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i
I. 공학박사 학위논문
Control of Biofouling in Membrane Bioreactor for
Wastewater Treatment by Quorum-Quenching
Bacteria Immobilized in Moving Beads
정족수감지 억제 세균이 고정화된 유동성 담체를 이용한
하폐수 처리용 분리막 생물반응기에서의
생물막오염 제어
2018년 8월
서울대학교 대학원
화학생물공학부
김 상 룡
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iii
Abstract
Control of Biofouling in Membrane Bioreactor for
Wastewater Treatment by Quorum-Quenching
Bacteria Immobilized in Moving Beads
Sang-Ryoung Kim
School of Chemical and Biological Engineering
The Graduate School
Seoul National University
Membrane bioreactor (MBR) is a widely used as an advanced wastewater
treatment process, but membrane biofouling is a chronic problem. Recently quorum
quenching (QQ) technology has attracted attention as a new solution to suppress
biofouling. The purpose of this study is to develop a moving carrier capable of
simultaneously anticipating both the physical cleaning and the quorum sensing
inhibition to prevent more effective biofouling.
Rhodococcus sp. BH4, known as QQ bacteria, was immobilized on a calcium-
alginate matrix and developed a new membrane fouling inhibitor, freely moving
bead, called a “cell entrapping beads (CEBs).” Because CEB has a specific gravity
similar to water, it moves freely by aeration, generates physical cleaning effect by
colliding with the membrane as well as effectively suppresses QS mechanism by
using QQ. Especially, when the QS of membrane fouling microorganisms is
iv
disturbed, extracellular polymeric substances (EPS) production is slowed, and
loosely bound biofilm is formed. Therefore, the physical cleaning effect of CEB
synergistically increased with the QQ effect. Through this combined effect, CEB
delayed membrane biofouling by 8 times slower than conventional MBR in a
continuous process.
As the following study, improvement of the physical and chemical stability of the
CEB have conducted to the application for actual MBR processes. The first is a “fluid
coating carrier (Macrocapsule),” which surrounds CEB with porous membrane using
a non-solvent induced phase separation technique. The porous membrane was
formed on the surface by a spontaneous phase inversion between the amphipathic
polymer solution and the contained water in CEB. Macrocapsule showed excellent
biofilm control effect for 80 days on laboratory scale continuous MBR process and
retained its QQ activity. The second is “W-bead,” which is prepared by double cross-
linking with polyvinyl alcohol and alginate. The W-bead showed excellent stability
and biofouling mitigation effect in the MBR fed with real wastewater. This study
suggests the free-moving beads as an efficient QQ bacteria carrier and showed
potential for actual MBR application.
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Keywords
Membrane bioreactor (MBR), Biofouling, Quorum sensing, Quorum-quenching
bacteria, Moving beads, Cell immobilization, Wastewater treatment
Student Number: 2007-23079
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Table of Contents
Abstract ....................................................................................... iii
List of Figures .............................................................................. x
List of Tables .......................................................................... xviii
I. Introduction .............................................................................. 1
I.1. Backgrounds ...................................................................................... 3
I.2. Objectives ........................................................................................... 4
II. Literature Review ................................................................... 5
II.1. MBR for Advanced Wastewater Treatment .................................... 7
II.1.1. Concept and Process ................................................................... 7
II.1.2. Development of MBR ................................................................. 9
II.1.3. Trends in MBR: Market and Research ................................... 13
II.1.4. Fouling Control in MBR Systems ............................................ 19
II.1.5. Biofilm in MBR ......................................................................... 25
II.2. Quorum Sensing (QS) Signaling in Bacteria ................................. 29
II.2.1. Definition and Mechanism ....................................................... 29
II.2.2. Gram-Negative Bacteria with AHLs: Type AI-1 System ....... 31
II.2.3. Gram-Positive Bacteria with AIPs .......................................... 38
II.2.4. Interspecies Communication: Type AI-2 System ................... 39
II.2.5. Other QS Systems ..................................................................... 41
II.2.6. QS Regulated Biofilm Formation ............................................ 42
vii
II.3. QS Control Strategy ........................................................................ 44
II.3.1. Three-Point of QS Inhibition Strategies ................................. 44
II.3.2. Quorum Sensing Inhibitor (QSI) for AI-1 Regulation .......... 45
II.3.3. Reporter Strain to Detect QS Signal and Screening QSI ...... 49
II.4. Immobilization Technique for Biocatalyst..................................... 52
II.4.1. Enzyme Immobilization Method ............................................. 53
II.4.2. Nanobiocatalysis ....................................................................... 58
II.4.3. Whole-Cell Immobilization Method ........................................ 59
II.4.4. Industrial Application Using Immobilization Technique ..... 62
II.5. Quorum Quenching (QQ) Application to MBR ............................ 64
II.5.1. Enzymatic QQ Application to MBR ........................................ 65
II.5.2. Bacteria Strains with QQ Enzyme .......................................... 66
II.5.3. Bacterial QQ Application to MBR .......................................... 68
II.5.4. Microbial Ecology in MBR ...................................................... 70
III. Control of Membrane Biofouling in MBR by QQ Bacteria Entrapping Alginate Beads ................................... 73
III.1. Introduction ..................................................................................... 75
III.2. Experimental Section ...................................................................... 76
III.2.1. Bioassay for Detecting AHL Molecules ................................... 76
III.2.2. Preparation of Cell Entrapping Beads (CEBs) ...................... 77
III.2.3. Measurement of QQ Activity ................................................... 78
III.2.4. Extraction and Analysis of AHLs using High-Pressure Liquid
Chromatography (HPLC) ........................................................ 79
III.2.5. MBR Operation ........................................................................ 80
III.2.6. Measurement of Loosely and Tightly Bound Biofilms .......... 82
viii
III.2.7. Straining of CEBs Image Analysis Using a Confocal Laser
Scanning Microscope (CLSM) ................................................. 84
III.2.8. Analytical Methods ................................................................... 84
III.3. Results and Discussion .................................................................... 84
III.3.1. Characterization of CEBs. ....................................................... 84
III.3.2. QQ Activity of Free BH4 and CEBs ........................................ 87
III.3.3. Application of CEBs to the Lab-Scale MBR. ......................... 88
III.3.4. Physical Washing Effect of CEBs ............................................ 90
III.3.5. QQ Effect of CEBs. ................................................................... 92
III.3.6. Inhibition of EPS Production by CEBs ................................... 93
III.3.7. Identification of Signal Molecules in MBRs ........................... 95
III.3.8. Visual Confirmation of the QQ Effect by CEBs .................... 98
III.3.9. Influence of CEBs on MBR Performance and Its Stability .. 99
III.4. Conclusions .................................................................................... 101
IV. Stability Enhancement of QQ Bacteria Entrapping Moving Bead and Its Application to MBR for Biofouling Control .................................................................................. 103
IV.1. Introduction ................................................................................... 105
IV.2. Experimental Section .................................................................... 106
IV.2.1. Microorganisms and Growth Conditions ............................. 106
IV.2.2. Preparation of Macrocapsules and W-beads ........................ 106
IV.2.3. Luminescence Method for Detecting AHL Molecules ......... 108
IV.2.4. Determination of QQ Activity ................................................ 109
IV.2.5. Measurement of Mechanical Strength .................................. 110
IV.2.6. Measurement of Chemical Stability (Macrocapsule) .......... 110
IV.2.7. Restoration of QQ Activity of Disintegrated Beads ............. 111
ix
IV.2.8. Measurement of Durability in Wastewater (W-bead) ......... 111
IV.2.9. MBR Operation Condition .................................................... 111
IV.2.10. Scanning Electron Microscopy (SEM) and Confocal Laser
Scanning Microscopy (CLSM)............................................... 113
IV.3. Results and Discussion .................................................................. 113
IV.3.1. Preparation and Characterization of Macrocapsules with
Various Polymeric Coatings ................................................... 113
IV.3.2. Characteristics of W-bead ...................................................... 117
IV.3.3. QQ Activities of PSf-Macrocapsules ..................................... 119
IV.3.4. QQ Activities of W-beads ....................................................... 122
IV.3.5. Stability of Macrocapsule in a Harsh Environment ............ 123
IV.3.6. Biofouling Inhibition by Macrocapsules in a Continuous
MBR Fed with Synthetic Wastewater ................................... 126
IV.3.7. Biofouling Inhibition by Macrocapsules in a Continuous
MBR Fed with Real Wastewater ........................................... 129
IV.3.8. Biofouling Inhibition by W-bead in a Continuous MBR ..... 134
IV.3.9. Stability of W-bead in Various Wastewater .......................... 137
IV.4. Conclusions .................................................................................... 139
V. Conclusions .......................................................................... 141
VI. Suggestions..…..…….……………………………………145
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List of Figures
Figure II-1. Schematic diagrams of wastewater treatment processes. (a)
Conventional activated sludge process and (b) membrane bioreactor
process. ......................................................................................................... 9
Figure II-2. Schematics of the (a) side-stream MBR and (b) submerged
MBR. ........................................................................................................... 11
Figure II-3. (a) MBR market revenue and installation capacity forecast in
China (Frost & Sullivan, 2011). (b) Global MBR market share on the
company (Environmental Leader, 2014). ................................................ 14
Figure II-4. Research trends in MBR. Data analyzed by Scopus. ................. 15
Figure II-5. Pore blocking and cake layer forming into/onto a membrane
(Source: University Tunku Abdul Rahman, www.utar.edu.my/).. ........ 17
Figure II-6. MBR fouling factors and operation & design characteristics
(Zhang et al., 2006a). ................................................................................. 18
Figure II-7. MBR fouling mechanisms–3 stages of fouling (Zhang et al.,
2006a). ......................................................................................................... 18
Figure II-8. The principle of the MCP flow field in a membrane tank with
a submerged flat sheet module and serrated weir as retention system
for plastic particles (Rosenberger et al., 2011). ....................................... 20
Figure II-9. A model of the stages of bacterial biofilm development (Annous
et al., 2009). ................................................................................................ 27
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Figure II-10. Representative signal molecules of bacteria QS. ...................... 30
Figure II-11. The molecular structure of each AHL autoinducer. ................. 33
Figure II-12. (a) Schematic diagram illustrating the general features of the
AHL biosynthetic pathway. SAM (1) and acyl-ACP bind the AHL
synthase (LuxI-type synthase), whereupon acylation and lactonization
reactions occur. The AHL is then released, along with the byproduct
holo-ACP and 5′-methylthioadenosineis. (b) Two SAM analogues, 2
and 3, they are known inhibitors of AHL synthesis in P. aeruginosa
(Parsek et al., 1999, Hentzer and Givskov, 2003) .................................... 34
Figure II-13. Model of acyl-homoserine-lactone mediated QS in a single
generalized bacterial cell (Fuqua and Greenberg, 2002). ...................... 35
Figure II-14. A general model for QS in gram-positive bacteria. The oval
represents a bacterial cell. The “P” in the circle represents the
phosphorylation cascade (Miller and Bassler, 2001). ............................. 39
Figure II-15. Chemical structures of representative AI-2 molecules. DPD
and its derivatives are possible in water and in the presence of borate
(Camilli and Bassler, 2006). ...................................................................... 40
Figure II-16. Epifluorescence and scanning confocal photomicrographs of
the WT and the lasI mutant Pseudomonas aeruginosa biofilms
containing the GFP expression vector pMRP9-1 (Davies et al., 1998). . 43
Figure II-17. Diagram of the P. aeruginosa biofilm-maturation pathway. ... 44
Figure II-18. Three strategies to control LuxI/R type QS system (Yeon,
xii
2009) ............................................................................................................ 45
Figure II-19. Possible linkage degraded by QQ enzymes in quorum sensing
molecule (a) N-acyl homoserine lactone and (b) corresponding
degradation mechanism of QQ enzymes (Chen et al., 2013) ................. 49
Figure II-20. Activation of an amine-bearing support with glutaraldehyde
followed by enzyme coupling .................................................................... 57
Figure II-21. (a) Assembly of enzyme aggregate coating on electrospun
nanofibers (Kim et al., 2008a) (b) Nano-in-Nano approach for enzyme
immobilization based on block copolymer (Auriemma et al., 2017)…..59
Figure II-22. Schematic diagram: (a) Preparing steps of LetiKats and (b)
its application method for wastewater treatment process (Source:
LentiKat`s Biotechnologies, http://www.lentikats.eu/en/). ..................... 64
Figure II-23. (a) Photograph and enlarged diagram of a microbial vessel
(Oh et al., 2012). (b) Concept of a quorum quenching-hollow cylinder
(Lee et al., 2016). ........................................................................................ 70
Figure II-24. (a) Comparison of major genus groups in mixed liquor and
biocake at each initial (M30, B30) and late biofouling stage (M70, B70).
The percentage was calculated from the pyrosequencing data (Lim et
al., 2012). (b) Proportions of Enterobacter, Pseudomonas, and
Acinetobacter at a genus level in biofilm samples of control and QQ
MBRs (Kim et al., 2013a). ......................................................................... 72
Figure III-1. Bioassay for measuring the AHL concentrations. .................... 77
xiii
Figure III-2. Schematic diagram for the preparation of cell entrapping
beads (CEBs). ............................................................................................. 78
Figure III-3. Schematic diagrams for three sets of operations of MBRs. ..... 82
Figure III-4. Schematic for the quantitative analysis of loosely and tightly
bound biofilms. .......................................................................................... 83
Figure III-5. Photographs of (a) individual CEBs and (b) CEBs in the MBR
with and without aeration. ........................................................................ 86
Figure III-6. SEM microphotographs of the beads: a cross-section of a
vacant bead (a) ×25, (b) ×1000, and (c) ×6000. Cross section of a CEB
(d) ×25, (e) ×1000, and (f) ×6000. ............................................................. 86
Figure III-7. Reconstructed CLSM images of a CEB cross-section; (a) alive
and (b) a dead cell stained with the BacLight Live-Dead staining kit.
Magnification x100. Image size 1212 μm x 1212 μm. ............................. 87
Figure III-8. (a) QQ activity of live and dead BH4 cells. (b) Quantitative
QQ activity of control, vacant beads, and CEBs. Error bar: standard
deviation (n=4). .......................................................................................... 88
Figure III-9. Comparison of TMP between (a) control and CEBs MBRs, (b)
control and vacant beads MBRs, and (c) vacant beads and CEBs
MBRs under the same operating conditions ........................................... 90
Figure III-10. Detached biomass from used membranes in the beaker with
and without a vacant bead. Error bar: standard deviation (n=5). ........ 92
Figure III-11. Identification of AHLs extracted from the biofilm formed on
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the used membrane by HPLC. (a) Chromatogram of standard and
extracted AHLs. (b) Bioassay of fractions (1 and 2) collected every 9
min for the MBR with vacant beads. (C) Bioassay of fractions (1 and
2) collected every 9 min for the MBR with CEBs. .................................. 97
Figure III-12. Reconstructed CLSM images of biofilm formed on the
membrane surface in (a) control MBR, (b) MBR with vacant beads,
and (c) MBR with CEBs after 48 h operation, stained with SYTO9
(cell; green). Magnification: ×100. Image size: 1212 μm × 1212 μm. .... 98
Figure III-13. (a) COD change of permeate during continuous MBRs
experiments. (b) QQ activity of CEBs during MBR operation. QQ
activity (%): Percent ratio of the degraded amount of the standard
C8-HSL for 30 min by the fresh or used CEBs to the initial amount of
the standard C8-HSL. Error bar: standard deviation (n=3). .............. 100
Figure IV-1. Preparation scheme of a macrocapsule coated with a
membrane layer through the phase inversion method. ........................ 108
Figure IV-2. Calibration curve for the quantification of AHLs by
luminescence method. Error bar: standard deviation (n=3). .............. 109
Figure IV-3. SEM microphotographs of an alginate bead: (a) Top and (b)
cross-section views of a fresh alginate bead, (c) Top and (d) cross-
section views of a used alginate bead after 60 days’ MBR operation.. 115
Figure IV-4. (a) Photographs of an alginate bead and PSf, PES, PVDF
coated macrocapsules. (b) SEM image of the outer surface, inner
xv
surface, and cross-section of each macrocapsule prepared with PSf,
PES and PVDF. ........................................................................................ 116
Figure IV-5. Comparison of mechanical strength between alginate beads
and three types of coated macrocapsules. Error bar: standard
deviation (n=20). ...................................................................................... 117
Figure IV-6. (a) Photographs of vacant W-bead and QQ bacteria
entrapping W-bead. (b) CLSM image of the live/dead cell distribution
in a W-bead (Green: Live, Red: Dead, Magnification: X100) ............. 118
Figure IV-7. Comparison of the AHL removal rate between alginate beads,
macrocapsules and restored macrocapsules. Error bar: standard
deviation (n=3). ........................................................................................ 120
Figure IV-8. CLSM image of the live/dead cell distribution in a
macrocapsule. Note that green and red colors appear in the PSf-
membrane layer because the fluorescence dyes were adsorbed onto the
membrane layer during the staining step. (Green: Live, Red: Dead,
Magnification: X100) .............................................................................. 121
Figure IV-9. CLSM image of the live/dead cell distribution after
reactivation (Green: Live, Red: Dead, Magnification: X100). ............ 122
Figure IV-10. QQ activity of vacant W-bead and QQ bacteria entrapping
W-bead. Error bar: standard deviation (n=4). ..................................... 123
Figure IV-11. Chemical stability and relative QQ activity of alginate beads
and macrocapsules. (a) Leakage of QQ bacteria in both beads after
xvi
chemical treatment using citrate buffer (30 mM EDTA, 55 mM sodium
citrate and 0.15 M sodium chloride). (b) Relative QQ activity of both
beads before and after chemical treatment and after restoration.
Relative QQ activity: the percentage of residual QQ activity to initial
QQ activity. Error bar: standard deviation (n=3). ............................... 125
Figure IV-12. (a) Effect of macrocapsules on the enhancement of
permeability in MBR. (b) Reconstructed CLSM images of biocakes
formed on the surface of hollow fiber membrane after 12 days
operation of the continuous MBR. ......................................................... 128
Figure IV-13. Relative C8-HSL degradation stability and mechanical
stability of macrocapsules during continuous MBR operation. Error
bar: standard deviation (n=3). ............................................................... 129
Figure IV-14. TMP profiles during the operation of continuous MBR fed
with real wastewater. In the 1st cycle, the vacant-macrocapsule and
macrocapsule with QQ bacteria were inserted in the Control and QQ
MBRs, respectively. At the end of the 2nd cycle, used membranes were
taken out of both MBRs for analyzing biocakes with CLSM and EPS
concentrations in biocakes. ..................................................................... 132
Figure IV-15. (a) Reconstructed CLSM images of biocakes formed on the
surface of hollow fiber membranes after the same operating period of
9 days in the control and QQ MBRs. The sampling was done on the
32nd day in the 2nd cycle in Figure IV-14. (b) Polysaccharide and
xvii
protein concentrations per unit membrane area in the biocakes at the
end of 9 days operation. The sampling was done on the 32nd day in
the 2nd cycle in Figure IV-14. Error bar: standard deviation (n=3). .. 133
Figure IV-16. (a) TMP profiles of the MBR with and without alginates
beads during continuous operation with real wastewater. (b) The
photograph of alginate beads in one MBR at the end of 26 days of
operation in (a). ........................................................................................ 134
Figure IV-17. (a) Biofouling inhibition of W-beads in continuous MBR. (b)
Reconstructed CLSM images of biocakes formed on the hollow fiber
membrane................................................................................................. 136
Figure IV-18. Change of shape of QQ bacteria entrapping W-beads in
synthetic and real wastewater. ................................................................ 137
Figure IV-19. Change of (a) average size of W-beads and (b) relative
activity of W-bead in synthetic and real wastewater environments.
Error bar: standard deviation (size data: n=10, QQ activity data: n=3)
................................................................................................................... 138
xviii
List of Tables
Table II-1. Membrane Module Products, Bulk Municipal Market (The
MBR site, http://www.thembrsite.com/features.php). ......................... 11
Table II-2. AHL-Dependent QS Systems in Gram-Negative Bacteria: The
Regulatory Phenotype and AHL (Matthew TG Holden, 2007) ............. 35
Table II-3. Bacterial Reporter Strains Used to Detect QS Signals. ............... 50
Table II-4. The Reactive Functional Group in the Enzyme. .......................... 54
Table II-5. Quorum-Quenching Enzymes Involved in the Degradation of
QS Signal AHLs. ........................................................................................ 66
Table III-1. The Composition of the Synthetic Wastewater in Continuous
MBR Operation. ........................................................................................ 81
Table III-2. Amount of TAB, EPS and Loosely and Tightly Bound Biofilms
in the Used Membrane Modules for the MBR with Vacant Beads and
MBR with CEBs ........................................................................................ 95
1
Chapter I
Introduction
Introduction
2
3
I.1. Backgrounds
Membrane bioreactor (MBR) has been proposed as one of the innovative options
in the advanced wastewater treatment technology. It is highly appropriate to an
urbanized place at present because it requires a smaller footprint and produces
treated water of higher quality comparing to a conventional activated sludge (CAS)
process (Lin et al., 2012, Le-Clech, 2010, Judd, 2004). However, because MBR
process is based on the solid-liquid separation mechanism biofouling is inevitably
occurred on the membrane surface. Membrane biofouling in MBR caused severe
flux decline, short membrane lifespan, and an increase of energy consumption
(Drews, 2010). Therefore, many scientists have tried to solve this issue through
engineering, material and chemical approaches. Although these approaches have
been proven to alleviate membrane biofouling, they have still limitations in
uprooting biofouling in MBR that is biological engineering process (Malaeb et al.,
2013, Lin et al., 2012).
