach for removing ammonium and phosphorus from municipal wastewater. Water Res. 38 (2004) 466-474. (14) S. X. Liu, S. W. Hermanowicz, M. Peng, Nitrate removal from drinking water through the use of encapsulated microorganisms in alginate beads. Environ Technol. 24 (2003) 1129-1134. (15) N. Mallick, Biotechnological potential of immobilized algae for wastewater N, P and metal removal: a review. Biometals 15 (2002) 377-390. (16) G. Orive, S. K. Tam, J. L. Pedraz, J-P. Halle, Biocompatibility of alginate-poly-Llysine microcapsules for cell therapy. Biomaterials 27 (2006) 3691-3700. (17) T. Chandy, D. L. Mooradian, G. H. Rao, Chitosan/polyethylene glycol-alginate microcapsules for oral delivery of hirudin. J Appl Polymer Sci. 70 (1998) 2143-2153. (18) Y. Y. Wong,; S. J. Yuan, C. Choong, Degradation of PEG and non-PEG alginatechitosan microcapsules in different pH environments. Polym Degrad Stabil. 96 (2011) 21892197. (19) J. M. Guisan, Immobilization of enzymes and cells, second ed., Humana press, New Jersey, 2006. (20) T. H. Young, L. P. Cheng, D. J. Lin, L. Fane, W. Y. Chuang, Mechanisms of PVDF membrane formation by immersion-precipitation in soft (1-octanol) and harsh (water)
nonsolvents. Polymer 40 (1999) 5315-5323. (21) Y. J. Won, J. Lee, D. C. Choi, H. R. Chae, I. Kim, C. H. Lee, I. C. Kim, Preparation and application of patterned membranes for wastewater treatment. Environ Sci Technol. 46 (2012) 11021-11027. (22) I. M. Wienk, R. M. Boom, M. A. M. Beerlage, A. M. W. Bulte, C. A. Smolders, H. Strathmann, Recent advances in the formation of phase inversion membranes made from amorphous or semi-crystalline polymers. J Membrane Sci. 113 (1996) 361-371. (23) J. Lee, H. R. Chae, Y. J. Won, K. Lee, C. H. Lee, H. H. Lee, I. C. Kim, J. M. Lee, Graphene oxide nanoplatelets composite membrane with hydrophilic and antifouling properties for wastewater treatment. J Membrane Sci. 448 (2013) 223-230. (24) W. C. Fuqua, S. C. Winans, A Luxr-Luxi type regulatory system activates Agrobacterium Ti plasmid conjugal transfer in the presence of a plant tumor metabolite. J Bacteriol. 176 (1994) 2796-2806. (25) C. Fuqua, S. C. Winans, Conserved cis-acting promoter elements are required for density-dependent transcription of Agrobacterium tumefaciens conjugal transfer genes. J Bacteriol. 178 (1996) 435-440. (26) K. M. Yeon, W. S. Cheong, H. S. Oh, W. N. Lee, B. K. Hwang, C. H. Lee, H. Beyenal, Z. Lewandowski, Quorum sensing: a new biofouling control paradigm in a membrane bioreactor for advanced wastewater treatment. Environ Sci Technol. 43 (2009) 380-385. (27) P. van de Witte, P. J. Dijkstra, J. W. A. vandenBerg, J. Feijen, Phase separation processes in polymer solutions in relation to membrane formation. J Membrane Sci. 117 (1996) 1-31. (28) M. R. M. Abed, S. C. Kumbharkar, A. M. Groth, K. Li, Ultrafiltration PVDF hollow
fibre membranes with interconnected bicontinuous structures produced via a single-step phase inversion technique. J Membrane Sci. 407 (2012) 145-154. (29) T. Kawaguchi, Y. P. Chen, R. S. Norman, A. W. Decho, Rapid screening of quorumsensing signal N-acyl homoserine lactones by an in vitro cell-free assay. Appl Environ Microb. 74 (2008) 3667-3671. (30) R. Geiger, E. Schneider, K. Wallenfels, W. Miska, A New ultrasensitive bioluminogenic enzyme substrate for beta-galactosidase. Biol Chem. H-S 373 (1992) 11871191. (31) F. Edwards-Levy, M. C. Levy, Serum albumin-alginate coated beads: Mechanical properties and stability. Biomaterials 21 (2000) 645-646. (32) D. C. Harris, Quantitative Chemical Analysis, eighth ed., W. H. Freeman and Company, New York, 2010. (33) H. J. Hauselmann, R. J. Fernandes, S. S. Mok, T. M. Schmid, J. A. Block, M. B. Aydelotte, K. E. Kuettner, E. J. M. A. Thonar, Phenotypic stability of bovine articular chondrocytes after long-term culture in alginate beads. J Cell Sci. 107 (1994) 17-27. (34) C. Yi, M. Le-He, W. Guo-Liang, L. Dong-Qiang, Y. Shan-Jing, Gelation conditions and transport properties of hollow calcium alginate capsules. Biotechnol Bioeng. 87 (2004) 228-233.
Figure 1. Preparation n scheme off a macrocappsule coatedd with a mem mbrane layeer through thhe phase inv version meth hod.
Figure 2.. Schematic diagram of the lab-scalee MBR in continuous opeeration.
(a)
(b) Figure 3.. (a) Photogrraphs of an alginate beadd and PSf, PES, PVDF coated macroocapsules. (bb)
SEM im mages of thee outer surfaace, inner ssurface and cross-sectioon of each macrocapsule coated with w PSf, PES S, and PVDF F, respectivelly.
Figure 4. Comparisoon of mechaanical strenggth between an alginatee bead and three t types of o coated macrocapsule m es. Error bar:: standard deeviation (n=220).
Figure 5.. Comparison n of the AHL L (C8-HSL) removal ratte (i.e., QQ activity) betw ween variouss types of beads. b Errorr bar: standarrd deviation (n=3).
Figure 6.. CLSM imaage of the liive/dead celll distribution n in a macroocapsule. No ote that greeen and red colors appeear in the PSf-membraane layer because b the fluorescencce dyes werre adsorbedd onto the meembrane layer during thee staining steep.
(a)
(b) Figure 7. Chemical stability andd relative quuorum quennching activiity of alginaate beads annd macrocap psules. (a) Leakage L of quorum quuenching baacteria in booth beads after chemical treatmentt using citraate buffer (3 30 mM EDT TA, 55 mM M sodium cittrate and 0.115 M sodium m chloride)). (b) Relativve QQ activ vity of bothh beads befo ore and afterr chemical treatment t annd
after resttoration. Rellative QQ acctivity: the percentage p of o residual Q QQ activity to initial QQ activity. Error E bar: standard deviaation (n=3).
Figure 8.. TMP profilles during th he operation of continuoous MBR fedd with real wastewater. w I In the 1st cyycle, the vaccant-macroccapsule and macrocapsu ule with QQ bacteria weere inserted in i the Contrrol and QQ MBRs, resppectively. Att the end of the 2nd cycle, used mem mbranes werre taken ou ut of both MBRs M for analyzing a bioocakes withh CLSM and EPS conccentrations in i biocakes.
Highlights z The first report on bacterial quorum quenching in MBR fed with real wastewater. z Macrocapsule mitigated biofouling in MBR through bacterial quorum quenching. z Microbial alginate bead was coated by membrane layer via phase inversion method. z Macrocapsule showed greater physicochemical stability than microbial alginate bead.
Alginate bead
Alginate matrix
Quorum quenching bacteria
Graphical Abstract (for review)
Coating step
Polymeric solution
Macrocapsule
Membrane coating layer