It is revealed that cell-cell communication which is called as quorum sensing is
involved in biofilm formation in MBR (Yeon et al., 2009a). Since then novel
biological approaches had been attempted to control biofouling using quorum
quenching (QQ, i.e., via disruption of quorum sensing) (Oh et al., 2012, Jahangir et
al., 2012, Yeon et al., 2009a). These approaches, however, had drawbacks such as
the high cost of enzyme extraction and purification as well as enzyme instability, or
the limited mass transfer of signal molecules from the mixed liquor to the inside of
the microbial vessel. Therefore, more effective and practical QQ methods are
required to develop for wastewater treatment.
4
I.2. Objectives
The objective of this study is to develop more effective biofouling strategy based
on bacterial quorum quenching (QQ) in MBR for wastewater treatment. Free-
moving beads entrapping QQ bacteria were prepared using with polymeric materials
to add physical cleaning efficiency to previously reported QQ based anti-biofouling
strategy.
(1) Control of Membrane Biofouling in MBR by QQ Bacteria Entrapping
Bead
Cell entrapping beads (CEBs) with highly interconnected microstructural pores
were prepared by entrapping QQ bacteria (Rhodococcus sp. BH4) into Ca-alginate
matrix of the beads. The CEBs were applied to MBR to test their QQ and anti-
biofouling activities.
(2) Enhancement of Chemical and Physical stabilities of CEBs
Macrocapsules were prepared by coating a polymeric membrane layer around the
CEBs by phase inversion and by fusion (W-beads). Those modified beads were
tested for their chemical, mechanical and biological stabilities.
5
Chapter II
Literature Review
II. Literature Review
6
7
II.1. MBR for Advanced Wastewater Treatment
II.1.1. Concept and Process
Membrane bioreactor (MBR) is the combination of a membrane process like
microfiltration (MF) or ultrafiltration (UF) with a conventional activated sludge
process and is now widely used for industrial and municipal wastewater treatment.
As illustrated in Figure II-1, MBR is similar to conventional activated sludge
systems (CAS) except the submerged membrane modules. As a result, the MBR has
many advantages over conventional wastewater treatment processes. MBR process
can produce effluent of high quality enough to be discharged to coastal, surface or
brackish waterways or to be reclaimed for urban irrigation. Other advantages of
MBR over CAS include small footprint, less sludge production and easy retrofit of
old wastewater treatment plants. MBR is possible to operate at higher mixed liquor
suspended solids (MLSS) concentrations compared to CAS, thus reducing the
reactor volume to achieve the same loading rate.
The advantages offered by MBRs over conventional wastewater treatment
processes are widely recognized and of these the ones most often cited are (Judd
and Judd, 2006):
(1) Production of high quality clarified and largely disinfected permeate product
in a single stage; The membrane has an adequate pore size <0.1 mm – significantly
smaller than the pathogenic bacteria and viruses in the sludge.
(2) Independent control of solids and hydraulic retention time (SRT and HRT,
respectively); In a CAS separation of solids is achieved by sedimentation, which then
relies on growth of the mixed liquor solid particles (of flocs) to sufficient size (>50
mm) to allow their removal by the settlement. In an MBR, the separation process
8
only be depended on membrane more size.
(3) Operation at higher mixed liquor suspended solids (MLSS) concentrations,
which reduces the required reactor size and promotes the development of specific
nitrifying bacteria, thereby enhancing ammonia removal.
(4) Reduced sludge production, which results from operation at long SRTs because
the longer the solids are retained in the bioreactor, the lower the waste solids (sludge)
production.
It is the intensity of the process and the high quality of treated product water that
is generally most important of these advantages for the effective wastewater
treatment process. MBR processes have some disadvantages compared with CAS,
primarily by:
(1) Greater process complexity; membrane separation demands additional
operational protocols relating to the maintenance of membrane cleanliness.
(2) Higher capital equipment and operating costs; the membrane component of
the MBR incurs a significant capital cost over and above that of a CAS and
maintaining membrane cleanliness demands further capital equipment (capex) and
operating costs (opex).
9
(a)
(b)
Figure II-1. Schematic diagrams of wastewater treatment processes. (a)
Conventional activated sludge process and (b) membrane
bioreactor process.
II.1.2. Development of MBR
The idea of combining CAS with membrane technology was conceived in the late
1960s by Dorr-Oliver (Stanford, Connecticut) (Figure II-2a). The Dorr-Oliver
system succeeded in establishing the principle of MBR to simultaneously
concentrate the biomass while generating a clarified, disinfected product. Thetford
System (Ann Arbor, Michigan), which later became part of Zenon (now owned by
GE), accomplished this in the early 1970s. Zenon developed an MBR process for
on-site treatment and recycling of wastewater. The system, called Cycle-Let® was
based on anaerobic – anoxic activated sludge process with tubular UF in a two-pump
10
feed and bleed loop. In the early 1980s, Cycle-Let® system was applied to larger
facilities such as major office buildings, shopping centers, industrial parks, sports
facilities and other buildings where recycled flush water was required to reduce
wastewater discharge.
The breakthrough for the MBR came in 1989 by Yamamoto and co-workers idea
of immersing the hollow fiber membranes in the bioreactor (Yamamoto et al., 1989)
(Figure II-2b). More than 90% COD removal, 80% nitrate removal was reported by
Yamamoto et al. using microfiltration hollow fiber membrane used as submerged
MBR. Most importantly, the power consumption was found to be as low as 0.007
kWh/m3. The concept was picked up by Japanese companies that continued the
development and commercialized the technology. Kubota Corporation developed
flat sheet panels, while Mitsubishi Rayon Corporation focused their efforts on
hollow fibers.
The ZeeWeed® immersed hollow fiber membrane was conceived by Zenon in the
early 1990s with simplify the MBR system and reduce energy consumption.
ZeeWeed® rapidly replaced tubular membranes in Cycle-Let® systems. By the mid-
1990s, the ZeeWeed® technology had been developed to the point where it could be
applied in municipal wastewater treatment. In 1997, the first Kubota municipal
wastewater treatment installed outside Japan was at Porlock in the United Kingdom.
Moreover, the first Zenon membrane-based plant installed outside of the USA was
the Veolia Biosep® plant at Perthes en Gatinais in France in 1999. Both these plants
have a peak flow capacity just below two megaliters per day (MLD) and represent
landmark plants in the development and implementation of immersed MBR
technology.
The first half of the 1990s saw the launch of only three major immersed MBR
membrane products, originating from just two countries (the US and Japan), the first
11
five years of the following decade saw the launch of at least 10 products originating
from seven countries. For 5 major suppliers as of 2018, there were existing or
planned MBR installations of more than 175 MLD capacity (Table II-1).
(a) (b)
Figure II-2. Schematics of the (a) side-stream MBR and (b) submerged MBR.
Table II-1. Membrane Module Products, Bulk Municipal Market
(The MBR site, http://www.thembrsite.com/features.php).
Installations Technology
Provider
Date of
Commissioning
(MLD)
ADF
(MLD)
Henrikdsal, Sweden SUEZ 2019-2026 864 536
Beihu WWTP, China OW 2019 800
Tuas WRP, Singapore TBC 2025 800
Seine Aval, France SUEZ 2016 357 224
12
Canton WWTP, USA Ovivo/Kubota 2015-2017 333 159
Water Affairs Integrative
EPC, China OW 307
Euclid, USA SUEZ 2018 250 83
9th and 10th WWTP,
China OW 2013 250
Shunyi, China SUEZ 2016 234 180
Wuhan Sanjintan
WWTP, China OW 2015 200
Jilin WWTP, China OW 2015 200
Caotan WWTP, China OW 200
Huhehaote Xinxinban,
China OW 2016 200
Weibei Industrial Park
Wanzi, China OW 2016 200
Brussels Sud, Belgium SUEZ 2017 190 86
Riverside, USA SUEZ 2014 186 124
Brightwater, USA SUEZ 2011 175 122
PDF: Peak daily flow, (Megalitres per day)
ADF: Average daily flow, (Megalitres per day)
OW: Beijing Origin Water
13
II.1.3. Trends in MBR: Market and Research
II.1.3.1. World Market
According to the WWI (Water & Wastewater International), the global market for
MBR systems grew to $838.2 million in 2011 and is projected to increase up to $3.44
billion by 2018. This represents a compound annual growth rate (CAGR) of 22.4%
over this period. Such impressive market growth can be seen as a reaction to global
megatrends such as urbanization and water stress, that are now clearly shaping our
future.
The global MBR market is growing due to its ability to meet stringent effluent
criteria along with its compact size and less operational cost as compare to other
systems and equipment which are used for wastewater treatment. According to WHO,
about one-fifth of the world's population resides in areas where water is physically
scarce, while one fourth face scarcities due to lack of infrastructure to transport water.
Governments have realized the importance of wastewater treatment, as a necessity
and also as means to improve the bottom line. Stringent legislations for the
implementation of the treatment facilities, combined with space and operational
advantages that MBRs provide, are expected to be key drivers of the market going
forward. The sophistication and high capital costs associated with the system could,
however, prove to be key obstacles.
Asia-Pacific holds major market share in MBR market. According to the report
published by MarketsandMarkets, Asia-Pacific nations lead the global MBR market
with a share of 38.7% regarding revenue in the year 2011. Among the Asia nations,
China is the world’s largest MBR wastewater market, which was initially spurred by
the 2008 Olympics build-out and continued urbanization, with more than 1.4 million
m3/d installed to date and 730,000 m3/d of additional capacity in the planning stages
14
(Global membrane bioreactor market: An emerging competitive
landscape). According to the 'Frost & Sullivan: The membrane bioreactor market in
China files high,' the Chinese MBR market is witnessing exponential growth and
was worth US$ 228 million in 2010. As shown in Figure II-3a, the MBR market of
China will reach $1.35 billion until 2017, six times larger than it is at present.
'Global Membrane Bioreactor Market: An Emerging Competitive Landscape'
reported that US and Chinese suppliers including GE Water, Beijing Origin Water,
Koch Membrane Systems, Beijing E&E and Tianjin Motimo had supplied more than
74% of the large-scale MBR capacity systems worldwide (Figure II-3b). GE Water
is the market leader in large-scale MBR systems with nearly 47 percent global
market share. While the company has strong supply ties in the US, UAE, South
Korea, and Australia, it has supplied a small 61,000 m3/d to the booming Chinese
market. However, Chinese suppliers such as Beijing Origin Water have emerged as
a primary threat to GE Water’s global MBR dominance based on its home market.
(a) (b)
Figure II-3. (a) MBR market revenue and installation capacity forecast in
China (Frost & Sullivan, 2011). (b) Global MBR market share on
the company (Environmental Leader, 2014).
15
II.1.3.2. Research: Membrane Fouling
The research trend in MBR technology was reviewed by the data on the number
of articles published since the year 1998. As shown in Figure II-4, the number of
published papers increased dramatically since 2005, which suggests growing
acceptance of MBR technology and escalating technical demand to overcome the
perceived drawbacks of MBR such as its complex and small-scale nature, high costs
and operator skill requirements.
The performance of MBR process is mainly deteriorated by the membrane fouling
which is the occlusion or blocking of membrane pores at the surface of the membrane.
This membrane fouling causes severe operation problems of flux decline, short
membrane life-span, and increases of energy consumption and therefore has been
regarded as the main obstacle restricting the development of MBR technology. As
clearly shown in Figure II-4, about 30% of MBR researches were dedicated to the
topic of fouling including investigation of the mechanism and development of the
control techniques.
Figure II-4. Research trends in MBR. Data analyzed by Scopus.
16
As mentioned Figure II-4, research studies on membrane fouling are extensive in
recent years. Membrane biofouling continuously decreases filtration performance
during MBR operating time. This is due to the accumulation of soluble materials and
microbial flocs (i.e., activated sludge) onto the membrane, attributed to increases in
the overall membrane resistance (Figure II-5). This major drawback and process
limitation has been under investigation since the early MBRs and remains one of the
most challenging issues facing further MBR development.
The degree of fouling in any membrane process will be determined by three basic
fouling factors, the nature of the feed, the membrane properties and the
hydrodynamic environment experienced by the membrane. In the MBR these factors
are more complicated than in most membrane applications. Figure II-6 described the
‘fouling factors,’ and this illustrates the complex nature of the feed and the features
of the hydrodynamic environment. The ‘reactor parameters’ determine the nature of
the potential foulants. (Zhang et al., 2006a, Zhang et al., 2006b, Zhang et al., 2006c).
Various reviews of MBR fouling encompassing fouling mechanisms have been
presented in the literature (Chang et al., 2002a, Chang et al., 2002b, Le-Clech et al.,
2006, Meng et al., 2009). MBR systems are operated under constant flux conditions
with convection of foulant towards the membrane surface. Since fouling rate
increases roughly with flux (Le Clech et al., 2003, McAdam et al., 2010b, McAdam
et al., 2010a, Monclus et al., 2010), sustainable operation dictates that MBRs should
be operated at modest fluxes and preferably below the critical flux. Even sub-critical
flux operation may lead to fouling according to a two-stage pattern (Wen, 2004,
Brookes et al., 2006, Pollice et al., 2005): a low trans-membrane pressure (TMP)
increase over an initial period followed by a rapid increase after a certain critical
period. Before these two filtration stages, a conditioning period is generally observed
17
(Zhang et al., 2006b). The three-stage process (conditioning fouling, slow steady
fouling and TMP rise-up) is summarized in Figure II-7.
Figure II-5. Pore blocking and cake layer forming into/onto a membrane
(Source: University Tunku Abdul Rahman, www.utar.edu.my/).
18
Figure II-6. MBR fouling factors and operation & design characteristics
(Zhang et al., 2006a).
Figure II-7. MBR fouling mechanisms–3 stages of fouling (Zhang et al.,
2006a).
19
II.1.4. Fouling Control in MBR Systems
As mentioned earlier, fouling on membranes is the biggest challenge for the
widespread application of MBR technology. Thus, various researchers have been
carried out over 30 years for fouling control. However, improvements in practice on
biofouling control and management have been remarkably slow. This slow progress
in the successful control of biofouling is largely attributed to the complex
interactions of the biological compounds involved, and the lack of representative-
for-practice experimental approaches to evaluate potential effective control
strategies (Drews, 2010). Biofouling is driven by microorganisms and their
associated extra-cellular polymeric substances (EPS) and microbial products.
Microorganisms and their products convene together to form matrices that are
commonly treated as a ‘black box’ in conventional control approaches (Malaeb et al.,
2013). Physical cleaning removes gross solids attached to the membrane surface,
generally termed ‘reversible’ or ‘temporary’ fouling, whereas chemical cleaning
removes more tenacious material often termed ‘irreversible’ or ‘permanent’ fouling.
II.1.4.1. Physical Approach
In MBRs, physical cleaning is normally achieved either by backflushing
(reversing the flow) or relaxation (ceasing permeation), while continuing to scour
the membrane with air bubbles. Aeration is an indispensable operating factor in
MBR to provide the oxygen required for the microbial growth and shake up the
membranes to inhibit solids from fouling the pores of the membranes (Meng et al.,
2009, Aslam et al., 2017, Lee et al., 2016).
Meanwhile, moving bead was applied to submerged type MBR for biofouling
inhibition on the membrane surface through physical friction. The experiments
reported are based on the mechanical cleaning process (MCP), developed by Krause
20
and co-worker, which consists primarily of the addition of plastic particles and the
establishment of a fluidized bed flow in a submerged flat sheet MBR (Rosenberger
et al., 2011, Siembida et al., 2010). Incorporation of moving bead in MBR as a
physical cleaning platform was one of the effective fouling control method
(Rosenberger et al., 2011, Lee et al., 2006). Furthermore, it might reduce the dosage
of strong cleaning chemical such as sodium hypochlorite, which affects the microbial
activity required for biological degradation of pollutant. As the eco-biofouling
inhibition strategy, the MCP was commercialized to BIO-CEL by Microdyn-Nadir
(Figure II-8).
Figure II-8. The principle of the MCP flow field in a membrane tank with a
submerged flat sheet module and serrated weir as retention
system for plastic particles (Rosenberger et al., 2011).
Physical cleaning with moving carrier can be affected by various factors such as
bead size, bead dosage, and aeration intensity, etc. These factors have reciprocal
action thus the conventional one-variable-at-a-time method is not an efficient
optimization approach. Shim et al. used the design of experiment (i.e., response
surface methodology) to optimize the engineering parameters of polymeric moving
carriers for the effective physical cleaning in MBR (Shim et al., 2015).
21
II.1.4.2. Chemical Approach
II.1.4.2.1. Chemical Cleaning
Physical cleaning is supplemented with chemical cleaning to remove residual and
irreversible fouling (Meng et al., 2009). Chemical cleaning is carried out with
mineral or organic acids, caustic soda or, sodium hypochlorite. Such type of cleaning
should be done on a weekly to monthly basis, designed to remove residual fouling
and intensive chemical cleaning (once or twice a year) to remove the irreversible
fouling.
II.1.4.2.2. Chemical Additives
The basic idea of this approach is to remove the significant foulants (small colloid
or biopolymers) through the addition of chemicals. Since pore size of microfiltration
(MF) membrane conventionally used in MBR varies from about 0.04 to 0.4 μm,
colloidal particles smaller than this cause pore plugging which increases the filtration
resistance. Therefore, the addition of coagulant can reduce membrane fouling.
Ferric chloride and aluminum sulfate (alum) have both been assessed for
membrane fouling amelioration. Small biological colloids (from 0.1 to 2 μm) have
been observed to coagulate and form a larger aggregate when alum is added to MBR
activated sludge. Ferric dosing of MBRs has been used for enhancing the production
of iron-oxidizing bacteria responsible for the degradation of gaseous H2S (Lee et al.,
2001).
Charged polymers have been reported to be effective in biofouling control by
reducing the biopolymers such as soluble microbial products (SMP) and
extracellular polymeric substances (EPS). For example, Nalco (US) has developed
the cationic polymers with the trademark of MPE (Membrane Performance
22
Enhancer). MPE was reported to reduce the level of polysaccharide (Yoon et al.,
2005, Yoon and Collins, 2006, Guo et al., 2008) and was successfully applied to
pilot- and full-scale MBRs (Collins et al., 2006).
Addition of adsorbents into biological treatment systems decreases the level of
organic compounds. Dosing with powdered activated carbon (PAC) produces
biologically activated carbon (BAC) which adsorbs and degrades soluble organics
and has been shown to be effective in reducing SMP and EPS levels (Kim and Lee,
2003)
II.1.4.3. Material Approach
Membrane fouling is an interfacial phenomenon between solid membrane surface
and mixed liquor containing various organic pollutants and microorganisms.
Therefore, increasing intrinsic fouling resistance of membrane surface can be a
simple and effective fouling control technique.
Futamura et al. reported that the transmembrane pressure (TMP) increase rate of
hydrophobic membranes was higher than that of hydrophilic ones. This suggests that
fouling resistance of the membrane can be enhanced by regulating the hydrophilicity
of the surface. Sainbayar et al. modified the surface of polypropylene membrane by
ozone treatment followed by graft polymerization with 2-hydroxy-ethyl
methacrylate. This modified membrane showed enhanced water permeability in the
anaerobic MBR.
II.1.4.4. Biological Approach
The biofouling process develops into a complex and difficult to control problem
so that conventional physical cleaning processes such as back-washing and back-
pulsing are no longer effective (Chu and Li, 2005, Malaeb et al., 2013, Le-Clech et
23
al., 2006). Some biological control strategies against biofouling have been reported,
including i) inhibition of quorum sensing, ii) nitric oxide-induced biofilm dispersal,
iii) enzymatic disruption of extracellular polysaccharides, proteins, and DNA, and
iv) disruption of biofilm by bacteriophage (Xiong and Liu, 2010). Biological-based
antifouling strategies are a promising constituent of an effective integrated control
approach since they target the essence of biofouling problems. However, biological-
based strategies are still in their developmental phase, and several questions need to
be addressed to set a roadmap for translating existing and new information into
sustainable and effective control techniques
II.1.4.4.1. Inhibition of Quorum Sensing (QS)
Microorganisms are known to coordinate their communal behaviors by QS, e.g.,
biofilm formation, swarming motility, production of extracellular polymeric
substances, etc. (Gonzalez and Keshavan, 2006). The QS-coordinated process is
achieved by producing, releasing, and detecting small signal molecules known as
autoinducers (AI). Increasing bacterial density gives rise to an accumulation of AIs.
Once the critical AI concentrations are achieved, the regulator proteins are triggered,
and target DNA sequences are induced, leading to transcription of QS-regulated
genes, followed by changes of bacterial social behaviors. AIs which have been
identified so far are as follows: oligopeptides, N-acyl homoserine lactones (AHL),
and autoinducer-2 (AI-2) synthesized by LuxS. Oligopeptides and AHL are involved
in the cellular communication of only gram-positive and gram-negative bacteria,
respectively, whereas AI-2 is universal for interspecies communication of both
gram-positive and gram-negative bacteria (Xavier and Bassler, 2003). Since 2009,
the concept of bacterial QS has been introduced to an MBR by Lee’s group. They
showed that membrane biofouling could be efficiently removed by the addition of
AHLs inhibitors (Yeon et al., 2009a). The biofouling control by inhibiting QS will
24
be described in more detail in the next section.
II.1.4.4.2. Enzymatic Disruption of EPS
Extracellular polymeric substances (EPS) secreted by bacteria can facilitate
attachment of bacteria to a solid surface such as membranes. EPS can decrease the
susceptibility of bacteria to antibiotics and thus act as a shelter to protect the bacteria
in the biofilm–EPS matrix. EPS have been believed to be irreversible foulants of
membrane fouling, which cannot be efficiently removed by traditional physical or
chemical cleaning methods. However, EPS could be hydrolyzed by some specific
enzymes, implying a novel means to control EPS-mediated microbial attachment and
membrane biofouling (Xiong and Liu, 2010). EPS are mainly composed of proteins,
polysaccharides and extracellular DNA. Therefore the enzymes such as protease,
polysaccharides, and DNase, which degrade those substances, can provide a feasible
and effective mean for controlling membrane biofouling. Poele and van der Graaf
used a protease to remove biofouling on ultrafiltration (UF) membrane for
wastewater treatment (Poele and van der Graaf, 2005). Compared to the traditional
cleaning method by alkaline, enzymatic cleaning by protease exhibited a much
higher efficiency in removing biofouling, leading to a high-efficiency recovery of
the permeate flux. Moreover, enzymatic cleaning of the fouled inorganic UF
membranes by proteins was also tested (Arguello et al., 2002, Arguello et al., 2003),
and results showed that over 90% of removal efficiency would be achievable.
II.1.4.4.3. Bacteriophage
Bacteriophage can infect the host bacteria by the rapid replication of virions to
cause lysis of the host cells or by incorporation into the host cell's genome (Xiong
and Liu, 2010). One important application of phage is to inhibit or disrupt biofilm
development on solid surfaces such as membranes. Goldman et al. (2009) employed
bacteriophage to control biofouling of UF membrane. Results showed that the
25
addition of phages could reduce microbial attachment to membrane surface by 40%
as average, and the performance of the MBR treating the effluents from the sewage
treatment plant was improved significantly regarding membrane permeability.
Although the study by Goldman et al. (2009) sheds lights on the potential application
of bacteriophage in mitigating membrane biofouling without the use of antimicrobial
agents, the specific-parasite characteristics of bacteriophage would eventually pose
a challenge on its application in large-scale wastewater treatment, which needs to be
taken into serious account in future investigations.
II.1.5. Biofilm in MBR
In the early stage of the MBR research, the fouling layer established on the
membrane surface was considered as a biocake layer. Therefore, the fouling
mechanism was investigated using physicochemical concepts such as adsorption of
organic foulants on the membrane surface, accumulation of mixed liquor suspended
solids, and compression of cake according to the operating pressure. However, many
reports have shown the importance of the biological factor on the permeability loss
in the MBR (Drews, 2010). As a result, recent MBR researchers placed their research
focus on the revealing of the fouling phenomena using the biological frame.
II.1.5.1. Fundamentals of Biofilm
Microbial biofilms may be defined as populations of microorganisms that are
concentrated at an interface and typically surrounded by an extracellular polymeric
slime matrix (Costerton et al., 1995). The development of a biofilm in vitro involves
the following 5 stages (Annous et al., 2009) (Figure II-9): Stage 1: reversible
attachment of bacterial cells to a surface, Stage 2: irreversible attachment mediated
by the formation of exopolymeric material, Stage 3: formation of microcolonies and
26
the beginning of biofilm maturation, Stage 4: formation of a mature biofilm with a
3-dimensional structure containing cells packed in clusters with channels between
the clusters that allow transport of water and nutrients and waste removal, and Stage
5: detachment and dispersion of cells from the biofilm and initiation of new biofilm
formation; dispersed cells are more similar to planktonic cells than to mature biofilm
cells. The biofilm may spread into uninfected areas as environmental conditions
allow and, occasionally, cells will detach from the biofilm and re-enter a planktonic
mode. These planktonic cells can repeat the cycle, infecting new surface. Essentially,
the biofilm may form on any surface exposed to bacteria and water. Once anchored
to a surface, biofilm microorganisms carry out a variety of detrimental or beneficial
reactions, depending on the surrounding environmental conditions.
Since the year of 2000, the concept of biofilm, ubiquitous surface microbial
system, started to be introduced in the interpretation of the biofouling in the MBR
and become the main research stream in the area of the MBR for advanced water
treatment. Biofouling is defined as the undesirable accumulation of microorganisms
at a phase transition interface (solid-liquid, gas-liquid or liquid-liquid), which may
occur by deposition, growth, and metabolism of bacteria cells or flocs on the
membranes. Several sequential steps are generally considered to be involved in the
progression of biofilm formation, including: (i) surface conditioning by formation of
a conditioning film (macromolecules, proteins, etc.); (ii) attachment of pioneer
planktonic cells onto surfaces; (iii) formation of microcolonies by primary adhesion;
and (iv) subsequently development of mature biofilms (Ghayeni et al., 1996, Guo et
al., 2012). Biofilms may or may not uniformly cover the membrane and consist of
multiple layers of living and dead microorganisms and their associated extracellular
products. Bacteria accumulate on the membrane by two processes: attachment
(adhesion and adsorption) and growth (multiplication) (Ivnitsky et al., 2005). The
27
adhesion step will depend on the properties of the microorganisms, the solution, and
the surface.
Figure II-9. A model of the stages of bacterial biofilm development (Annous
et al., 2009).
II.1.5.2. Biofilm Formation in MBR
A previous study (Park and Lee, 2005) has experimentally proved that the cake
layer formed on the membrane surface was able to biodegrade a certain percentage
of the soluble COD. This COD removal fraction increased in proportional to the
growth of the cake, which implies that the fouling layer has characteristics of biofilm.
Based on these results, Lee (Lee et al., 2008a) proposed the concept of biocake which
summarize both the physical deposition of the mixed liquor suspended solids (MLSS)
in mixed liquor and biofilm growth on the membrane surface.
Among various biofilm themes, structure, defined as the distribution of biomass
in the space occupied by the biofilm, gained the attention of the MBR research. It is
because the different biofilms have different structures, and the structure of the same
biofilm varies over time. In addition, biofilm structure reflects the precise function
of the biofilm such as filtration resistance in the case of the MBR.
28
Yun et al. (2006) analyzed the structure of the membrane-biocake in the different
dissolved oxygen (DO) level in terms of the internal porosity using the ‘biofilm
structure analysis technique’ consists of fluorescent staining of biocake components
such as bacterial cell and EPS. They found that high DO concentration condition
induced larger porosity compared to low DO condition, which resulted in the
enhanced water permeability in membrane-biocake. Kim et al. (2006) compared the
distribution of EPS in the membrane-biocake in different high and low DO
environment using the same techniques and showed that not only the amount of EPS
but also its spatial distribution affects the water permeability of the membrane-
biocake.
This biofilm structure analysis technique also provided a more exact
understanding of conventional fouling control techniques. For example, Hwang et al.
(2007) monitored the porosity change when conventional fouling reducing cationic
polymer was added and found that this chemical induced the more heterogeneous
structure with high porosity, which is the main reason for the performance
enhancement by addition of the cationic polymer. Lee et al. (2009) analyzed the
spatial distribution of biocake porosity on the hollow fiber membrane immersed in
MBR using biofilm structure analysis technique and investigated a correlation
between the biocake porosity and the flux at every local membrane position. Based
on these results, they finally suggested the optimum position of an aerator in the
reactor to obtain minimal membrane biofouling
29
II.2. Quorum Sensing (QS) Signaling in Bacteria
II.2.1. Definition and Mechanism
Quorum sensing (QS) is a system of stimulus and response correlated to
population density and communicate with each other using small signaling
molecules called autoinducers. QS was discovered and described over 35 years ago
in two luminous marine bacterial species, Vibrio fischeri and Vibrio harveyi (Nealson
and Hastings, 1979). In both species the enzymes responsible for light production
are encoded by the luciferase structural operon luxCDABE (Engebrecht and
Silverman, 1984, Miyamoto et al., 1988), and light emission was determined to occur
only at high cell-population density in response to the accumulation of secreted
autoinducer signaling molecules (Nealson and Hastings, 1979). The detection of a
minimal threshold stimulatory concentration of an autoinducer leads to an alteration
in gene expressions. Bacteria use QS communication circuits to regulate a diverse
array of physiological activities. These processes include symbiosis, virulence,
competence, conjugation, antibiotic production, motility, sporulation, and biofilm
formation (Miller and Bassler, 2001, Waters and Bassler, 2005).
Gram-negative bacteria is mediated by N-acyl homoserine lactones (AHLs) with
various moieties distinguishing signals among intraspecies, which is called as AI-1
type QS (Fuqua et al., 2001). In Gram-positive bacteria, intraspecies QS is mostly
facilitated through autoinducing peptides (AIPs) (Merritt et al., 2003). More recently
discovered interspecies communication has been linked to autoinducer-2 (AI-2), a
furanosyl borate diester (Chen et al., 2002). A recent review of cell-to-cell signaling
in Escherichia coli and Salmonella enterica has a concise description of AI-2
signaling (Ahmer, 2004, March and Bentley, 2004). At least, two additional QS
system have been identified in gram-negative bacteria. These include autoinducer-3
30
(AI-3), which is associated with virulence regulation in EHEC O157:H7 and the
Pseudomonas quinolone signal (PQS), which is associated with Pseudomonas
aeruginosa (Mashburn and Whiteley, 2005). AI-3 is associated with luxS homologs
in EHEC O157:H7, but this signal itself is hydrophobic and thus chemically distinct
from the polar AI-2 signals. AI-3 is also biologically distinct from AI-2. During
EHEC pathogenesis, both AI-3 and host epinephrine, but not AI-2 stimulates
expression of the locus of enterocyte effacement gene and thus provide evidence of
bacteria and host cross-talk during this infection (Walters and Sperandio, 2006).
Generally, QS Systems can be divided into three general classes based on the type
of autoinducer signal and the apparatus used for its detection (Figure II-10);
[1] Gram-negative bacteria with AHLs
[2] Gram-positive bacteria with AIPs
[3] AI-2 for the interspecies communication
Figure II-10. Representative signal molecules of bacteria QS.
31
II.2.2. Gram-Negative Bacteria with AHLs: Type AI-1 System
Acyl-homoserine lactones (AHLs) are a major autoinducer signal used by Gram-
negative bacteria for intraspecies QS. AHLs are composed of homoserine lactone
(HSL) rings carrying acyl chains of C4 to C18 in length (Fuqua et al., 2001). These
side chains harbor occasional modification at the third position or unsaturated double
bonds, as shown in Figure II-11 (Matthew TG Holden, 2007). The first AHL
autoinducer and its cognate regulatory circuit were discovered in the bioluminescent
marine bacterium Vibrio fischeri, which colonizes the light organ of the Hawaiian
Bobtail Squid Euprymna scolopes (Ruby, 1996, Ng and Bassler, 2009). In these
Gram-negative bacteria, QS circuits contain, at a minimum, homologs of two
regulatory protein called LuxI and LuxR. These two proteins are essential for QS
control of bioluminescence in V. fischeri. LuxI is the synthase of the QS autoinducer
N-3-(oxo-hexanoyl)-homoserine lactone (Schaefer et al., 1996b).
AHL synthesis by LuxI-homologue synthases generally proceeds via a
sequentially ordered reaction mechanism utilizing S-adenosylmethionine (SAM, 1)
as the amino donor for the formation of the homoserine lactone ring moiety and an
acylated carrier protein (ACP) as the precursor to the acyl side chain (Figure II-12).
(Galloway et al., 2011) The majority of studies on the chemical modulation of AHL
synthesis to date are based on the use of various analogues of SAM; for example, S-
adenosyl-homocysteine (SAH, 2), sinefungin (3), and butyryl SAM have proved to
be potent inhibitors of the P. aeruginosa AHL synthase RhlI in vitro, presumably
acting directly at the level of the synthase (Hentzer and Givskov, 2003, Parsek et al.,
1999). The general mechanism of LuxI/LuxR type QS system of gram-negative
bacteria is depicted in Figure II-13 (Fuqua and Greenberg, 2002). The LuxI/R
systems have been identified in over 25 species of Gram-negative bacteria, and these
bacteria were summarized at (Table II-2).
32
The most widely known LuxI/LuxR signaling bacteria is Pseudomonas
aeruginosa. Two pairs of LuxI/LuxR homologs, LasI/LasR (Passador et al., 1993)
and RhlI/RhlR (Brint and Ohman, 1995), exist in Pseudomonas aeruginosa. Both
LasI and RhlI are autoinducer synthases that catalyze the formation of 3-oxo-C12-
HSL (Pearson et al., 1994) and C4-HSL (Pearson et al., 1995), respectively. The two
regulatory circuits act in tandem to control the expression of a number of
Pseudomonas aeruginosa virulence factors. The Pseudomonas aeruginosa QS
circuit functions as follows. At high cell density, LasR binds its cognate AHL
autoinducer, and together they bind at promoter elements immediately preceding the
genes encoding a number of secreted virulence factors that are responsible for host
tissue destruction during initiation of the infection process. These pathogenicity
determinants include elastase, encoded by lasB; a protease encoded by lasA;
ExotoxinA, encoded by toxA; and alkaline phosphatase, which is encoded by the
aprA gene (Davies et al., 1998). Analogous to the Vibrio fischeri LuxI/LuxR circuit,
LasR bound to autoinducer also activates lasI expression, which establishes a
positive feedback loop (Seed et al., 1995). The LasR-autoinducer complex also
activates the expression of the second QS system of Pseudomonas aeruginosa.
Specifically, expression of rhlR is induced. RhlR binds the autoinducer produced by
RhlI; this complex induces the expression of two genes that are also under the control
of the LasI/LasR system, lasB, and aprA. Additionally, the RhlR-autoinducer
complex activates the second class of specific target genes. These genes include rpoS,
which encodes the stationary phase sigma factor; rhlAB, which encodes
rhamnosyltransferase and is involved in the synthesis of the biosurfactant/hemolysin
rhamnolipid; genes involved in pyocyanin antibiotic synthesis; the lecA gene, which
encodes a cytotoxic lectin; and the rhlI gene. Again, similar to LasI/LasR and
LuxI/LuxR, activation of rhlI establishes an autoregulatory loop.
33
Figure II-11. The molecular structure of each AHL autoinducer.
34
Figure II-12. (a) Schematic diagram illustrating the general features of the
AHL biosynthetic pathway. SAM (1) and acyl-ACP bind the AHL
synthase (LuxI-type synthase), whereupon acylation and
lactonization reactions occur. The AHL is then released, along
with the byproduct holo-ACP and 5′-methylthioadenosineis. (b)
Two SAM analogs, 2 and 3, they are known inhibitors of AHL
synthesis in P. aeruginosa (Parsek et al., 1999, Hentzer and
Givskov, 2003).
35
Figure II-13. Model of acyl-homoserine-lactone mediated QS in a single
generalized bacterial cell (Fuqua and Greenberg, 2002).
Table II-2. AHL-Dependent QS Systems in Gram-Negative Bacteria: The
Regulatory Phenotype and AHL (Matthew TG Holden, 2007).
Organism Phenotype Major AHLs
Aeromonas hydrophila Biofilms, exoproteases,
virulence
C4-HSL, C6-HSL
Aeromonas salmonicida Exoproteases C4-HSL, C6-HSL
Agrobacterium tumefaciens Plasmid conjugation 3-oxo-C8-HSL
Agrobacterium vitiae Virulence C14:1-HSL, 3-oxo-C16:1-
HSL
36
Burkholderia cenocepacia Exoenzymes, biofilm
formation,
Swarming motility,
siderophore,
virulence
C6-HSL, C8-HSL
Burkholderia pseudomallei Virulence, exoproteases C8-HSL, C10-HSL,
3-hydroxy-C8-HSL,
3-hydroxy-C10-HSL,
3-hydroxy-C14-HSL
Burkholderia mallei Virulence C8-HSL, C10-HSL
Choromobacterium violaceum Exoenzymes, cyanide,
pigment
C6-HSL
Erwinia carotovora Carbapenem, exoenzymes,
virulence
3-oxo-C6-HSL
Pantoea (Erwinia) stewartii Exopolysaccharide 3-oxo-C6-HSL
Pseudomonas aeruginosa Exoenzymes, exotoxins,
protein secretion, biofilm,
swarming motility, secondary
metabolites, 4-quinolone
signalling, virulence
C4-HSL; C6-HSL,
3-oxo-C12-HSL
Pseudomonas aureofaciens Phenazines, protease, colony
morphology, aggregation, root
colonization
C6-HSL
Pseudomonas chlororaphis Phenazine-1-carboxamide C6-HSL
Pseudomonas putida Biofilm formation 3-oxo-C10-HSL, 3-oxo-
C12-HSL
37
Table II-2. (Continued)
Organism Phenotype Major AHLs
Pseudomonas syringae Exopolysaccharide, swimming
motility, virulence
3-oxo-C6-HSL
Rhizobium leguminosarum
bv. Viciae
Root nodulation/symbiosis,
plasmid transfer, growth
inhibition, stationary phase
adaptation
C14:1-HSL, C6-HSL, C7-
HSL, C8-HSL, 3-oxo-C8-
HSL, 3-hydroxy-C8-HSL
Rhodobacter sphaeroides Aggregation 7-cis-C14-HSL
Serratia spp. ATCC 39006 Antibiotic, pigment,
exoenzymes
C4-HSL, C6-HSL
Serratia liquefaciencs MG1 Swarming motility,
exoprotease, biofilm
development, biosurfactant
C4-HSL, C6-HSL
Serratia marcescens SS-1 Sliding motility, biosurfactant,
pigment, nuclease,
transposition frequency
C6-HSL, 3-oxo-C6-HSL,
C7-HSL,
C8-HSL
Serratia proteamaculans B5a Exoenzymes 3-oxo-C6-HSL
Sinorhizobium meliloti Nodulation efficiency,
symbiosis, exopolysaccharide
C6-HSL, C12-HSL,
3-oxo-C14-HSL,
3-oxo-C16:1-HSL, C16:1-
HSL,
C18-HSL
Vibrio fischeri Bioluminescence 3-oxo-C6-HSL
Yersinia enterocolitica Swimming and swarming
motility
C6-HSL, 3-oxo-C6-HSL,
3-oxo-C10-HSL, 3-oxo-
C12-HSL, 3-oxo-C14-HSL
Yersinia pseudotuberculosis Motility, aggregation C6-HSL, 3-oxo-C6-HSL,
C8-HSL
38
II.2.3. Gram-Positive Bacteria with AIPs
Gram-positive bacteria also regulate a variety of processes in response to
increasing cell-population density. However, in contrast to gram-negative bacteria,
which use AHL autoinducers, gram-positive bacteria employ secreted peptides as
autoinducers for QS. In general, the autoinducing peptides (AIPs) are secreted via a
dedicated ATP-binding cassette (ABC) transporter as an autoinducer for QS. A
general model for QS in gram-positive bacteria is shown in Figure II-14 (Miller and
Bassler, 2001). In gram-positive bacteria, a peptide signal precursor locus is
translated into a precursor protein (black and white diamonds) that is cleaved (arrows)
to produce the processed peptide autoinducer signal (black diamond). Generally, the
peptide signal is transported out of the cell via an ABC transporter (gray protein
complex).
When the extracellular concentration of the peptide signal accumulates to the
minimal stimulatory level, a histidine sensor kinase protein of a two-component
signaling system detects it. The sensor kinase autophosphorylates on a conserved
histidine residue (H), and subsequently, the phosphoryl group is transferred to a
cognate response regulator protein. The response regulator is phosphorylated on a
conserved aspartate residue (D). The phosphorylated response regulator activates the
transcription of the target gene(s). Note that the lengths of the precursor and
processed peptides are not meant to signify any specific number of amino acid
residues.
39
Figure II-14. A general model for QS in Gram-positive bacteria. The oval
represents a bacterial cell. The “P” in the circle represents the
phosphorylation cascade (Miller and Bassler, 2001).
II.2.4. Interspecies Communication: Type AI-2 System
AHLs and peptides represent the two major classes of known bacterial QS
molecules, used by Gram-negative and Gram-positive bacteria, respectively, for
intraspecies communication. A family of molecules generically termed autoinducer-
2 (AI-2) has been found (Chen et al., 2002). It has been proposed that AI-2 is an
interspecies signal molecule among Gram-negative and Gram-positive bacteria
(Figure II-15).
Bassler and co-workers first identified AI-2-based QS mechanism of Vibrio
harveyi in the early 1990s (Bassler et al., 1993, Bassler et al., 1994). It was observed
that an AHL-deficient strain of the bacterium remained capable of producing
bioluminescence even in the absence of the AHL autoinducer. This suggested that a
second QS pathway, employing a different signaling molecule, was operating. This
novel autoinducer, whose structure at the time was unknown, was termed AI-2. It
was subsequently shown that cell-free culture fluids from some bacterial species
40
were capable of stimulating activity in a V. harveyi AI-2 reporter strain (Bassler et
al., 1997). This suggested that the AI-2 signal may be produced by numerous
bacterial species. Later work demonstrated that the same gene was responsible for
AI-2 biosynthesis in V. harveyi, E. coli, and S. typhimurium (Surette et al., 1999).
This gene, designated luxS, has since been found in over 70 bacterial species
(Lowery et al., 2008a). These observations have led to the proposal that AI-2 is a
universal signaling molecule for interspecies communication. It should be noted that
the product of the luxS gene, the enzyme LuxS, is thought to have a metabolic role
in cells, in addition to being responsible for AI-2 biosynthesis (Lowery et al., 2008a,).
This may provide an alternative explanation for the widespread conservation of luxS.
Despite this controversy, there is a growing body of evidence that AI-2 does indeed
represent a universal language for interspecies communication.
DPD: 4,5-dihydroxy-2,3-pentanedione
DHMF: 2,4-dihydroxy-2-methyldihydrofuran THMF: 2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran
Figure II-15. Chemical structures of representative AI-2 molecules. DPD and
its derivatives are possible in water and in the presence of
borate (Camilli and Bassler, 2006).
41
II.2.5. Other QS Systems
At least, two additional QS systems have been identified in gram-negative bacteria.
These include autoinducer 3 (AI-3), which is associated with virulence regulation in
EHEC O157:H7 and the Pseudomonas quinolone signal (PQS), which is associated
with Pseudomonas aeruginosa (Mashburn and Whiteley, 2005).
AI-3 is associated with luxS homologs in EHEC O157:H7, but this signal itself is
hydrophobic and thus chemically distinct from the polar AI-2 signals. AI-3 is also
biologically distinct from AI-2. During EHEC pathogenesis, both AI-3 and host
epinephrine, but not AI-2 stimulates expression of the ‘locus of enterocyte
effacement (LEE)’ genes and thus provide evidence of bacteria and host cross-talk
during this infection (Walters and Sperandio, 2006).
A novel, additional autoinducer has been demonstrated to be involved in QS in
Pseudomonas aeruginosa. This signal is noteworthy because it is not of the
homoserine lactone class. Rather, it is 2-heptyl-3-hydroxy-4-quinolone (denoted
PQS for Pseudomonas quinolone signal) (Pesci et al., 1999). PQS partially controls
the expression of the elastase gene lasB in conjunction with the Las and Rhl QS
systems. The expression of PQS requires LasR, and PQS, in turn, induces
transcription of rhlI. These data indicate that PQS is an additional link between the
Las and Rhl circuits. The notion is that PQS initiates the Rhl cascade by allowing
the production of the Rhl-directed autoinducer only after the establishment of the
LasI/LasR signaling cascade. PQS molecules are quite hydrophobic and have been
shown to be transported between cells by outer membrane vesicles. There is also
strong evidence that the PQS induces the formation of these vesicles through
interference with Mg2+ and Ca2+ ions in the outer membrane. In a recent review, it
was suggested that membrane vesicles might represent a mechanism for
interkingdom signaling in the plant rhizosphere.
42
II.2.6. QS Regulated Biofilm Formation
In general, biofilm cells encounter much higher local cell densities than free-
floating, planktonic cell populations. An obvious consequence of this is the elevated
levels of metabolic by-products, secondary metabolites and other secreted or
excreted microbial factors that biofilm cells encounter. Of particular interest is
intercellular signaling or QS molecules. Because biofilm generally consists of
aggregates of cells, one could argue that they present an environmentally relevant
context for QS. This idea that the biofilm is optimum sites for expression of
phenotypes regulated by QS has led to numerous studies of QS mechanism in the
bacterial biofilm including its various phenotypes. The maturation of a biofilm
community occurs downstream of adherence. Several factors have been shown to
influence biofilm architecture, including motility, homogeneity of microorganisms,
extracellular polymeric substance matrix production and rhamnolipid production
(Klausen et al., 2003, Hentzer et al., 2001, Davey et al., 2003).
AHL-based QS has been shown to influence biofilm maturation for the gram-
negative bacterium Serratia liquefaciens (Labbate et al., 2004). QS Regulates
swarming motility in S. liquefaciens (Eberl et al., 1996). Wild-type S. liquefaciens
biofilms are heterogeneous, consisting of cell aggregates and long filaments of cells.
A mutation in the AHL synthesis gene, swrI, resulted in thin biofilms that lacked
aggregates and filaments. Two regulated genes, bsmA, and bsmB, were implicated
in biofilm development. The AhyI/R AHL QS system of Aeromonas hydrophila has
also been shown to be required for biofilm formation (Lynch et al., 2002). A strain
harboring an ahyI mutation formed biofilms that were structurally less differentiated
than the wild-type strain. For all three of the systems mentioned, the functional
consequence of this altered architecture is unclear. According to Pseudomonas
aeruginosa biofilm inhibition test, it has revealed that QS is crucial for proper
43
biofilm formation. Specifically, Pseudomonas aeruginosa lasI mutants do not
develop into mature biofilms. Rather, they terminate biofilm formation at the micro-
colony stage (Davies et al., 1998). These mutants can be complemented to wild-type
biofilm production by the exogenous addition of the LasI-dependent 3-oxo-C12-
HSL autoinducer (Figure II-16).
There is growing evidence that QS constitutes a global regulatory system in many
different parts. Many studies have shown that QS affects the biofilm development of
several bacterial species. For example, In Pseudomonas aeruginosa (Parsek and
Greenberg, 2000) (Figure II-17), Burkholderia cepacia, and Aeromonas hydrophila,
are known to require a functional AHL-mediated QS system for formation of
biofilms (Davies et al., 1998, Huber et al., 2001, Lynch et al., 2002). The biofilm
formation control by inhibiting QS signal molecules will be described in more detail
in the next section.
Figure II-16. Epifluorescence and scanning confocal photomicrographs of
the WT and the lasI mutant Pseudomonas aeruginosa biofilms
containing the GFP expression vector pMRP9-1 (Davies et al.,
1998).
44
Figure II-17. Diagram of the P. aeruginosa biofilm-maturation pathway.
Unattached cells that approach a surface may attach.
Attachment involves specific functions. Attached cells will
proliferate on a surface and use specific functions to actively
move into micro-colonies. The high-density micro-colonies
differentiate into mature biofilms by a 3-oxo-C12-HSL-dependent
mechanism (Parsek and Greenberg, 2000).
II.3. QS Control Strategy
II.3.1. Three-Point of QS Inhibition Strategies
Bacteria can use QS to coordinate their group behaviors which are biofilm
formation, swarming, motility, production of extracellular polysaccharides, etc. (Li
et al., 2007, Ng and Bassler, 2009, Lowery et al., 2008a). Also, QS can occur within
single species bacteria community as well as interspecies bacteria community. The
QS mechanism is achieved by producing, releasing, and detecting small signal
molecules known as AHLs, AIPs, and AI-2. These signal molecules are synthesized
by generator protein which is called LuxI homolog: AHLs, Precursor protein: AIPs
and LuxS: AI-2. Moreover, the signal molecule made this way is perceived receptor
proteins LuxR homolog: AHLs, Sensor kinase: AIPs and LuxP homolog: AI-2.
Therefore, QS systems generally offer three points of attack: the signal generator,
the signal molecule and the signal receptor (Rasmussen and Givskov, 2006, Roy et
45
al., 2011, Yeon, 2009). Therefore, QS inhibition strategy can be divided into
blockage of signal molecule synthesis, interference with signal receptor and
inactivation of signal molecule. Figure II-18 shown three strategies to control the
AHL type QS system.
Figure II-18. Three strategies to control LuxI/R type QS system (Yeon, 2009).
II.3.2. Quorum Sensing Inhibitor (QSI) for AI-1 Regulation
II.3.2.1. Blockage of AHL Synthesis: Inhibition of Signal Generator
Parsek et al. have found that analogs of AHL building blocks such as holo-ACP,
L/D-S-adenosyl homocysteine, sinefungin and butyryl-S-adenosyl methionine
(butyryl-SAM) were able to block AHL production in vitro (Parsek et al., 1999).
However, none of them has been tested on bacteria in vivo and how these analogs of
AHL building block, SAM, and acyl-ACP, which are also used in central amino acid
and fatty acid catabolism, would affect other cellular functions is presently unknown.
Also, Rudrappa et al. have reported that curcumin inhibits PAO1 virulence factors
such as biofilm formation, pyocyanin biosynthesis, elastase/protease activity, and
46
AHL production. However, the exact inhibition mechanism of curcumin was not
revealed (Rudrappa and Bais, 2008).
II.3.2.2. Interference with Signal Receptor
The most widely used interference method is to block the receptor with an analog
of the AHL. AHL analogs can be substituted in either the side chain or the ring moiety.
Analogs of the 3-oxo-C6 HSL molecule with a different substituent in the side chain
can displace the native signal from the LuxR receptor. These compounds exhibit
agonists effects which limit their use as QS inhibitor (Schaefer et al., 1996a). If the
C-2 atom in the side chain is replaced by a sulfur atom, it will produce a potent
inhibitor of both the lux and las systems (Persson et al., 2005). Likewise, if the C-1
atom is replaced by a sulfonyl group, a QS inhibitor is also generated (Castang et al.,
2004). Another strategy to modify the AHL signal molecules is to place atoms or
groups at the end of the side chain. Substituting secondary alkyl groups at the C6
atom of 3-oxo-C6 HSL gives rise to agonists, whereas positioning of a secondary
aryl group on that location gives rise to an antagonist. Instead of substitutions at acyl
side chain, the entire ring can be exchanged with another cyclic structure. For
example, 3-oxo-C12-(2-aminocyclohexanone) is an inhibitor of the LasR-based QS
system. It can down-regulate LasR dependent expression of LasI AHL synthase
(Reverchon et al., 2002). Ishida et al. (Ishida et al., 2007) have synthesized a series
of structural analogs of N-octanoyl cyclopentylamide with 4~12 carbon. They also
have reported that N-decanoyl cyclopentylamide inhibited production of virulence
factors, including elastase, pyocyanin, rhamnolipid, and biofilm formation without
affecting the growth of Pseudomonas aeruginosa PAO1.
In case of natural product, vanillin (4-hydroxy-3-methoxybenzaldehyde) extracted
from vanilla beans was reported to inhibit QS signaling systems by interference with
47
AHL receptor. This compound was found to inhibit the short- and long-chain AHL-
mediated QS systems, leading up to 46.3% reduction of biofilm of Aeromonas
hydrophila isolated from a biologically fouled RO membrane on the polystyrene
surface. These, in turn, indicate that vanillin would be able to prevent RO membrane
from biofouling (Ponnusamy et al., 2009, Kappachery et al., 2010).
In a recent screening of 50 Penicillium species grown on different growth media,
a remarkably high fraction, 66% were found to produce secondary metabolites with
QS inhibition activity. Two of the compounds were identified as penicilic acid and
patulin produced by Penicillium radicicola and Penicillium coprobium, respectively.
Also, many plant species secrete mimic AHL signals. Interestingly, plant-derived
AHL mimics include substances that both stimulate and inhibit bacterial QS systems.
For instance, components of pea seedling exudates inhibited AHL induced violacein
synthesis in Chromobacterium violaceum, induced swarming activity in Serratia
liquefaciens MG44, which is defective in AHL synthesis, and induced luminescence
in Escherichia coli reporters containing plasmids encoding either LuxR from Vibrio
fischeri, AhyR from Aeromonas hydrophila, or LasR from Pseudomonas aeruginosa
(Teplitski et al., 2000). In addition, extracts from rice, soybean, tomato, crown vetch,
and Medicago truncatula all contain AHL mimics (Mathesius et al., 2003, Teplitski
et al., 2004), (Gao et al., 2003). The unicellular green alga Chlamydomonas
reinhardtii also produces substances that interfere with bacterial QS systems
(Teplitski et al., 2004).
II.3.2.3. Inactivation of AHL Signal Molecules
Three quorum-quenching enzymes (QQ) are known to interfere with bacterial QS
molecules. It is presumable that four potential cleavage sites in the QS signal
molecule AHLs are likely to cut off enzymatically based on the AHL structure, as
48
shown in Figure II-19 (Chen et al., 2013). The enzymes catalyzing AHL degradation
can be divided into two groups: one that leads to the degradation of the homoserine
lactone ring mediated by lactonase or decarboxylase and one that causes the cleavage
of AHL to a homoserine lactone and a free fatty acid moiety by acylase or deaminase.
Only two enzyme families in the microorganism have the capability of cutting AHL
structures; the AiiA-like AHL-lactonases and the AiiD-like AHL-acylases have been
demonstrated to be involved in the real cleavage of the QS signal molecules,
although a large diversity of QQ microbes have been identified (Dong et al., 2000,
Leadbetter and Greenberg, 2000, Oh et al., 2013). The other two types of enzymes
have not been identified. An oxidoreductase was included in QQ by substituting the
oxo-group at C3 with the hydroxyl group, which may successively be degraded by
amidohydrolase to form homoserine lactone and hydroxydecanoic acid (Uroz et al.,
2005, Chen et al., 2013). Although the role of those QQ enzymes in their native
environments is not always clear, their QQ ability and utility in potential industrial
and therapeutic applications are promising.
Another simple way to achieve inactivation of the AHL signal molecules is by
increasing the pH to above 7.0 (Yates et al., 2002). This causes ring opening of the
AHL (lactonolysis). A number of higher organisms employ this strategy in defense
against invading QS bacteria. When some plants are infected with Erwinia
carotovora, causing the tissue-macerating plant pathogen, the plants will increase
pH as a first response for attacking the virulence microorganisms by inactivation of
QS signal molecules and blocking expression of QS-controlled genes (Byers et al.,
2002).
49
Figure II-19. Possible linkage degraded by QQ enzymes in quorum sensing
molecule (a) N-acyl homoserine lactone and (b) corresponding
degradation mechanism of QQ enzymes (Chen et al., 2013).
II.3.3. Reporter Strain to Detect QS Signal and Screening QSI
One of the most important issues for developing QSI is its detection. Therefore,
AHL reporter strains were developed over a period of time by many researchers (
Table II-3). These reporter strains allow sensitive, quantitative and real-time
detection of QS signals such as AHLs. In most of the reporter strains known so far,
the QS-regulated promoter is fused to the lux operon or lacZ. Although these reporter
strains have a functional regulator protein, they lack the AHL synthase enzyme. The
promoter activity gets induced by exogenous QS signals. Thus, the receptor gets
activated by the presence of AHLs which binds to its cognate LuxI promoter and
50
initiates the expression of certain genes. The expression of the relevant genes is
proportional to the concentration of the signal molecules (Winson et al., 1998, Swift
et al., 1997).
Chromobacterium violaceum has high sensitivity for QS signal compounds with
4-6 carbon acyl side chains, E. coli harboring pSB410 is effective for 6-8 carbon side
chains and pSB1075 is sensitive for detecting AHLs with 10-14 carbon side chains
lengths (Winson et al., 1998, McClean et al., 1997). The inability of C. violaceum
CV026 biosensor to detect 3-hydroxy derivatives of AHL can prove helpful in
elucidating potential cases where P. fluorescens may be present (Cha et al., 1998).
Another equally effective biosensor for long-chain AHL inhibitor screening is
Agrobacterium tumefaciens NT1 (traR, tra::lacZ749). It contains a lacZ fusion in the
tra1 gene of pTiC58, which is induced to produce the enzyme β-galactosidase. The
degradation of X-gal results in the appearance of blue color. The best part of this
biosensor strain is its ability to respond to a wide range of AHLs at very low
concentrations (Shaw et al., 1997).
Table II-3. Bacterial Reporter Strains Used to Detect QS Signals.
Reporter Strain QS signal detected Phenotype
Agrobacterium tumefaciens A136
[traI-lacZ fusion
(pCF218)(pCF372)]
C6-HSL to C14-HSL β-galactosidase
activity
51
A. tumefaciens strain NT1
(pDCI41E33 containing a
traG::lacZ fusion)
AHLs with 3-oxo-, 3-
hydroxy-, and 3-unsubstituted
side chains of all lengths,
(C6-HSL to C14-HSL) with
the exception of C4-HSL
β-galactosidase
activity
Chromobacterium violaceum strain
CV026–CviR receptor Wide host range of AHLs
Violacein
pigment
production
Escherichia coli plasmid carrying
a luxCDABE cassette activated by
AhyRI' receptor of Aeromonas
hydrophila (pSB536)
C4-HSL Bioluminescent
E. coli plasmid carrying a
luxCDABE cassette activated by
AhyR receptor of A. hydrophila
(pSB403)
Wide host range of AHLs Bioluminescent
E. coli JM109 plasmid carrying a
luxCDABE cassette activated by
LuxR receptor of Vibrio fischeri
(pSB401)
C6-HSL Bioluminescent
E. coli JM109 plasmid carrying a
luxCDABE cassette activated by
the LasR receptor of Pseudomonas
aeruginosa (pSB1075)
C12-HSL Bioluminescent
E. coli JM109 plasmid carrying a
luxCDABE cassette activated by
RhlR receptor of P. aeruginosa
C4-HSL Bioluminescent
52
Pseudomonas aurofaciens strain
30-84I C6-HSL
Phenazine
antibiotic
production
Pseudomonas putida 117(pAS-
C8)-CepR receptor C8-HSL
Green
Fluorescent
Protein
P. putida IsoF∕gfp 3-oxo-C12-HSL Fluorescence
Serratia liquefaciens strain MG44 C4-HSL Biosurfactant
production
S. liquefaciens strain PL10 -
LuxAB reporter C4-HSL Bioluminescent
Sinorhizobium meliloti Rm41
sinI::lacZ (pJNSinR) C16-HSL to C20-HSL
β-galactosidase
activity
II.4. Immobilization Technique for Biocatalyst
Modern developments in biotechnology have paved the way for the widespread
application of biocatalysis in industrial synthesis. Especially, DNA recombinant
technique has been possible to produce the most enzyme or probiotic bacteria for a
commercially acceptable price. Nonetheless, practical application is often hampered
by a lack of long-term stability and difficult recovery of the enzyme or probiotic
bacteria. These drawbacks can often be overcome by immobilization. Therefore, the
main purpose of an enzyme or whole-cell immobilization is the reuse of enzymes for
repetitive reaction cycle. This can greatly improve the economics of a process.
53
II.4.1. Enzyme Immobilization Method
Improvement of enzyme property has to be strongly associated with the design of
protocols for enzyme immobilization (Guisán, 2006). Despite their excellent
catalytic properties, enzymes are not suitable for their use in industrial engineering:
low stability, substrates and products inhibition, low activity and recovery of waste
materials (Cassidy et al., 1996). The simple enzyme immobilization method was
studied to overcome these unsuitable characteristics of the enzyme. Also, enzyme
immobilization is to increase enzyme activity or stability especially under denaturing
conditions (Clark, 1994, Klibanov, 1979). Thermal stability can often be improved
by many orders of magnitude compared to the soluble enzyme (Kawamura et al.,
1981, Mozhaev et al., 1983, Mozhaev, 1993). Another important advantage of
immobilization is the possibility to apply various pH conditions. Lastly, enzyme
immobilization is that it enables the use of enzymes in multi-enzyme and chemo-
enzymatic cascade processes (Sheldon, 2007). Three conventional methods of
enzyme immobilization can be distinguished, binding to a support binding,
entrapment (encapsulation) and cross-linking.
II.4.1.1. Support Binding
Support for binding can be van der Waals interactions, ionic bonding, or covalent
bonding. In this case, the support can mostly use a synthetic resin, a biopolymer or
an inorganic polymer such as mesoporous silica or zeolite. The properties of
supported enzyme preparations are governed by the properties of both the enzyme
and the carrier material. The interaction between the two provides an immobilized
enzyme with specific chemical, biochemical, mechanical and kinetic properties.
Enzymes contain a number of functional groups capable of covalently binding to
supports. Table II-4 lists these groups along with their relative frequency in a typical
54
protein. Of the functional groups of enzymes listed, -NH2, -CO2H, and -SH are most
frequently involved in covalent immobilization.
Acrylic resins such as Eupergit C are widely used as supports. Eupergit C is a
macroporous copolymer of N,N’-methylene-bi-(methacrylamide), glycidyl
methacrylate, allyl glycidyl ether and methacrylamide with epoxy groups can be
rendered inactive by capping using a variety of reagents to prevent any undesired
support-protein reaction. Due to the high density of oxirane groups on the surface of
the beads enzymes are immobilized at various sites of their structure. This “multi-
point-attachment” is largely responsible for the high operational stability of enzymes
bound to Eupergit C (Yildirim et al., 2013).
Various porous acrylic resins, such as Amberilte XAD-7, are used to immobilize
enzymes via simple adsorption without covalent attachment. For instance, the widely
used enzyme C. antarctica lipase B is commercially available in immobilized form
as Novozym 435 which consists of the enzyme adsorbed on a macroporous acrylic
resin (Giraldo et al., 2007). A disadvantage of immobilization in this way is that,
because it is not covalently bound, the enzyme can be leached from the support in
an aqueous medium (Sheldon, 2007).
Table II-4. The Reactive Functional Group in the Enzyme.
Reactive group Chemical structure
ε-Amino of lysine and N-terminus
Carboxylate of glutamic acid, aspartic
acid, and C-terminus
Thiol of cystein
NH2
COOH
SH
55
Phenolic of tyrosine
Guanidino of arginine
Imidazole of histidine
Disulfide of cystine
Indole of tryptophan
Thioether of methionine
Hydroxyl of serine and threonine
II.4.1.2. Entrapment (Encapsulation)
Entrapment is the inclusion of an enzyme in a polymer network such as an organic
polymer, a silica sol-gel or a microcapsule. The physical restraints generally are too
weak, however, to prevent enzyme leakage entirely. Hence, the additional covalent
attachment is often required. Generally, entrapment requires the synthesis of the
polymeric network in the presence of the enzyme.
Enzymes can be immobilized by entrapment in sol-gel matrices formed by
hydrolytic polymerization of metal alkoxides. Immobilization in silica sol-gels
prepared by hydrolytic polymerization of tetraethoxysilane, in the presence of the
OH
NH
C
NH
NH2
NN
S S
N
CH2
S
CH2OH
56
enzyme, was pioneered by Avnir and co-workers and has been used for the
immobilization of a wide variety of enzymes (Sertchook and Avnir, 2003).
Enzymes can also be entrapped in silicone elastomers and polydimethylsiloxane
membranes. Kobayashi and co-workers have described a novel polymer-
incarceration methodology for immobilizing enzymes (Kobayashi et al., 1992).
Also, entrapment can be achieved by mixing an enzyme with a polyionic polymer
material and then crosslinking the polymer with multivalent cations in an ion-
exchange reaction to form a lattice structure that traps the enzymes/cells (ionotropic
gelation) (Bonilla et al., 1983). Temperature change is a simple method of gelation
by phase transition using 1% to 4% solutions of agar or gelatin. However, the gels
formed are soft and unstable. A significant development in this area has been the
introduction of x-carrageenan polymers that can form gels by ionotropic gelation and
by temperature-induced phase transition, which has introduced a greater degree of
flexibility in gelation systems for immobilization.
II.4.1.3. Cross-linking
Cross-linking method used bifunctional reagents. There is an increasing interest
in carrier-free immobilized enzymes to prevented mass-transfer resistant, such as
cross-linked enzyme aggregates. This approach offers clear advantages: highly
concentrated enzyme activity in the catalyst, high stability and low production costs
owing to the exclusion of an additional (expensive) carrier.
In the early 1960s, studies of solid phase protein chemistry led to the discovery
that cross-linking of dissolved enzymes via reaction of surface -NH2 groups with a
bifunctional chemical cross-linker, such as glutaraldehyde, afforded insoluble cross-
linked enzymes with retention of catalytic activity (Sheldon, 2007). Mechanical
stability and ease of handling could be improved by cross-linking the enzyme in a
57
gel matrix or on a carrier, but this led to the disadvantageous dilution of activity
mentioned above. Consequently, in the late 1960s, the emphasis switched to carrier-
bound enzymes, which became the most widely used industrial methodology for
enzyme immobilization for the next three decades.
In detail, the most common way is through the use of difunctional reagents such
as diimidate ester, diisocyanates, and dialdehyde. Glutaraldehyde (GA) is often used
as it is one of the lease expensive difunctional reagents available in bulk. This reagent
reacts complexly to form Schiff bases with amine groups on the support and
produces pendent Aldehydes and α,β–unsaturated carbonyl functionalities through
which enzymes may attach. Enzyme attachment is simply accomplished by mixing
the enzyme with the activated support. A simplified example of this was shown in
Figure II-20. The acid-labile Schiff based can be reduced to more stable secondary
amine bonds with sodium borohydride to increase the stability of the enzyme-support
linkage.
Figure II-20. Activation of amine-bearing support with glutaraldehyde
followed by enzyme coupling.
H H
O O
NH2 + HN
O
HN
O
NN
Activated supportAmine containing support
Immobilized enzyme
NH2Enzyme
Enzyme
H H
O O
NH2 + HN
O
HN
O
HN
O
HN
O
NN
Activated supportAmine containing support
Immobilized enzyme
NH2EnzymeNH2EnzymeEnzyme
EnzymeEnzyme
58
II.4.2. Nanobiocatalysis
‘Nanobiocatalysis’ can be defined as the incorporation of the enzymes into
nanostructured materials such as nanoporous materials, electrospun nanofibers and
magnetic nanoparticles (Kim et al., 2008a, Auriemma et al., 2017) (Figure II-21).
This application gathered attention by immobilizing enzymes onto a high surface
area of nanostructured materials. This large surface area resulted in improved
enzyme loading, which in increasing the apparent enzyme activity per unit mass or
volume compared to that of enzyme systems immobilized onto conventional
materials.
Most techniques for obtaining nanoparticles that contain enzymes have been based
on the so-called ‘nano entrapment’ approach using the water-in-oil microemulsion
system (reverse micelles), which leads to discrete nanoparticles through
polymerization in the water phase or water–oil interface (Yang et al., 2004). In 2003,
a new synthetic approach was reported under the name of ‘single enzyme
nanoparticles (SENs),’ in which an organic-inorganic hybrid polymer network of a
thickness of less than a few nanometers was built up from the surface of the enzyme
(Kim and Grate, 2003).
Enzyme aggregate coating combines covalent enzyme attachment on various
nanomaterials with enzyme crosslinking, leading to an increase in enzyme loading,
overall enzyme activity, and enzyme stability. Figure II-21 schematically shows the
assembly of such an enzyme coating on the surface of electrospun polymer
nanofibers. In a first step, enzyme molecules are covalently attached to the surface
of nanofibers and serve as ‘seed’ sites. The second step involves the addition of
further enzyme molecules and their crosslinking to the seed enzyme molecules,
thereby leading to a crosslinked enzyme aggregate coating. This approach has
successfully been applied for various nanomaterials, including nanofibers (Nair et
59
al., 2007), carbon nanotubes (Fischback et al., 2006) and magnetic nanoparticles
(Lee et al., 2008b). These nanomaterials provide a large surface area for enzyme
immobilization, leading to high enzyme loading, which is further increased by one
to two orders of magnitude by multiple-layer enzyme coating. The high degree of
enzyme stabilization observed suggests that the enzyme coating is tightly bound to
the surface of nanomaterials and resistant to washing, even under rigorous shaking
conditions, thereby providing excellent operational stability. The intrinsic enzyme
stability was also improved by multiple covalent linkages, preventing the enzyme
denaturation (Govardhan, 1999).
(a)
(b)
Figure II-21. (a) Assembly of enzyme aggregate coating on electrospun
nanofibers (Kim et al., 2008a). (b) Nano-in-Nano approach for
enzyme immobilization based on block copolymer (Auriemma et
al., 2017).
II.4.3. Whole-Cell Immobilization Method
The immobilized whole-cell technique is an alternative to enzyme immobilization.
Unlike enzyme immobilization, where the enzyme is attached to a solid support, in
60
immobilized whole-cell technique, the target cell is immobilized. Whole-cell
immobilization methods may be applied when the enzymes required are difficult or
expensive to extract, an example being intracellular enzymes. Furthermore, the
growth of live cells, when immobilized, can be of value in some instances such as
MBR process. Therefore, whole cell immobilization may be used for convenience in
an industrial process. Most of the immobilized cells have been used in bioreactors
and production of useful compounds such as amino acids, organic acids, antibiotics,
steroids (Brodelius, 1987). Various whole cell techniques (Akin, 1987) and the many
applications (Akin, 1987, Coughlan and Kierstan, 1988) possible have been
examined. Whole-cell immobilization describes many different forms of cell
attachment or entrapment. These different forms include flocculation, adsorption on
surfaces, covalent bonding to carriers, cross-linking of cells, encapsulation in a
polymer-gel, and entrapment in a matrix.
In these things, the whole-cell immobilized in a hydrogel matrix can be protected
from harsh environmental conditions such as pH, temperature, an organic solvent,
and poison. Also, immobilized cells can be handled more easily and recovered from
the solution without difficulty.
II.4.3.1. Bead Entrapment
Cells have generally been entrapped in the hydrogel matrix through which
substrates and products diffuse easily. The hydrogel matrix is composed of agar,
agarose, carrageenan, collagen, alginate, chitosan and cellulose (Jen et al., 1996,
Lother and Oetterer, 1995). Calcium alginate is widely used for the entrapment of
animal cells, microbial cells, mitochondria, chloroplasts, protoplasts and red blood
cells (Jen et al., 1996). Calcium alginate gelled by ionic bond swells in the solution
and dissolves in a solution containing a chelating agent such as phosphate. Post-
61
treatment of the calcium alginate bead with triethylenetetramine and glutaric
dialdehyde increases mechanical strength and prevents the bead from swelling and
dissolving (Yotsuyanagi et al., 1990).
Polyvinyl alcohol, which is cheap and non-toxic to cells, is not mechanically
strong. Cross-linking the bead of polyvinyl alcohol with boric acid solution makes
the bead strong and durable but damages cells in a lengthy cross-linking mechanism
(de Melo et al., 2007, Wu and Wisecarver, 1992). However, a drawback of the
method for preparing conventional PVA–boric acid beads is that microorganisms
enclosed in the PVA matrix are drastically damaged by boric acid during the bead
preparation process (Wu and Wisecarver, 1992). In the present study, sodium sulfate
as an inducer for cross-linkage of PVA was utilized to avoid the drastic decrease in
cell viability caused by the saturated boric acid solution. (Hill et al., 2002, Lim et al.,
2000). Addition of some alginate or phosphate to the boric solution during the cross-
linking procedure prevents the polyvinyl alcohol beads from aggregation (Wu and
Wisecarver, 1992).
II.4.3.2. Encapsulation inside porous membrane carrier
The second major category of cell immobilization is encapsulation within a porous
matrix which is a semi-permeable membrane. Cells entrapped in beads leak, escape
from the gel matrix, and grow in the medium solution (Kuhn, 1988, Celik et al.,
2017). Cells grow mostly on the surface and in the pore of the matrix where the space
available for the cell growth. The cells immobilized in a large bead proliferate only
in the side-line of the bead because of the substrate and oxygen limitation (Park and
Chang, 2000). The maximum cell loading in the entrapped beads is limited to 25%
by volume because of weak mechanical strength (Buchholz and Klein, 1987). To
overcome these problems, cells were encapsulated in porous polymer materials.
62
Encapsulation techniques have become attractive since Lim developed the pre-gel
dissolving two-step method in 1980 (Lim and Sun, 1980). Mammalian cells have
been successfully grown in polylysine enclosed droplets suspended in the culture
broth. The semi-permeable membrane allows nutrients to diffuse to the cells but
retains the cells and some of the higher molecular weight products produced by the
cells. It has been reported that bacterial cells have been similarly immobilized.
According to Cheong et al., the ultimate dry cell density in the capsule reached 310
g/L by the inner volume of the capsule (Cheong et al., 1993). The monoclonal
antibodies produced by hybridoma cells entrapped in the alginate beads leaked from
the hydrogel, but those from the encapsulated cells stayed inside the capsule without
leaking (King et al., 1987, Duff, 1985). The encapsulated cells grew, and the dry cell
weight reached a limiting value, but the cells entrapped in the gel matrix burst the
beads at the end (Duff, 1985).
II.4.4. Industrial Application Using Immobilization Technique
Over the last 30 years, a number of protocols for the immobilization of cells and
enzymes have been reported in scientific literature. However, only very few
protocols are simple and useful enough to improve significantly the functional
properties of enzymes and cells, activity, stability and selectivity. In addition, the
case of industrial application may be as few as these limitations of carrier supports.
In the early 1980s, encapsulation technology was applied to animal cells as the
commercial ENCAPCEL process (Encapsulation of biological material, Lim F., US
Patent 4-352-883, 1982). The molecular weight cut-off of the capsule membrane
produced by Lim’s two-step method (commercial name ENCAPCEL process) was
60,000, and the cells and monoclonal antibodies were enclosed inside the capsule
(Lim and Sun, 1980). The concentration of the monoclonal antibodies accumulated
63
inside the capsule reached up to 1,250 g/mL, and the cell density was 23,107
cells/mL. Conventional suspension culture required a large reactor of 5000 L to
produce 20 g of antibody, but the ENCAPCEL microencapsulation process needed
only a small 40 L reactor to produce the same quantity of antibody in 2 or 3 weeks.
In case of entrapment in hydrogel particles process (using polyvinylalcohol bead),
LentiKats is known to the successful industrial application (Figure II-22) (Guisan,
2006). The LentiKats process is shown by some examples of successful
immobilization of whole-cells and enzymes. In detail, bioconversion of raw glycerol
to 1,3-propanediol, bioethanol production from nonsterile molasses, reduction of
energy consumption for sewage treatment, and production of (R)-cyanohydrin by
using entrapped (R)-oxynitrilase in LentiKats. Correctly stabilized LentiKats tolerate
a maximum temperature of 50 to 55 °C, and pH values between 3.1 and 8.5 were
tested for several days or weeks without signs of disintegration of LentiKats.
64
(a)
(b)
Figure II-22. Schematic diagram: (a) Preparing steps of LetiKats and (b) its
application method for wastewater treatment process (Source:
LentiKat`s Biotechnologies, http://www.lentikats.eu/en/).
II.5. Quorum Quenching (QQ) Application to MBR
The concept of bacterial QS has been proposed to novel biofouling inhibition
strategy in MBR (Yeon et al., 2009a). QS-based strategies are reported to offer the
advantages of higher efficiency, lower toxicity, more sustainability and less bacterial
resistance over other conventional biofouling control approaches. Since 2009, many
researchers have started to study on the biofouling control by QQ in membrane
process for water treatment.
65
II.5.1. Enzymatic QQ Application to MBR
Yeon et al. showed that membrane biofouling could be efficiently removed by the
addition of porcine kidney acylase I (Yeon et al., 2009a). Moreover, the enzyme
prevents MBR biofouling by quenching AHL autoinducers. In detail, the study
demonstrated the evidence of QS signal molecules in MBR and could correlate QS
with membrane fouling. The enzyme can inactivate AHL by amide bond cleavage as
an enzymatic QQ method as shown in Figure II-19. After having proven the
feasibility of QQ by free enzymes in a lab-scale MBR, they have overcome the
technological limitations of using free enzymes by applying magnetic enzyme
carriers (MEC) (Yeon et al., 2009b). When MEC was applied to MBR in a
continuous operation, it enhanced the membrane permeability to a large extent
compared with a conventional MBR with no enzyme. Later studies confirmed that
MEC prevented the membrane biofouling in different MBR operating conditions
(Kim et al., 2013a). They investigated the changes in population dynamics and gene
expression in the MBR with MEC by using pyrosequencing and proteomics.
Meanwhile, using enzymatic QQ approach is that it only influences sludge
characteristics and biofouling, while not impacting pollutant degradation. Jiang et al.
showed that enzymatic QQ application enhanced membrane permeability with no
apparent effects on effluent quality of MBR. Also, QQ reduces the production of
polysaccharides and proteins and reduced viscosity and relative sludge
hydrophobicity (Jiang et al., 2013).
As other enzymatic QQ applications to membrane process, AHL-acylase directly
immobilized onto nanofiltration (NF) membrane by Kim et al. (Kim et al., 2011).
Acylase immobilized NF membrane shown to prohibit the formation of mushroom-
shaped mature biofilm due to reduced EPS secretion.
66
II.5.2. Bacteria Strains with QQ Enzyme
By development in the science of genetics, several AHLs degrading enzymes have
been identified from many of bacteria species. Subsequent database searches for the
homologs of the QQ enzyme in complete bacterial genomes have shown the
existence of related enzymes in a wide range of species.
Type of AHL-acylase such as AiiD is produced by Ralstonia sp. XJ12B and P.
aeruginosa PAO1 (Lin et al., 2003, Huang et al., 2003). In addition, AhlM, PvdQ,
QuiP, and AiiC were identified from Streptomyces sp., Pseudomonas aeruginosa
PAO1, and Anabaena sp. PCC7120, respectively (Romero et al., 2008, Huang et al.,
2006, Park et al., 2005). The acylases HacA and HacC produced by P. syringae
B728a have shown to degrade QS signal AHLs (Shepherd and Lindow, 2009).
The other QQ enzyme AHL-lactonases have been reported from various bacteria.
The most promising bacteria producing AHL-lactonase are strains belonging to
diverse Bacillus sp. such as B. cereus, B. subtilits and B. thuringiensis (Huma et al.,
2011, Chan et al., 2010, Dong et al., 2002). The other bacterial lactonase was AiiA
from Bacillus sp. 240B1 (Dong et al., 2000), AiiB from A. tumefaciens (Liu et al.,
2007) and QIcA from Acidobacteria sp. (Riaz et al., 2008) respectively. A list of the
known QQ enzymes from bacteria was constructed in Table II-5 (Chen et al., 2013).
Table II-5. Quorum-Quenching Enzymes Involved in the Degradation of QS
Signal AHLs.
Enzyme Host Substrate
AHL-acylase
Acylase I Porcine (Kidney) C4-HSL, C6-HSL, C8-HSL, 3-oxo-C10-HSL, 3-oxo-C12-HSL
C4-HSL, C6-HSL, C8-HSL
AiiD Ralstonia sp. XJ12B
3-oxo-C8-HSL, 3-oxo-C10-HSL, 3-oxo-C12-HSL
67
Ralstonia eutropha C8-12-HSL
AiiC Anabaena sp. PCC7120
C4-HSL ͠ C14-HSL
PvdQ Pseudomonas sp. strain PAI-A
C7-12-HSL with or without C3-substitution
HacA Pseudomonas syringae
C8-HSL, C10-HSL, C12-HSL
HacB
Pseudomonas syringae Variovorax sp Variovorax paradoxusTenacibaculum maritimum Comomonas sp. D1 Rhodococcus erythropolis W2
C6-12-HSL with or without C3-substitution Broad Broad C10-HSL C4-16-AHL with or without C3-substitution C10-HSL
Aac Ralstonia solanacearum
Chain length more than C6
Shewanella sp. strain MIB015
Broad but prefer long chain
AhlM Streptomyces sp. strain M664
C8-HSL, C10-HSL, 3-oxo-C12-HSL
QuiP Pseudomonas aeruginosa
C7-14-HSL with or without C3-substitution
AHL-lactonase
AttM Agrobacterium tumefaciens
3-oxo-C8-HSL, C6-HSL
AiiA Bacillus sp. 240B1 C8-HSL
B. anthracis C6-HSL, C8-HSL, C10-HSL
B. cereus and B. mycoides
C6-HSL, C8-HSL, C10-HSL
B. thuringiensis Bacillus mycoides
C6-HSL, 3-oxo-C6-HSL, C8-HSL C8-HSL
AiiB A. tumefaciens C58 Broad
68
AiiB A. tumefaciens C4-HSL, 3-oxo-C6-HSL, C6-HSL, 3-oxo-C8-HSL, C8-HSL, C10-HSL
AiiS Agrobacterium radiobacter K84
Broad
GKL Ge. kaustophilus strain HTA426
C6-HSL, C8-HSL, C10-HSL, 3-oxo-C8-HSL, 3-oxo-C12-HSL
AiiM M. testaceum StLB037C6-HSL, C7-HSL, C8-HSL, C10-HSL
MCP My. avium subsp. paratuberculosis K-10
C7-HSL, C8-HSL, 3-oxo-C8-HSL, C10-HSL, C12-HSL
PPH My. tuberculosis C4-HSL, 3-oxo-C8-HSL, C10-HSL
AidH Ochrobactrum sp. T63C4-HSL, C6-HSL, 3-oxo-C6-HSL, 3-oxo-C8-HSL, C10-HSL
AhlS So. silvestris StLB046 C10-HSL
SsoPox Sul. solfataricus strain P2
3-oxo-C8-HSL, C8-HSL, 3-oxo-C10-HSL, 3-oxo-C12-HSL
QsdA Rhodococcus erythropolis W2
C6-14-HSL with or without C3-substitution
QIcA Acidobacteria sp. C6-8-HSL
AhlD Arthrobacter sp. IBN110
Broad
AhlK Klebsiella pneumoniae
C6-8-HSL
Oxidoreductase
P450BM3 Bacillus megateriumCYP102 A1
Oxidizes; C12-HSL, 3-oxo-C12-HSL, C14-HSL, 3-oxo-C14-HSL, C16-HSL, C18-HSL, C20-HSL
II.5.3. Bacterial QQ Application to MBR
To avoid practical issues of cost and stability of enzymes, Oh et al. (2012)
proposed that QQ might be more feasible, has a longer lifespan and does not require
enzyme purification (Oh et al., 2012). They encapsulated Recombinant Escherichia
coli producing N-acyl homoserine lactonase or Rhodococcus sp. BH4 isolated from
69
a real MBR inside hollow fiber membrane as shown in Figure II-23. The microbial
vessel (MV) could successfully control biofouling (Jahangir et al., 2012; Oh et al.,
2012) in continuous MBR system. The strain BH4 degraded AHL molecules
intracellularly by hydrolyzing the lactone ring of AHLs. The AHL–lactonase gene of
strain BH4 showed a high degree of identity to qsdA in R. erythropolis W2 (Oh et
al., 2013). It degraded a wide range of AHLs with various degradation rates
depending on AHL, which is not always the same compared to other reported AHL–
lactonase-producing strains belonging to the Rhodococcus genus (Oh et al., 2013).
In addition, the QQ effect was largely dependent on the recirculation rate of the
mixed liquor between the bioreactor and the membrane tank in external submerged
type MBR (Jahangir et al., 2012). Higher recirculation rates facilitated transport of
signal molecules from the biofilm into the bulk mixed liquor and then to the MV.
Cheong et al. reported that indigenous bacterium (Pseudomonas sp. 1A1)
demonstrated QQ activity against AHLs (Cheong et al., 2013). Pseudomonas sp.
1A1 produces extracellular QQ enzyme activity and excretes them out of the cell.
Also, he proposed QQ MBR with ceramic microbial vessel (CMV) which was
designed to overcome the extremely low F/M ratio inside an MV (Cheong et al.,
2014). The CMV also showed its potential with effective biofouling control over the
long-term operation of the QQ MBR.
As shown in Figure II-23, Lee et al. reported anti-biofouling performance of QQ-
hollow cylinder (QQ-HC) with Rhodococcus sp. BH4 (Lee et al., 2016). The MBR
with QQ-HCs was approximately 4.5 times less prone to membrane fouling
compared to the conventional-MBR that lacked media. When only the QQ activity
of the QQ-HCs was taken into account, the biological QQ effect alone reduced
membrane fouling by half.
70
(a) (b)
Figure II-23. (a) Photograph and enlarged diagram of a microbial vessel (Oh
et al., 2012). (b) Concept of a quorum quenching-hollow
cylinder (Lee et al., 2016).
II.5.4. Microbial Ecology in MBR
In the MBR, biofouling and QS mechanism are an intrinsically natural biological
process of undefined mixed cultured communities on the membrane surface.
Therefore, it can be expected that information about microbial ecology is a
prerequisite to fully understanding and successful QQ application to MBR. Although
the genetic basis of biofilm formation has been investigated for several bacterial
species, studies of mixed culture are very limited such as MBR environment.
In fact, microbial community in MBR has been investigated by using various
analytical techniques like fluorescence in-situ hybridization (FISH) and denaturing
gradient gel electrophoresis (DGGE) (Pala et al., 2008, Hong et al., 2013, Chen and
LaPara, 2006). These approaches have been successfully conducted to get some
information on the microbial community. However, none of them is sufficient to find
and compare dominant microbial groups in broth and biofilm in MBR due to
experimental limitation. Innovative sequencing method was developed and applied
71
to the analysis of microbial community in mixed culture such as MBR environment
(Lim et al., 2012). Lim et al. studied that microbial community structure in activated
sludge and membrane biocake were analyzed using pyrosequencing technique at
different fouling phase in MBR (Lim et al., 2012). As a result, specific microbial
groups such as the genera Enterobacter and Dyella were found to be dominantly
present in the biocake of initial and later fouling stage respectively, which implicate
that only a few major players in the whole microbial community could be the main
target of fouling control in MBR (Figure II-24a). At the same time, QS active
microbes (i.e., E. cancerogenus) were confirmed and isolated from MBR process.
Later, Kim et al. tried to elucidate the mechanism of biofouling inhibition by
magnetic enzyme carrier (MEC) in MBR. They investigated the changes in
population dynamics and gene expression in the MBR with MEC by using
pyrosequencing and proteomics (Kim et al., 2013a). According to Kim`s result, the
microorganisms which play QS (Enterobacter, Pseudomonas, Acinetobacter,
Bradyrhizobium, and Cytophagales_uc_g) were found in the biofilm in MBR, and
their portion in biofilm was about 40% (Figure II-24b). Moreover, enzymatic QQ
with acylase decreased the portion of these bacteria in the biofilm from 40% to 26%.
The reason for decreasing the portion of QS microorganism is the number of EPS.
At the same study, the total amount of EPS data showed that biofilm in the QQ MBR
contained less protein and polysaccharide comparing to control MBR. Thus
microorganisms in QQ MBR had difficulties in attaching on the membrane surface.
72
(a) (b)
Figure II-24. (a) Comparison of major genus groups in mixed liquor and
biocake at each initial (M30, B30) and late biofouling stage (M70,
B70). The percentage was calculated from the pyrosequencing
data (Lim et al., 2012). (b) Proportions of Enterobacter,
Pseudomonas, and Acinetobacter at a genus level in biofilm
samples of control and QQ MBRs (Kim et al., 2013a).
73
Chapter III
Control of Membrane Biofouling
in MBR by QQ Bacteria
Entrapping Alginate Beads
III. Control of Membrane Biofouling in MBR by QQ
Bacteria Entrapping Alginate Beads
74
75
III.1. Introduction
Although membrane bioreactors (MBRs) have been in commercial use for more
than two decades, membrane biofouling caused by the formation of biocakes
(deposited microbial flocs plus biofilm) on the membrane surface still remains a
bottleneck that limits their widespread use (Drews, 2010, Le-Clech, 2010). Novel
biological approaches have been attempted to control biofouling using enzymatic
quorum quenching (QQ), that is, via disruption of quorum sensing (QS) (Yeon et al.,
2009a). QS is the cell to cell communication among bacteria, which determines
phenotypes such as biofilm formation, secretion of extracellular polymeric
substances (EPS) and virulence. These applications, however, have drawbacks, such
as the high cost of enzyme extraction and purification as well as enzyme instability
(Yeon et al., 2009b). As an alternative to enzymatic quenching, Oh et al. isolated
bacteria that produce QQ enzymes and also developed a microbial vessel in which
QQ bacteria (Rhodococcus sp. BH4) were encapsulated (Oh et al., 2012).
Rhodococcus sp. BH4 has proven its potential to inhibit biofouling in various
conditions of MBR (Cheong et al., 2014, Oh et al., 2012, Jahangir et al., 2012).
However, all of the previous research depended solely on biological strategy for
biofouling inhibition without additional biofouling control strategies such as
physical strategy. Furthermore, in their study, QQ bacteria were confined within a
small vessel that was submerged in a fixed place in the MBR so that they could
degrade only soluble signal molecules that were able to diffuse into the vessel. As
such, the mass transfer of signal molecules from the mixed liquor to the inside of the
microbial vessel was limited.
In this study, QQ based anti-biofouling strategy was combined with mechanical
cleaning strategy to create more effective biofouling inhibition. We prepared free-
76
moving beads using alginate and entrapped Rhodococcus sp. BH4 into highly
interconnected microstructural pores of the beads, which will be called cell
entrapping beads (CEBs) throughout this article. We placed CEBs directly into a
submerged MBR and allowed them to move freely together with other
microorganisms in the mixed liquor as well as to contact the biofilm on the filtration
membrane to catch up signal molecules in the biofilm more easily. It is thought that
CEBs inhibit biofilm formation through QQ as well as physical washing through
their collisions against the membrane surface.
III.2. Experimental Section
III.2.1. Bioassay for Detecting AHL Molecules
All the AHLs were purchased from Sigma-Aldrich (U.S.). AHLs were detected
using the indicating agar plate, which was made by mixing an overnight culture of
Agrobacterium tumefaciens A136 (AHL biosensor) and LB agar in the ratio of 1:9
(Fuqua and Winans, 1996, Yeon et al., 2009a) with X-gal. If the sample produces or
contains AHLs, they diffuse into the indicating agar, developing the blue color as a
result. The samples were loaded into the each well of the indicating agar plate and
the amounts of AHLs were calculated using relationship equations based on the color
zone size and known amounts of AHLs (Dong et al., 2000, Oh et al., 2012, Yeon et
al., 2009a) (Figure III-1).
77
Figure III-1. Bioassay for measuring the AHL concentrations.
III.2.2. Preparation of Cell Entrapping Beads (CEBs)
A Sodium alginate (Sigma-Aldrich), a nontoxic substance to bacteria, was used as
a cell immobilization material. The isolated Rhodococcus sp. BH4 (KCTC 33122)
were inoculated in Luria−Bertani (Miller, US) broth at 30 °C for 24 h. The BH4
culture was centrifuged (12,000g, 15 min), washed with water, and resuspended in 3
mL of water. The BH4 suspension (200 mg BH4/mL of water) was gently mixed
with 97 mL of the sterile sodium alginate suspension to make a 4% (w/v)
BH4−alginate suspension. The BH4−alginate suspension was dripped into 3% (w/v)
CaCl2 solution through a nozzle at a rate of 1.6 mL/min. As depicted in Figure III-2,
the dripping device consisted of a nozzle, fluid line, and pump with a velocity
controller. The CEBs were formed and left in CaCl2 solution for 3 h before being
washed three times with distilled water and dried at room temperature. The average
size and density of CEBs were approximately 3.5 mm and 1.5 g/mL, respectively.
The BH4 content of the CEBs was 6.0 mg BH4/g sodium alginate. Because the size
and mechanical properties of the CEBs can be easily controlled by changing the
diameter of the nozzle or the concentration of CaCl2, this method offers advantages
for the preparation of diverse CEBs suitable for various types of MBRs.
78
Figure III-2. Schematic diagram for the preparation of cell entrapping beads
(CEBs).
III.2.3. Measurement of QQ Activity
The QQ activity and stability of CEBs were evaluated by the degradation rate of
standard C8-HSL (N-octanoyl-DL-homoserine lactone) (Sigma-Aldrich, USA),
which is one of the dominant signal molecules (autoinducers) in the MBR for
wastewater treatment. The degradation rate of C8-HSL was measured according to
the method described by previous studies (Yeon et al., 2009a, Yeon et al., 2009b).
C8-HSL was added to 50 mM Tris−HCl buffer (pH 7.0, 30 mL) to a final
concentration of 200 nM. Twenty individual CEBs were then added to the Tris−HCl
buffer containing C8-HSL. The remaining concentrations of C8-HSL were measured
using bioassay. The activity of the CEBs was measured via the decrease in the C8-
HSL concentration with time. The stability of the CEBs was measured from the
decrease in the C8-HSL concentration for 30 min and was monitored 13 times during
79
continuous MBR operation over 30 days. The CEBs were periodically removed from
the mixed liquor but were returned to the MBR following activity measurement. In
the activity test of the whole cell, C8-HSL was added at a final concentration of 0.2
M to the overnight bacterial culture, which was diluted to an optical density at 600
nm (OD600) of 1.0.
III.2.4. Extraction and Analysis of AHLs using High-Pressure
Liquid Chromatography (HPLC)
Standard AHLs and AHL extracts were analyzed by high-pressure liquid
chromatography (HPLC, Waters, USA). AHL was extracted from the biofilm on the
used membrane as follows: The used membrane was placed in 400 mL of deionized
water, and the biofilm was detached by backwashing and sonication. After the
membrane was removed the biofilm in suspension was shaken with 100 mL of
acidified ethyl acetate (0.1% acetic acid) for 2 h (Bertani and Venturi, 2004).
After the organic layer was separated from the water layer using a separating
funnel, it was dried in a vacuum evaporator. The residue was dissolved in 200 μL of
methanol for HPLC analysis.
Commercially available standard AHLs were dissolved in methanol to obtain 1
mg/mL solutions. Aliquots (20 μL) of each of these 1 mg/mL solutions of C4-HSL,
3-oxo-C4-HSL, C5-HSL, C6-HSL, 3-oxo-C6-HSL, C7-HSL, C8-HSL, 3-oxo-C8-
HSL,C10-HSL, 3-oxo-C10-HSL, C12-HSL and C14-HSL were added to 980 μL of
methanol-water (35:65, v/v) with 0.1% formic acid, to obtain a stock mixture
solution containing 20 μg/mL of each of the twelve AHLs. Extracted AHLs, as well
as standard AHLs, were analyzed by high-performance liquid chromatography
(HPLC). A1525 Binary HPLC pump and 717 plus Auto-samplers were used (Waters,
80
USA). The column used in the HPLC was Gemini C18, 50 mm x 2 mm, 5 μm particle
size. AHLs standard mixtures (25 μL), blanks (HPLC grade methanol) or samples in
mobile phase (methanol/water 35:65, v/v) with 0.1% formic acid, were injected at a
flow rate of 0.25 mL/min. An isocratic profile of methanol/water (35:65, v/v) for 5
min, followed by a linear gradient from 35% to 95% methanol in water over 33 min
was applied to separate mixed AHLs. A subsequent linear gradient from 95% to 35%
methanol in water over 2 min and an isocratic profile of methanol/water (35:65, v/v)
for 5 min were applied for flushing the column for the following run. The HPLC was
connected to a fraction collector (Waters, USA). Fractions were collected every 9
min into a test-tube, reduced in volume, then loaded for the bioassay of AHL
molecules using the indicating agar plate (Kumari et al., 2008)
III.2.5. MBR Operation
Two laboratory-scale MBRs, each with a 1.6 L working volume, were operated in
parallel. Three sets of operating schemes were designed for two MBRs, depending
on the presence of either vacant beads or CEBs in each MBR (Figure III-3): set 1,
control and CEBs (with BH4 cells); set 2, control and vacant beads (without BH4
cells); set 3, vacant beads and CEBs. The number of vacant beads or CEBs inserted
into each MBR was 40, and each MBR was always operated under a constant flux
of 28.7 L/(m2 h). Hydraulic retention times and sludge retention times were set to
5.3 h and 25 d, respectively. The submerged hollow fiber membrane was made of
polyvinylidene fluoride (ZeeWeed 500, GE-Zenon, USA) with an effective area of
134 cm2. Mixed liquor suspended solids (MLSS) concentration of the MBR was
maintained at 12.5 to13.0 g/L. Activated sludge was taken from a wastewater
treatment plant (Tancheon, Korea). The detailed composition of the synthetic
wastewater is given in Table III-1. In this study, following the results of preliminary
81
experiments, 40 of CEBs had been used for optimized physical cleaning effect. The
optimum condition of CEB was further studied as a separated research because the
analyzing is dependent on various factors associated with moving beads (Shim et al.,
2015). In the study, the correlation between the detachment efficiency of biocake and
three design parameters (i.e. bead diameter, bead number, and aeration rate) was
established using Box-Behnken methodology and analyzed optimum operating
condition of moving bead (Shim et al., 2015).
Table III-1. The Composition of the Synthetic Wastewater in Continuous MBR
Operation.
Components Concentration (mg/L)
Glucose 306.75
Peptone 115
Yeast extract 14
(NH4)2SO4 104.75
KH2PO4 21.75
MgSO4 7H2O 32
FeCl3 6H2O 0.125
CoCl2 6H2O 1.25
CaCl2 H2O 3.25
MnSO4 5H2O 2.875
NaHCO3 255.5
82
Figure III-3. Schematic diagrams for three sets of operations of MBRs.
III.2.6. Measurement of Loosely and Tightly Bound Biofilms
For the qualitative and quantitative analysis of biofilm formed on the surface of
membranes, biofilms were detached from the used membranes and were classified
into two types: loosely bound biofilm (LB biofilm) or tightly bound biofilm (TB
biofilm). The former was defined as a biofilm that can be detached only by air
scouring at a fixed aeration time and rate, whereas the latter, by sonication and
subsequent air-scouring (Figure III-4). The used membranes covered with biofilm
83
were submerged in an aeration tank filled with 1 L of water. The LB biofilm was
obtained after 80 min of aeration at a rate of 3 L/min. The remaining biofilm on the
same used membrane was further sonicated for 10 min, followed by an additional 20
min of aeration to obtain the TB biofilm. The dry weight of each biofilm in
suspension was measured. The total weight of LB and TB biofilms was regarded as
the total attached biomass (TAB). Extraction of EPS from the biofilm in suspension
was carried out using an ion-exchange resin method (Frolund et al., 1996). A cation
exchange resin in sodium form (CER, Dowex Marathon, Sigma-Aldrich) was
washed for 1 h in phosphate buffer and was added to the suspension of each biofilm
(10 g CER/g detached biofilm). The mixed suspension was stirred at 300 rpm for 2
h and then centrifuged at 4000g for 20 min. A pellet composed of cells and CER
formed in the bottom of the tube, and the supernatant contained EPS. The weight of
cells was determined by subtracting the weight of CER from the weight of the pellet.
The number of EPS was determined by subtracting the cell weight from the TAB.
Figure III-4. Schematic for the quantitative analysis of loosely and tightly
bound biofilms.
84
III.2.7. Straining of CEBs Image Analysis Using a Confocal
Laser Scanning Microscope (CLSM)
The live (green) or dead (red) cells inside the CEBs as well as free BH4 cells were
stained using the Live-Dead BacLight Bacterial Viability kit (Molecular Probes,
Eugene, OR) according to the manufacturer’s instructions. After careful washing
with PB solution, the stained CEBs were observed with a Confocal Laser Scanning
Microscope (CLSM, C1 plus, Nikon, Japan) with an optical lens (x10). A Z-section
image stack (Slice thickness: 1 μm) of each red and green channel was reconstructed
using IMARIS software (Bitplane AG, Switzerland). An Image Structure Analyzer
(ISA-2) was used to observe the cells inside the CEBs in three dimensions.
III.2.8. Analytical Methods
MLSS and chemical oxygen demand (COD) was determined according to
standard methods (Franson et al., 1998). Extracellular polymeric substances (EPS)
were extracted from the biocake using the cationic ion exchange resin method
(Frolund et al., 1996). The mean particle size was measured using particle size
analyzer (S3500, Microtrac, U.S.). The optical density at a wavelength of 600 nm
was measured using spectrophotometer (Optizen pop, Mecasys, South Korea).
III.3. Results and Discussion
III.3.1. Characterization of CEBs.
The vacant beads were almost spherical, with a smooth surface and uniform size.
Entrapment of QQ bacteria (BH4) into the beads did not result in any significant
change in either the shape or the size of beads (Figure III-5a). The size of CEBs was
∼3.5 mm, and their density was roughly 1.5 g/mL. The CEBs were able to circulate
85
in the mixed liquor under aeration (Figure III-5 b).
The cross-sectional SEM image showed the morphologies of the vacant beads and
CEBs as well as the differences between the two. The vacant beads and CEBs possess
a porous microstructure, with a high degree of interconnectivity (Figure III-6a and
d). Since there are many pores and the pore diameter in CEBs is around 300 μm,
CEBs appear to provide enough space for BH4 colonization as well as low mass
transfer resistance. Inside the pore, no BH4 were observed in vacant beads (Figure
III-6b), whereas BH4 were spread on the alginate matrix surface (Figure III-6e) in
CEBs. The BH4 appear as short rods with an approximate size of 1.2−2.0 μm in
length and 0.5 μm in width (Figure III-6e).
To investigate the viability of BH4 during entrapment, CLSM images of CEBs
were taken after viability staining. Before entrapment, the proportion of free live
BH4 was ∼80% (±3%), on the basis of the ISA image. After entrapment, the BH4
appeared densely packed and evenly dispersed in the microstructure of the CEBs
(Figure III-7). From the images, the mean percentage of living cells entrapped in
CEBs was calculated to be 65% (±5%). The damage to living cells during entrapment
indicates that cell immobilization had a negative effect on cell viability. It is possible
that BH4 near the surface of CEBs were killed by contact with the CaCl2 solution,
but the decrease in the amount of live BH4 was not substantial (Figure III-7). The
SEM and CLSM analysis confirmed that CEBs were successfully constructed by the
combination of alginate, Ca+ and BH4.
86
Figure III-5. Photographs of (a) individual CEBs and (b) CEBs in the MBR
with and without aeration.
Figure III-6. SEM microphotographs of the beads: a cross-section of a vacant
bead (a) ×25, (b) ×1000, and (c) ×6000. Cross section of a CEB
(d) ×25, (e) ×1000, and (f) ×6000.
87
Figure III-7. Reconstructed CLSM images of a CEB cross-section; (a) alive
and (b) a dead cell stained with the BacLight Live-Dead staining
kit. Magnification x100. Image size 1212 μm x 1212 μm.
III.3.2. QQ Activity of Free BH4 and CEBs
The QQ activity of isolated free BH4 was tested using a bioassay with C8-HSL,
which was most abundant in the biofilm-formed membranes in MBRs (Yeon et al.,
2009a). Live BH4 readily degraded C8-HSL, whereas dead BH4 hardly reduced C8-
HSL levels, despite its potential physicochemical adsorption (Figure III-8a)
The QQ activity of CEBs was tested using the same method as for free BH4. As
shown in Figure III-8b, CEBs degraded 91% of C8-HSL, whereas the vacant beads
removed less than 10% of the C8-HSL in 60 min. The removal of vacant beads was
attributed to its physicochemical adsorption because vacant beads have neither QQ
bacteria nor QQ enzyme. A control was also conducted to check the potential
removal of C8-HSL by its adsorption onto the surface of a glass beaker, but the
adsorption was negligible.
88
(a) (b)
Figure III-8. (a) QQ activity of live and dead BH4 cells. (b) Quantitative QQ
activity of control, vacant beads, and CEBs. Error bar: standard
deviation (n=4)
III.3.3. Application of CEBs to the Lab-Scale MBR.
CEBs were applied to submerged MBRs to test their potential to inhibit biofouling
in MBRs (set 1 in Figure III-3). Two lab-scale MBRs in continuous mode were
operated in parallel under identical operating conditions except for the addition of
CEBs to one MBR at the start of the operation. The rise of the profiles
transmembrane pressure (TMP) of the control and CEB MBRs were compared to
evaluate the inhibition of biofouling by CEBs. As shown in Figure III-9a, it took 1.8
d for the TMP to reach 70 kPa in the first cycle of the control MBR, whereas it took
18.8 d for the first cycle of the CEB-treated MBR. Thus, CEBs mitigated the
formation of biofilm and extended the time required to reach the TMP of 70 kPa by
∼10-fold, compared with the control MBR. From a practical point of view, this is
important because the delay in the rise of TMP is closely associated with a saving of
energy in the operation of the MBR.
The remarkable effect of CEBs to reduce biofouling is better than that reported by
89
Oh et al (Cheong et al., 2013, Jahangir et al., 2012, Oh et al., 2012). They
encapsulated the same QQ bacteria, BH4, into a microbial vessel, applied it to a
submerged MBR, and observed that the microbial vessel resulted in much lower
biofouling compared with a conventional MBR. Although a direct comparison
between results obtained from CEBs and the microbial vessel is difficult, CEBs
appear to be superior to the microbial vessel in terms of reducing membrane fouling.
The excellent performance of CEBs could be attributed either to the inhibition of
biofilm formation by QQ or to the sloughing of biofilm from the membrane surface
by a collision between moving CEBs and the submerged membrane in the MBR. To
verify this, two consecutive MBR operations were carried, out as depicted in sets 2
and 3 in Figure III-3.
90
Figure III-9. Comparison of TMP between (a) control and CEBs MBRs, (b)
control and vacant beads MBRs, and (c) vacant beads and CEBs
MBRs under the same operating conditions.
III.3.4. Physical Washing Effect of CEBs
To confirm a physical washing effect, the control MBR and the MBR with the
vacant beads were run in parallel under the same operating conditions (set 2 in Figure
III-3). As shown in Figure III-9b, it took 1.8 d to reach the TMP of 70 kPa in the
control MBR, whereas it took 3.1 d to reach the same TMP in the MBR with vacant
beads. Although the vacant beads with porous microstructures contain no BH4, they
continuously circulate in the mixed liquor and collide with the surface of submerged
membranes in the MBR. This collision could facilitate the detachment of biocake
already deposited or formed on the membrane surface. A physical washing effect by
moving media in MBRs has been previously reported (Lee et al., 2006, Rosenberger
91
et al., 2011).
To quantitatively evaluate the physical washing effect, a separate experiment was
designed. Two control MBRs with neither vacant beads nor CEBs were run until the
TMP reached 70 kPa. The used hollow fiber membrane module was then removed
from each control MBR, and each used membrane module was immersed in two
separated beakers containing 1 L of water. Forty vacant beads were added to only
one beaker, and each beaker was then aerated for 80 min (3 L/min) to assess how
much biomass would be detached from each used module and to compare each
experiment. Further sonication following aeration also made it possible to determine
the TAB of each used membrane module. For the five repeating measurements, 0.64
g (87% of TAB) of biomass was detached in the beaker with the vacant beads,
whereas 0.52 g (72% of TAB) of biomass was detached in the beaker without the
vacant beads (Figure III-10). On the other hand, the average TAB of both membrane
modules was similar: 0.74 (±0.05) g in the beaker with the beads and 0.73 (±0.04) g
in the beaker without beads, with a 5% relative standard deviation of each.
Consequently, it can be concluded that the vacant beads facilitated the detachment
and, thus, increased the amount of detached biomass by ∼15% through their
collision with the membrane.
92
Figure III-10. Detached biomass from used membranes in the beaker with
and without a vacant bead. Error bar: standard deviation (n=5)
III.3.5. QQ Effect of CEBs.
To confirm the QQ effect, one MBR with vacant beads and the other MBR with
CEBs were run under the same operating conditions (set 3 in Figure III-3). During
the operation of each MBR, a used membrane module was replaced by a new one
when the TMP exceeded 70 kPa, and TMP monitoring was reperformed with the new
membrane. As shown in Figure III-9c, it took 17 d to reach the TMP of 70 kPa for
the MBR with CEBs, whereas it took only 2 or 3 d to reach the same TMP for the
MBR with the vacant beads. Eight cycles were thus repeated in the MBR with the
vacant beads during the only cycle with the one with CEBs. Expressed differently,
CEBs extended the time required for the MBR to reach the TMP of 70 kPa by about
7-fold. It is worth noting that assuming that the physical washing effects of the vacant
beads and CEBs were identical because the same amount of vacant beads or CEBs
were added to each MBR, the large difference in the rate of the TMP rise (set 3 in
Figure III-3) is attributable only to the BH4 in the porous microstructural CEBs.
93
III.3.6. Inhibition of EPS Production by CEBs
To investigate the mechanisms of QQ, biofilms were detached from the used
membrane modules after 75 h of operation in both the MBR with vacant beads and
the MBR with CEBs. Biofilms from both used modules were analyzed in terms of
EPS, TAB, and adhesiveness and then compared with each other. After 75 h of
operation, the TMP reached 70 kPa in the MBR with vacant beads, whereas only 7.9
kPa was reached in the MBR with CEBs. This coincided well with the greater TAB
in the former (0.77 g) compared with the latter (0.24 g), as shown in Table III-2: less
than one-third of the biomass developed on the membrane surface in the MBR with
QQ bacteria during the same operation period.
To characterize the detached biomass, the TAB was further divided into two
components: EPS and cells (Chang and Lee, 1998). Not only the total amount but
also the proportion of EPS was much lower in the MBR with CEBs (0.07 g, 29%)
than in the MBR with vacant beads (0.36 g, 47%). It is known that QS regulates the
production of EPS through the transcription of target genes and determines the
physiology of the microbial community (Fuqua et al., 1996). Previous studies
confirmed that enzymatic QQ decreases EPS production in the biofilm (Kim et al.,
2011, Yeon et al., 2009a, Yeon et al., 2009b). Rhodococcus sp. has been reported to
generate an enzyme (lactonase) that can degrade AHLs (Uroz et al., 2008, Oh et al.,
2012). Consequently, the application of CEBs (i.e. BH4) to MBRs inhibits QS
between cells by reducing the concentration of AHLs and thus decreasing EPS
production in the biofilm. The attached biomass was also classified into two types:
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LB biofilm or TB biofilm. As illustrated in Table III-2, one portion of the TAB
biofilm (TB biofilm) requires more vigorous conditions for detachment than the
other (LB biofilm), indicating that TB biofilm has stronger cohesive or adhesive
forces (or both) than LB biofilm. In Table III-2, both the total amount and the portion
of the TB biofilm was substantially lower in the MBR with CEBs (0.02 g, 11%) than
in the MBR with vacant beads (0.24 g, 32%). This could be attributed to the lower
production of EPS, the key element for the construction of biofilm due to QQ by
CEBs (Ahimou et al., 2007).
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Table III-2. Amount of TAB, EPS and Loosely and Tightly Bound Biofilms in
the Used Membrane Modules for the MBR with Vacant Beads and
MBR with CEBs.
MBR with
vacant beads MBR with CEBs
TMPa at the operating time of 75 h 70.3 kPa 7.9 kPa
TABb 0.77 g 0.24 g
TAB EPS 0.36 g
(47% of TAB)c
0.07 g
(29% of TAB)c
Cell 0.41 g 0.17 g
TAB Loosely bound
biofilm
0.53 g
(68% of TAB)d
0.22 g
(89% of TAB)d
Tightly bound
biofilm
0.24 g
(32% of TAB)d
0.02 g
(11% of TAB)d
aTMP: transmembrane pressure.
bTAB: total attached biomass.
cEPS: percentage = (EPS/TAB) x100.
dBound biofilm percentage = (bound biofilm/TAB) X 100.
III.3.7. Identification of Signal Molecules in MBRs
An important point concerns the demonstration of the destruction of signal
molecules by CEBs. For this purpose, both MBRs in this study were run for 48 h,
and the extracts from biofilm formed on the membrane surfaces from MBRs with or
without CEBs were then analyzed by HPLC and a bioassay. The extract from the
MBR with vacant beads showed four peaks (blue line in Figure III-11a). Two of the
96
four peaks appeared with a retention time of 3.2 and 11.8 min and were identified as
3oxoC8-HSL and C8-HSL, respectively, by comparison with the peaks of two
standard signal molecules (green and yellow lines in Figure III-11a). In the extract
from the MBR with CEBs (red line in Figure III-11a), however, no AHL was detected.
To ensure that the destruction of AHLs occurred by CEBs, we collected two HPLC
fractions, each after 9 min: fraction 1 was eluent collected for the first 9 min, and
fraction 2 was eluent collected for the second 9 min. Both fractions were analyzed
by bioassay with A136 as a reporter strain. The two fractions from the MBR with
vacant beads showed blue colors, indicating the presence of AHLs (Figure III-11b),
whereas those from the MBR with CEBs showed no blue color, indicating the
absence of AHLs (Figure III-11c).
97
(a)
Figure III-11. Identification of AHLs extracted from the biofilm formed on the
used membrane by HPLC. (a) Chromatogram of standard and
extracted AHLs. (b) Bioassay of fractions (1 and 2) collected
every 9 min for the MBR with vacant beads. (C) Bioassay of
fractions (1 and 2) collected every 9 min for the MBR with CEBs.
98
III.3.8. Visual Confirmation of the QQ Effect by CEBs
The QQ effect of CEBs was confirmed visually using CLSM. Figure III-12
represents the reconstructed CLSM images of biofilm formed on the membrane
surfaces which were removed from the MBR operated for 48 h. The amount of
biofilm formed in the MBR with CEBs was the least, whereas that in the control
MBR was the greatest, and that in the MBR with vacant beads was intermediate. In
summary, CEBs induced a physiological change in microorganisms, including a
decrease in EPS production through the disruption of AHLs. Consequently, the
cohesion between cells or the adhesion between cells and membrane was weakened,
and thus, less biomass was attached to the membrane with the CEBs. In short, CEBs
can inhibit biofilm formation by QQ effect.
Figure III-12. Reconstructed CLSM images of biofilm formed on the
membrane surface in (a) control MBR, (b) MBR with vacant
beads, and (c) MBR with CEBs after 48 h operation, stained
with SYTO9 (cell; green). Magnification: ×100. Image size:
1212 μm × 1212 μm.
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III.3.9. Influence of CEBs on MBR Performance and Its
Stability
In addition to variation in TMP, the quality of the permeated water is another
important factor in MBR performance. We monitored the removal efficiencies of
COD in three MBRs on the basis of their feed and permeate concentrations. Although
the influent COD to three reactors was around 500 mg/L, three reactors exhibited
similar COD concentrations in the permeate with more than 96% of COD removal:
6.2−19.1 mg/L for the control, 6.3−16.4 mg/L with vacant beads, and 6.9−14.2 mg/L
for CEBs (Figure III-13a). There was no significant difference in the operational
parameters among three MBRs throughout the entire experimental period. Moreover,
taking into account the volume of CEBs added to the MBR was less than 0.63% of
the working reactor volume, the adsorption of COD on CEBs would be negligible.
Therefore, it has been concluded that QQ with CEBs mitigates membrane biofouling
but does not decrease microbial activity, at least for the degradation of organic matter
in the MBRs. The structural integrity and QQ activity of CEBs were monitored
during the operation of continuous MBR for 30 days. The QQ activity of CEBs
increased by ~3% after 30 days, which is highly favorable for practical MBR
application. (Figure III-13b).
100
(a)
(b)
Figure III-13. (a) COD change of permeate during continuous MBRs
experiments. (b) QQ activity of CEBs during MBR operation.
QQ activity (%): Percent ratio of the degraded amount of the
standard C8-HSL for 30 min by the fresh or used CEBs to the
initial amount of the standard C8-HSL. Error bar: standard
deviation (n=3)
101
III.4. Conclusions
The purpose of this chapter was to investigate the potential of CEBs for efficient
biofouling control via both physical washing and QQ effects. Based on the results of
this study, the following conclusions were made:
● QQ bacteria were entrapped in alginate moving beads, and their QQ activity
was successfully maintained after immobilization due to highly porous
microstructure and biocompatibility of alginate matrix.
● When the CEB was applied to the continuous lab-scale MBR process, the
membrane biofouling was inhibited by both physical washing and biological (i.e.,
QQ) effect.
● In the continuous MBR operation, insertion of CEBs into the MBR
substantially delayed the TMP rise-up (i.e., membrane biofouling) without any
deterioration of wastewater treatment performance. Furthermore, it was found that
QQ effect of CEBs suppresses EPS production from bacteria thereby weakening the
structural integrity of the biofilm. This resulted in more effective detachment of
biofilm by physical washing effect of CEB.
102
103
Chapter IV
Stability Enhancement of QQ
Bacteria Entrapping Moving
Bead and Its Application to
MBR for Biofouling
IV. Stability Enhancement of QQ bacteria entrapping
Moving Bead and Its Application to MBR for
Biofouling Control
104
105
IV.1. Introduction
In the previous chapter, the QQ bacteria entrapping alginate beads (CEBs) are very
efficient to control biofouling in a membrane bioreactor (MBR) for wastewater
treatment (Kim et al., 2013b). However, alginate beads (i.e. calcium alginate) are
chemically unstable when chelators such as citrate and cations such as magnesium
ions are present. Alginate bead was reported to be susceptible to disintegration in the
presence of excess monovalent ion and Ca2+ chelating agents, which is the main
obstacle in its wider application (Smidsrod and Skjakbraek, 1990, Thorsen et al.,
2000). Some efforts have been made to increase in chemical and physical stability
of alginate matrix in the biological environment through coating polyelectrolyte such
as polylysine (Kendall et al., 2000), poly(ethylene glycol) (Liu et al., 2008), chitosan
(Wong et al., 2011), and polyvinylamine (Guisan, 2006). However, these coating
methods suffered from a relatively high cost or complex formation steps; they have
limitations to be used for wastewater treatment from the practical point of view.
Therefore, we need to find out a new QQ bacteria entrapping bead which have long-
term stability in the MBR environment. To overcome stability problem of alginate
bead, QQ bacteria were encapsulated in the porous membrane (i.e. Macrocapsule)
and entrapped with polyvinyl alcohol (i.e. W-bead).
Firstly, we examined various macrocapslue for the prevention of membrane
biofouling. QQ bacteria encapsulating macrocapsule was prepared according to the
previous study (Kim, 2014). Porous membrane layer was formed on the CEBs using
various commercial polymers of poly(vinylidene) fluoride (PVDF),
polyethersulfone (PES) and polysulfone (PSf), respectively. Then, characterization
of each membrane coated QQ beads was conducted in term of morphology, AHL
degrading activity, physical strength and chemical stability. Finally, the operational
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feasibility of polymer coated QQ-bead was evaluated through continuous operation
of lab-scale MBR fed with real wastewater.
Secondly, the W-beads also characterized its microbial viability and QQ activity.
And then, the beads were applied to continuous MBR to confirm biofouling
inhibition. Moreover, to see the possibility of practical application, W-beads were
measured in their change of shape, mechanical stability, and QQ activity in synthetic
and real wastewater environment.
IV.2. Experimental Section
IV.2.1. 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%). The quorum-quenching
Rhodococcus sp. BH4 strain was previously isolated from the MBR wastewater
treatment plant (Ok-Cheon, Korea) and was cultured in the Luria-Bertani medium at
30°C (Kim et al., 2013b, Oh et al., 2012).
IV.2.2. Preparation of Macrocapsules and W-beads
The overall preparation scheme of macrocapsules is graphically depicted in Figure
IV-1. First, QQ bacteria (Rhodococcus sp. BH4) were entrapped in the alginate
matrix (alginate beads with QQ bacteria, CEBs) (Step 1 in Figure IV-1).
Subsequently, cultured Rhodococcus sp. BH4 was centrifuged (12,000 g, 15 min)
and resuspended in 10 ml of deionized water. This Rhodococcus sp. The BH4
suspension was mixed with 90 ml of the alginate solution. The alginate concentration
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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.
Microporous membrane layers were formed on the surface of alginate beads using
the phase inversion technique. 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 IV-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 IV-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.
The overall preparation step of W-bead was similar to that of CEBs. To increase
of mechanical stability, high molecular weight polyvinyl alcohol (PVA, JUNSEI,
Japan) was used as W-bead core. In detail, 2 g of PVA was dissolved with 98 g of
hot water. The alginate powder was gently mixed with 100 mL of the sterile PVA
solution. The BH4 suspension (200 mg BH4/mL of water) was gently mixed with 97
mL of the sterile polymer solution. The suspension was dripped into 3% (w/v) CaCl2
solution through a nozzle. The beads were formed and left in CaCl2 solution for 2 h
before being washed three times with distilled water. The formed beads were then
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dropped into sodium sulfate solution and cross-linked for 12 hr. Finally, the W-beads
were washed three times with distilled water and dried at room temperature.
Figure IV-1. Preparation scheme of a macrocapsule coated with a membrane
layer through the phase inversion method.
IV.2.3. Luminescence Method for Detecting AHL Molecules
In this chapter, the concentrations of AHL molecules were measured using
luminescence method which also uses A. tumefaciens A136 as the reporter strain
likewise the bioassay method described in the previous chapter. The AHL level was
quantitatively determined using a bioassay with the luminescence substrate Beta-Glo
(Promega, USA). Briefly, 5 μl of AHL samples were loaded into a 96-well-plate with
95 μl of an overnight culture of A. tumefaciens A136. After incubation for 90 minutes
at 30°C, 30 μl of Beta-Glo were added to each well. This generated the luminescence
of oxyluciferin, whose intensity is proportional to the amount of beta-galactosidase
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
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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 chapter (Figure IV-2). Each bioassay was conducted in triplicate
to assess repeatability.
Figure IV-2. Calibration curve for the quantification of AHLs by luminescence
method. Error bar: standard deviation (n=3)
IV.2.4. Determination of QQ Activity
The quorum quenching (QQ) activities of macrocapsules and W-beads were
quantitatively determined by the reduction rate of C8-HSL standard solution. 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 A. tumefaciens A136
bioluminescence assay. The “QQ activity” of beads (i.e. alginate beads or
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macrocapsules) was defined by the rate of AHL (C8-HSL) 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. The relative QQ activity
was defined as the percentage ratio of residual QQ activity to initial QQ activity.
IV.2.5. Measurement of Mechanical Strength
The mechanical resistance of macrocapsules and W-beads were determined using
a texture analyzer (CT3 4500, Brookfield, USA). The mechanical deformation tests
were performed at a mobile probe (TA44) speed of 0.5 mm/s until bursting of the
bead matrix was observed. 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.
IV.2.6. Measurement of Chemical Stability (Macrocapsule)
To evaluate the chemical stability of any type of beads under a harsh chemical
environment, a buffered EDTA solution was selected to simulate a harsh
environment (Smidsrod and Skjakbraek, 1990). This is because EDTA is well known
to be a strong complexing agent with a calcium ion and thus is expected to easily
disintegrate alginate bead matrix containing calcium ion. 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
(Hauselmann et al., 1994). 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 for it
correlates with the chemical stability of beads.
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IV.2.7. Restoration of QQ Activity of Disintegrated Beads
Restoration of beads were conducted as the following: 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. 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.
IV.2.8. Measurement of Durability in Wastewater (W-bead)
To measure the durability of W-bead, 80 W-bead were contained with synthetic
and real wastewater during 84 days. Each sample of W-beads was recorded their
mechanical strength using texture analyzer. At the same time, the beads were
measured QQ activities.
IV.2.9. MBR Operation Condition
Two lab-scale MBRs (one control & one QQ MBR) were constructed in a similar
way to those described by other researchers and were operated in parallel. To
evaluate fouling control only by the QQ activity of macrocapsules, a control MBR
was also operated with vacant macrocapsules without Rhodococcus sp. BH4.
Activated sludge from a wastewater treatment plant (Si-Hwa, Korea) was inoculated
into the MBR after being acclimated with real wastewater for 6 months. The MBR
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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) as 155
cm2 of the surface area. 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. The working volume and the mixed liquor suspended solids
(MLSS) concentration of each reactor were 2.5 L and ~5,500 mg/L, respectively. For
the long-term stability test of macrocapsuel in the continuous MBR operating,
activated sludge from a wastewater treatment plant (Si-Hwa, Korea) was inoculated
into the MBR after being acclimated with synthetic wastewater with the previous
chapter. The working volume and the mixed liquor suspended solids (MLSS)
concentration of each reactor were 2.7 L and ~10,000 mg/L, respectively.
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. Hydraulic retention time (HRT) and sludge retention
time (SRT) was set 7 hrs and 20 days, respectively.
Meanwhile, the QQ bacteria entrapping W-bead were applied to MBR under the
similar operating condition. The working volumes of each reactor were 4.5 L and the
permeate flux was set to 28 LMH. Membrane modules were made of poly(vinylidene)
fluoride hollow fiber (ZeeWeed500). To evaluate fouling control only by the QQ
activity of macrocapsules, a control MBR was also operated with vacant
macrocapsules. MLSS in both reactors were maintained within the range of 8000 ~
12,000 mg/L. The permeate flux was set to 28 LMH.
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IV.2.10. Scanning Electron Microscopy (SEM) and Confocal
Laser Scanning Microscopy (CLSM)
The morphology of the macrocapsules and W-bead were 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 with 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.
The three-dimensional structure of beads and the membrane-biocake was visually
monitored with confocal laser scanning microscope (CLSM, C1 plus, Nikon, Japan).
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 μm) of each channel were reconstructed
using IMARIS software (Bitplane AG, Switzerland).
IV.3. Results and Discussion
IV.3.1. Preparation and Characterization of Macrocapsules
with Various Polymeric Coatings
In the previous study, QQ bacteria entrapping alginate bead (CEBs) were very
efficient at controlling biofouling in an MBR for wastewater treatment (Kim et al.,
2013b). However, we observed the gradual decomposition of the calcium alginate
matrix during long-term operation of the MBR (Figure IV-3). Alginate has been
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reported to be susceptible to disintegration in the presence of excess monovalent ions
and Ca2+ chelating agents. In this chapter, we prepared synthetic polymer coating
bead using phase inversion method (Kim, 2014). Three kinds of microporous
polymeric layers (i.e. PSf, PES, and PVDF) were formed on the surface of alginate
beads. As shown in Figure IV-4a, 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
IV-4b). 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 μm. 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 IV-5). 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 beads
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. As the PSf macrocapsule had the highest
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mechanical strength, it was adopted for subsequent tests of QQ activity and
biofouling control efficiency in continuous MBR operation.
Figure IV-3. SEM microphotographs of an alginate bead: (a) Top and (b)
cross-section views of a fresh alginate bead, (c) Top and (d)
cross-section views of a used alginate bead after 60 days’ MBR
operation.
(a) (b)
(c) (d)
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(a)
(b)
Figure IV-4. (a) Photographs of an alginate bead and PSf, PES, PVDF coated
macrocapsules. (b) SEM image of the outer surface, inner surface,
and cross-section of each macrocapsule prepared with PSf, PES
and PVDF.
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Figure IV-5. Comparison of mechanical strength between alginate beads and
three types of coated macrocapsules. Error bar: standard
deviation (n=20)
IV.3.2. Characteristics of W-bead
The vacant W-beads were almost spherical, with a smooth surface and uniform
size. The shape of W-bead was depended on the viscosity of the synthetic polymer.
In this study, QQ bacteria were entrapped for the portion of ~2.2 mg BH4/g polymer
solution. Entrapment of QQ bacteria into the beads did not result in any significant
change in either the shape or the size of beads (Figure IV-6a). The size of W-beads
was 4.0 ~ 4.5 mm, and their density was roughly 1.17 g/mL. Therefore, the W-beads
were able to circulate in the mixed liquor under aeration.
To investigate the viability of QQ bacteria during entrapment, CLSM images of
W-beads were taken after Live/dead staining. Before entrapment, the proportion of
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free live BH4 was ∼75% (±7%), on the basis of the ISA image. After entrapment,
the BH4 appeared densely packed and evenly dispersed in the microstructure of the
W-beads (Figure IV-6b). The percentage of living cells entrapped in W-bead was
calculated to be 62% (±9%). The damage to living cells during entrapment indicates
that cell immobilization had a negative effect on cell viability.
Figure IV-6. (a) Photographs of vacant W-bead and QQ bacteria entrapping
W-bead. (b) CLSM image of the live/dead cell distribution in a
W-bead (Green: Live, Red: Dead, Magnification: X100).
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IV.3.3. QQ Activities of PSf-Macrocapsules
The QQ activity of PSf-macrocapsules was evaluated using the standard C8-HSL
as a representative signal molecule (Kim et al., 2013b, Yeon et al., 2009b). 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 IV-7, 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 IV-8, 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 an 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 IV-7. It is
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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 IV-9). As a consequence of the
proliferation of live QQ bacteria, the C8-HSL degradation rate of macrocapsules
after reactivation was increased to 0.084 nmol C8-HSL/min, which is about 140%
higher than the initial level (0.059 nmol C8-HSL/min).
Figure IV-7. Comparison of the AHL removal rate between alginate beads,
macrocapsules and restored macrocapsules. Error bar: standard
deviation (n=3)
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Figure IV-8. CLSM image of the live/dead cell distribution in a macrocapsule.
Note that green and red colors appear in the PSf-membrane
layer because the fluorescence dyes were adsorbed onto the
membrane layer during the staining step (Green: Live, Red: Dead,
Magnification: X100).
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Figure IV-9. CLSM image of the live/dead cell distribution in a macrocapsule
after reactivation (Green: Live, Red: Dead, Magnification: X100).
IV.3.4. QQ Activities of W-beads
The QQ activity of W-beads was evaluated using the standard C8-HSL with A136
bioassay (Oh et al., 2013, Kim et al., 2013b). The QQ bacteria content of W-beads
was 2.0 mg QQ bacteria/g polymeric solution. The decomposition rate of C8-HSL
by QQ bacteria entrapping W-bead was 0.116 nmol C8-HSL/min after 60 minutes
(Figure IV-10). However, the adsorption of C8-HSL by vacant W-beads was not
significant, i.e., less than 10% in 120 minutes.
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Figure IV-10. QQ activity of vacant W-bead and QQ bacteria entrapping W-
bead. Error bar: standard deviation (n=4)
IV.3.5. Stability of Macrocapsule in a Harsh Environment
Taking into account their 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 two kinds of QQ media
(macrocapsules and alginate beads) 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 media was evaluated
quantitatively by measuring the optical density (OD600) in the solution (Figure
IV-11a). 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 media and interfered with the measurement of the cell
concentration in the suspension. Fortunately, the contributions from both vacant
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media to the OD600 were too small to induce significant interference with the
measurement of QQ bacteria. 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 activity change in both alginate beads and
macrocapsules through a cycle of chemical treatment and restoration. The chemical
treatment was carried out in citrate buffer for 10 minutes, as described above. The
restoration step was conducted by placing chemically treated alginate beads and
macrocapsules in Luria-Bertani medium and shaking them for 12 hours. Next, both
restored media were washed with deionized water before the QQ activity test. As
clearly shown in Figure IV-11b, 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 an MBR plant fed with real wastewater. On the
contrary, the macrocapsules displayed a continuous increase in QQ activity up to 115%
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 protect the QQ bacteria and 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
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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 (Chai et al., 2004).
(a)
(b) Figure IV-11. Chemical stability and relative QQ activity of alginate beads and
macrocapsules. (a) Leakage of QQ bacteria in both beads after
chemical treatment using citrate buffer (30 mM EDTA, 55 mM
sodium citrate and 0.15 M sodium chloride). (b) Relative QQ
activity of both beads before and after chemical treatment and
after restoration. Relative QQ activity: the percentage of residual
QQ activity to initial QQ activity. Error bar: standard deviation
(n=3)
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IV.3.6. Biofouling Inhibition by Macrocapsules in a Continuous
MBR Fed with Synthetic Wastewater
The long-term stability of QQ macrocapsules was tested through the parallel
operation of two lab-scale MBRs (Figure IV-12a). To test this, 500 vacant
macrocapsules containing no QQ bacteria (Rhodococcus sp. BH4) were put into one
MBR (i.e. the control MBR), while 500 QQ macrocapsules were put into the other
MBR (i.e. the QQ MBR). The COD removal efficiencies were maintained around
96-99% based on the permeate for both the control and QQ MBRs over the operation
period of 80 days. It took 12-15 days to reach a TMP of 40 kPa in the control MBR
in which only the physical cleaning effect would be expected through collisions
between moving vacant macrocapsules and hollow fibers. On the other hand, it took
25-30 days to reach the same TMP of 40 kPa in the QQ MBR, in which both physical
and biological (i.e. quorum quenching) effects would be expected. In summary, in
the long term operation of a continuous MBR, the QQ effect of macrocapsules was
pronounced, such that the rate of TMP rise-up was delayed by about two-fold. Taking
into account that the rate of TMP rise-up is directly linked to the energy consumption
of an MBR, macrocapsules are expected to play an important role in the design of a
future MBR with energy savings.
In order to visually confirm the inhibition of biofouling with macrocapsules, the
used membrane modules were taken out of both MBRs after the same operating
period to measure total attached biomass (TAB) as well as to visualize the biocakes
that formed on the surface of the membrane. The CLSM images clearly show that
the biocake that formed on the used membrane in the QQ MBR was much smaller
than in the control MBR (Figure IV-12b). Furthermore, the amount of TAB deposited
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on the used membrane in the control MBR was 244 (±87) mg, whereas that in the
QQ MBR was only 72 (±54) mg.
During continuous overall MBR filtration up to 80 days, the operation stability of
macromolecules was periodically tested in terms of mechanical strength and QQ
activity. Figure IV-13 showed the stable maintenance of QQ activity with negligible
deterioration during overall operation periods of both MBR runs. Interestingly, the
hardness work gradually decreased during operation periods in both filtration runs
(Figure IV-13). This correlation between mechanical strength and AHL quenching
activity indicate that although deterioration of alginate matrix was unavoidable
during long-term operation, PSf membrane layer effectively prevents the loss of
active biomass from the beads.
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(a)
(b)
Figure IV-12. (a) Effect of macrocapsules on the enhancement of
permeability in MBR. (b) Reconstructed CLSM images of
biocakes formed on the surface of hollow fiber membrane
after 12 days operation of the continuous MBR.
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Figure IV-13. Relative C8-HSL degradation stability and mechanical stability
of macrocapsules during continuous MBR operation. Error bar:
standard deviation (n=3)
IV.3.7. 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 lab-scale MBRs fed with real wastewater generated from a local restaurant
(Figure IV-14). 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 14. In the first cycle,
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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 (Kim et al., 2013b). 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 three-
fold overall in Figure IV-14.
At the end of the first cycle, the used filtration membranes from both MBRs were
cleaned with 1000 ppm NaOCl4 solution and then reinserted into each MBR for the
second cycle in Figure IV-14. Just before the second cycle started, activated sludges
were taken out from both control and QQ MBRs and remixed together and
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 number of biocakes [25.8 (±1.2)
mg] formed on the used membrane in the QQ MBR was much less than that [13.3
(±1.2) mg] in the control MBR (Figure IV-15a). 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 IV-15b).
It is worth noting that when the alginate beads with neither polymeric coating
layer nor QQ bacteria were 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 IV-16a). It was because the alginate beads became disintegrated
from the early stage of operation in the MBR fed with real wastewater and thus
131
physical cleaning effect of the alginate beads disappeared. As a matter of fact, we
observed most of the alginate beads were dissolved like porridge at the end of the
run (Figure IV-16b).
Since 2009, QQ effects in MBR for wastewater treatment have been reported
successively(Cheong et al., 2014, Kim et al., 2013b, Kim et al., 2013a, Jiang et al.,
2013, Oh et al., 2012, Jahangir et al., 2012, Yeon et al., 2009b). However, they made
successful QQ applications 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 an MBR, the delay of TMP rise-up in MBR with QQ
macrocapsules is very encouraging from the viewpoint of energy saving in the MBR
operation. Consequently, macrocapsules might play an important role in the design
of a future MBR with energy saving.
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. Both MBRs generated similar COD
concentrations in permeates with more than 93% of COD removal efficiencies:
6.7−12.5 mg/L with vacant macrocapsules and 5.3−11.4 mg/L with QQ-
macrocapsules. In addition, the change of average microfloc size in mixed liquor did
not make any significant difference between the two MBRs over the entire operating
period.
132
Figure IV-14. TMP profiles during the operation of continuous MBR fed with
real wastewater. In the 1st cycle, the vacant-macrocapsule and
macrocapsule with QQ bacteria were inserted in the Control
and QQ MBRs, respectively. At the end of the 2nd cycle, used
membranes were taken out of both MBRs for analyzing
biocakes with CLSM and EPS concentrations in biocakes.
133
(a)
(b)
Figure IV-15. (a) Reconstructed CLSM images of biocakes formed on the
surface of hollow fiber membranes after the same operating
period of 9 days in the control and QQ MBRs. The sampling
was done on the 32nd day in the 2nd cycle in Figure IV-14. (b)
Polysaccharide and protein concentrations per unit membrane
area in the biocakes at the end of 9 days operation. The
sampling was done on the 32nd day in the 2nd cycle in Figure
IV-14. Error bar: standard deviation (n=3)
134
(a)
(b)
Figure IV-16. (a) TMP profiles of the MBR with and without alginates beads
during continuous operation with real wastewater. (b) The
photograph of alginate beads in one MBR at the end of 26
days of operation in (a).
IV.3.8. Biofouling Inhibition by W-bead in a Continuous MBR
The biofouling inhibition effect by QQ bacteria entrapping W-bead was tested
through the parallel operation of two lab-scale MBRs. To test this, 500 vacant
macrocapsules containing no QQ bacteria were put into one MBR (i.e. the control
135
MBR), while 500 QQ macrocapsules were put into the other MBR (i.e. the QQ
MBR). The COD removal efficiencies were maintained around 97-98% based on the
permeate for both the control and QQ MBRs over the operation period of 30 days
(Figure IV-17a). It took 4 - 10 days to reach a TMP of 40 kPa in the control MBR in
which only the physical cleaning effect would be expected through collisions
between moving vacant W-bead and hollow fiber membranes. On the other hand, it
took 18 days to reach the same TMP of 40 kPa in the QQ MBR, in which both
physical and biological (i.e. quorum quenching) effects would be expected. In
summary, in the continuous MBR, the QQ effect of W-bead was pronounced, such
that the rate of TMP rise-up was delayed by about two-fold.
In order to visually confirm the inhibition of biofouling with macrocapsules, the
used membrane modules were taken out of both MBRs after the same operating
period to visualize the biocakes that formed on the surface of the membrane (Control
MBR: ~44 kPa, QQ MBR: ~8kPa). The CLSM images clearly show that the biocake
that formed on the used membrane in the QQ MBR was much smaller than in the
control MBR (Figure IV-17b). Furthermore, the amount of total attached biomass in
the control MBR was 134 (±24) mg, whereas that in the QQ MBR was only 68 (±27)
mg.
136
(a)
(b)
Figure IV-17. (a) Biofouling inhibition of W-beads in continuous MBR. (b)
Reconstructed CLSM images of biocakes formed on the hollow
fiber membrane.
137
IV.3.9. Stability of W-bead in Various Wastewater
It was shown that W-beads were able to inhibit biofouling for up to 30 days in the
continuous lab-scale MBR fed with synthetic wastewater. Also, to confirm their
potential for practical applications, QQ bacteria entrapping W-beads were measured
the change of shape, mechanical stability, and QQ activity in synthetic and real
wastewater. During 84 day in synthetic wastewater, these W-beads did not change
any shape as shown in (Figure IV-18). The average size of W-beads in each
environment was not significantly changed as shown in Figure IV-19a. In addition,
QQ activity of QQ bacteria in the W-bead, maintained after 84 days of both synthetic
and real wastewater environment (Figure IV-19b).
Figure IV-18. Change of shape of QQ bacteria entrapping W-beads in
synthetic and real wastewater.
138
(a)
(b)
Figure IV-19. Change of (a) average size of W-beads and (b) relative activity
of W-bead in synthetic and real wastewater environments. Error
bar: standard deviation (size data: n=10, QQ activity data: n=3)
139
IV.4. Conclusions
The purpose of this chapter was to enhance the mechanical stability of QQ
bacteria entrapping moving beads. Based on above results, the following
conclusions were made:
● As a new bead manufacturing coating platform (i.e., Macrocapsule), we
successfully coated alginate beads by a polymeric membrane layer using a phase
inversion method, which is well-known in the conventional preparation of
asymmetric membrane. Also, the other manufacturing platform (i.e., W-bead) was
constructed using high molecular weight synthetic polymer.
● During the preparation of macrocapsule, the decrease of QQ activity was
caused by the membrane resistance and dead bacteria in the surface of the microbial
bead. However, the decreased QQ activity overcame through the reactivation
process.
● Membrane layer of macrocapsule prevented QQ bacteria from leaking
outside in harsh chemical environment. Moreover, QQ bacteria within
macrocapsules were able to restore QQ activity by intensive culturing in LB broth.
● When the macrocapsules were applied to continuous MBR fed with real
wastewater, it successfully alleviated membrane biofouling resulting in less biocake
on the membrane surface.
● CLSM analysis confirmed that the QQ bacteria were entrapped in W-bead.
140
Moreover, the W-bead profitably inhibited biofouling in continuous MBR system.
● The mechanical stability and QQ activity of W-beads maintained in
synthetic and real wastewater condition for 84 days. It is concluded that the bead will
be successfully applied to practical MBR application.
141
Chapter V
Conclusions
V. Conclusions
142
143
In this study, as a novel approach to biofouling inhibition in MBR, we developed
three types of the cell entrapping bead (CEB) with quorum quenching (QQ)
microorganism which produces AHL-lactonase enzyme. All beads showed
excellent biofouling control in batch and continuous MBRs fed with synthetic or
real wastewater. Based on the experimental results, the following conclusions were
made:
● QQ bacteria (i.e., Rhodococcus sp. BH4) were entrapped in alginate moving
beads (CEBs), and QQ activity against autoinducer-1 (AI-1) was successfully
maintained due to the highly porous microstructure of alginate matrix.
● In the continuous lab-scale MBR process, membrane biofouling was
delayed 10 times by both physical cleaning (i.e., the collision of free-moving bead)
and biological inhibition effect (i.e., QQ).
● As a new bead manufacturing coating platform (i.e., Macrocapsule), we
successfully coated CEB (or alginate bead) by a polymeric membrane layer using a
phase inversion method. CEB surface moisture and PSf solution due to the non-
solvent phase separation phenomenon, the PSf membrane could be stably formed on
the CEB surface without complex process.
● After exposure to chemical treatment condition, the macrocapsule retained
QQ activity, but original CEB decreased. After the restoration process with LB
media, the CEB`s QQ activity of macrocapsule has increased by about 200% and
the original CEB has decreased to about 50%.
144
● The macrocapsule demonstrated successful performance by ~300%
retardation of membrane biofouling in continuous MBR fed with real wastewater
for 35 days.
● As strong manufacturing platform, W-bead was prepared by using high
molecular weight polyvinyl alcohol with alginate matrix. The alginate matrix formed
a spherical shape rapidly in the ionic solution, and then entrapped polyvinyl alcohol
was cross-linked by sodium sulfate to form physically/chemically stable W-bead.
● The W-bead showed successful performance by ~200% retardation of
membrane biofouling in continuous MBR fed for 25 days.
● As a long-term stability test, we confirmed W-bead had mechanical stability
and QQ activity for over 80 days even in real wastewater.
145
Chapter VI
Suggestions
146
147
Membrane bioreactors (MBR) has continued to improve on the basis of
technological advances, but biofouling is an inevitable problem. Thus effective
solution would be necessary to mitigate operating and maintenance cost. The
technique of immobilization of quorum quenching (QQ) bacteria on the moving bead
developed in this study showed a significantly improved performance than the
previous method. Therefore, we suggest the research direction to develop the next-
generation QQ-moving carrier as the final chapter.
In the MBR system, there are many types of signal molecules that microorganisms
use such as acyl-homoserine lactones (AHLs), oligopeptide, autoinducer-2,
autoinducer-3, or indole, etc. Therefore, the QQ method to prevent the biofouling
using only Rhodococcus sp. BH4 is a restricted application technology. The
lactonase enzyme, BH4 produces, can decompose one type of signal molecule
known as AHLs that gram-negative bacteria use. Of course, pyrosequencing reveals
that certain genus (Enterobacter and Dyella), which are gram-negative bacteria, are
dominantly involved in biofilm formation among microbial communities present in
MBR. However, even considering this fact, it is hard to say that bacteria population
and composition are same in all MBR because of the diversity of feed water and
operating conditions. Thus, the QQ technique is not always an applicable solution to
all biofouling problem, and the technology mentioned in this paper would apply to
the MBR which predominantly exists the microorganism who play quorum sensing
(QS) with AHLs. To develop all-around QQ technology, further studies should be
followed on identifying QS signal molecules present in the various MBR and
148
securing QQ enzymes (or microorganism that produce it) that can decompose the
molecules.
There is room to improve the physical and chemical stability of the moving carrier
in terms of material. The beads used in this study was selected based on its
biocompatibility, specific gravity, the efficiency of mass transfer, and manufacturing
availability. Alginate, cellulose, PVA carrageenan, agar, and chitosan were the
general option for cell immobilization, herein alginate was chosen because of its
superior biocompatibility, specific gravity for circulating, and reproducibility for
scientific analysis. In the macrocapsule study, although PVDF, PES, and PSf could
form membrane layer, PSf mainly used for MBR experiments because of it fast phase
inversion, chemical stability, and inexpensive cost. In the W-bead`s early study,
PVA which was fabricated by physical cross-linking was not enough reliable and
rigid. In subsequent studies, we could achieve high durability and reproducibility via
the chemical double cross-linking method. However, in this study, we fabricated cell
immobilization carrier with the only polymeric material. The composite moving
carrier can be a next-generation study since composite material (i.e.,
inorganic/organic composite structure) is more efficient on biocompatibility, control
of density, inner-space construction, and physical/chemical stability.
Therefore, I believe that research on recent QS studies and newly discovered
composite materials will be an excellent opportunity to open up practical possibilities
for inhibition of biofouling by using moving carriers in actual MBR plants.
149
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국 문 초 록
정족수감지 억제 세균이 고정화된 유동성 담체를 이용한
하폐수 처리용 분리막 생물반응기에서의
생물막오염 제어
서울대학교 대학원
화학생물공학부
김상룡
분리막 생물반응기 (Membrane bioreactor, MBR)는 고도 하폐수처리 공정의
하나로서 널리 사용되고 있지만, 생물막 오염 (biofouling)이란 고질적 문제를 안
고 있다. 최근 정족수감지 억제 (Quorum quenching) 기술이 생물막 오염을 억제
하기위한 새로운 해결책으로 주목 받고 있다. 본 연구의 목적은 생물막 오염을
보다 효과적으로 방지하기 위해서 물리세정 효과와 정족수감지 억제 효과를 동
시에 기대할 수 있는 유동성 담체를 개발하는데 두었다.
정족수감지 억제 미생물로 알려진 Rhodococcus sp. BH4를 칼슘-알지네이트
매트릭스에 고정화하여 “유동성 담체 (Cell entrapping bead, CEB)”라고 부르는
새로운 막오염 억제제를 개발하였다. 유동성 담체의 비중은 물과 비슷하기 때문
에 MBR내의 폭기에 의해서 자유롭게 유동하여 분리막 표면에 충돌하여 물리세
정 효과를 발생시킬 뿐만 아니라 효과적으로 막오염 미생물의 정족수 감지를
억제한다. 특히, 막오염 미생물들의 정족수 감지가 억제될 때 세포외 고분자 물
질의 생성이 교란되어 느슨하게 결합된 생물막이 형성된다. 그러므로, 유동성
담체의 물리 세정 효과가 동반 상승되었다. 이를 통해 연속공정 시스템에서 기
존 생물막 반응기 대비 8배 이상 막오염을 지연시킬 수 있었다.
175
후속 연구로 CEB를 실제 분리막 생물반응기 공정에 적용하기 위해서 물리적
및 화학적 안정성을 향상시키는 실험을 수행하였다. 그 첫 번째는, 상전이 기술
(Non-solvent induced phase separation)을 이용하여 유동성 담체를 다공성 분리
막으로 둘러싼 “유동성 코팅 담체 (Macrocapsule)”이다. 다공성 분리막은 양친매
성 고분자 용액과 수분을 포함하고 있는 유동성 담체 사이에서 자발적 상전이
현상으로 인해서 담체 표면에 형성되었다. 유동성 코팅 담체는 실험실 규모의
연속공정 분리막 생물반응기에서 80일 동안 우수한 생물막 제어 효과를 보였으
며, 정족수감지 억제 활성을 유지하였다. 두 번째는 polyvinyl alcohol (PVA)과
알지네이트를 이중 가교 결합하여 제조한 “W-bead”이다. W-bead는 실폐수를
처리하는 분리막 생물반응기에 적용되어 우수한 막오염 억제 효과와 정족수감
지 억제 미생물의 활성을 유지함을 보여주었다. 본 연구는 효과적인 정족수 감
지 억제 미생물 보관체로 유동성 담체를 제안했으며, 실제 분리막 생물반응기에
활용할 수 있는 잠재력을 보여주었다.
주요어: 분리막 생물반응기, 정족수감지, 분리막 오염, 정족수감지 억제 세균, 유
동성 담체, 세포 고정화, 폐수처리
학 번: 2007-23079
