Bacillus Sp. T5 AND Delftia - T6 On Microbial Community

ISTANBUL TECHNICAL UNIVERSITY  GRADUATE SCHOOL OF SCIENCE
ENGINEERING AND TECHNOLOGY
ISOLATION OF NOVEL QUORUM QUENCHING BACTERIA FOR THE

CONTROL OF MEMBRANE BIOFOULING AND THE EFFECT OF
Bacillus sp. T5 AND Delftia sp. T6 ON MICROBIAL COMMUNITY
STRUCTURE IN MEMBRANE BIOREACTOR
Ph.D. THESIS
Bahar YAVUZTÜRK GÜL
Department of Environmental Engineering
Environmental Biotechnology Programme
MAY 2018
ISTANBUL TECHNICAL UNIVERSITY  GRADUATE SCHOOL OF SCIENCE
ENGINEERING AND TECHNOLOGY

Ph.D. THESIS

(501102800)
Department of Environmental Engineering
Environmental Biotechnology Programme
Thesis Advisor: Prof. Dr. İsmail KOYUNCU
MAY 2018
ISTANBUL TEKNİK ÜNİVERSİTESİ  FEN BİLİMLERİ ENSTİTÜSÜ
MEMBRAN BİYOTIKANMASININ KONTROLÜ AMACIYLA YENİ

QUORUM QUENCHİNG BAKTERİLERİNİN İZOLASYONU VE
Bacillus sp.T5/Delftia sp. T6 TÜRLERİNİN MEMBRAN BİYOREAKTÖRDEKİ
MİKROBİYAL KOMÜNİTEYE ETKİSİNİN BELİRLENMESİ
DOKTORA TEZİ

(50102800)
Çevre Mühendisliği Anabilim Dalı
Çevre Biyoteknolojisi Programı
Tez Danışmanı: Prof. Dr. İsmail KOYUNCU
MAYIS 2018
Bahar YAVUZTÜRK GÜL, a Ph.D. student of İTU Graduate School of Science
Engineering and Technology student ID 501102800, successfully defended the
thesis/dissertation entitled “ISOLATION OF NOVEL QUORUM QUENCHING
BACTERIA FOR THE CONTROL OF MEMBRANE BIOFOULING AND THE
EFFECT OF Bacillus sp. T5/Delftia sp. T6 ON MICROBIAL COMMUNITY
STRUCTURE IN MEMBRANE BIOREACTOR”, which she prepared after fulfilling
the requirements specified in the associated legislations, before the jury whose
signatures are below.
Thesis Advisor : Prof. Dr. İsmail KOYUNCU ..............................

İstanbul Technical University
Jury Members : Prof. Dr. Süleyman ÖVEZ .............................

Prof. Dr. Mehmet KİTİŞ ..............................

Süleyman Demirel University
Prof. Dr. Bülent İNANÇ ..............................

Prof. Dr. Halil HASAR ..............................

Fırat University
Date of Submission : 21 March 2018

Date of Defense : 10 May 2018
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To my lovely family,
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FOREWORD
First and foremost, I express my profound gratitude to my advisor Prof. Dr. İsmail
KOYUNCU for his wide knowledge, patient and complaisant guidance and giving me
the opportunity to be a part of MEMTEK (National Research Center on Membrane
Technologies) family. He has shed light on my own path by offering me unique
opportunities and the freedom that I need on my modest journey in the academic world.
Without his support and encouragement, the completion of this work would not have
been possible.
I would like to thank my professors Prof. Dr. Süleyman ÖVEZ and Prof. Dr. Mehmet
KİTİŞ sincerely for the constructive and helpful comments and suggestions they have
made in order to improve my researches during the thesis. I am really grateful to Assoc.
Prof. Dr. Derya İMER for helping me comprehend the field of environmental
engineering and her guidance on writing scientific reports. I also wish to thank Dr.
Börte KÖSE MUTLU very much for sharing her precious experiences with me on the
subject of Quorum Quenching. Special thanks to Serkan GÜÇLÜ, M. Emin
PAŞAOĞLU, Çağrı ŞAHİN and Reyhan ŞENGÜR TAŞDEMİR for their help at
SEM, HPLC and confocal analysis, to Türker TÜRKEN and Recep KAYA who
contributed to successful installation of MBR system, and also to Gülsüm Melike
ÜRPER who has corrected my academic writings.
I specially would like to thank each member of the MEMTEK family for their sincere
friendship, kindness and help. My special and deepest thanks to my colleagues Ayşe
YÜKSEKDAĞ, Ayşegül Derya ALTINAY Bahriye ERYILDIZ, Başak KESKIN,
Burcu SAYINLI, Elifnur GEZMIŞ YAVUZ, Enise PEKGENÇ, Meltem AĞTAŞ,
Sevde KORKUT and Öykü MUTLU SALMANLI for their supports, warm
friendships, cheerful conversations and original ideas.
Thanks to The Scientific and Technological Research Council of Turkey (TÜBITAK)
(Project number: 114Y706) and Istanbul Technical University Scientific Research
Found. No: 39549 for their supports.
Most importantly, I express my thanks from the bottom of my heart to my father Vezir
YAVUZTÜRK and my mother Rezan YAVUZTÜRK, my sisters Gonca
TAVUKÇUOĞLU, Demet TOPÇU, Esma YAVUZTÜRK, my brother Yunus Emre
YAVUZTÜRK, that I cannot remunerate and who offer me a warm family atmosphere
which is mixed with love, compassion and care, and never refrain to their endless
supports.
I would like to express my thanks and endless love to my beloved husband Bilal GÜL,
who always makes his envelope, protective and affectionate heart felt in my heart. I
would not be able to complete this thesis without his limitless patience, encouraging
support and huge efforts. My eternal love and thanks go to my beloved sons Ahmet
Yusuf GÜL and Ali Selim GÜL, who are the meaning of my life and the ones that I
get strength from their pure love when it is hard to cope with the difficulties.
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All the praises and thanks be to almighty Allah (swt) whose blessings are countless
and who is the Lord of the universes, and peace and blessings be upon our beloved
Prophet Muhammad, the Messenger of Allah.
March, 2018 Bahar YAVUZTÜRK GÜL

(M.Sc. Biologist)
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TABLE OF CONTENTS
Page
FOREWORD ............................................................................................................. ix
TABLE OF CONTENTS.......................................................................................... xi
ABBREVIATIONS ................................................................................................. xiii
LIST OF TABLES ................................................................................................... xv
LIST OF FIGURES ............................................................................................... xvii
SUMMARY ............................................................................................................. xix
ÖZET ............................................................................................................. xxiii
INTRODUCTION .................................................................................................. 1
Purpose of Thesis ............................................................................................... 1
Unique Aspect .................................................................................................... 2
Organization of the Thesis ................................................................................. 2
LITERATURE REVIEW...................................................................................... 5
Quorum Quenching Application in Membrane Bioreactors .............................. 5
2.1.1 Enzymatic quorum quenching .................................................................... 5
2.1.2 Bacterial quorum quenching ....................................................................... 7
2.1.3 Fungal quorum quenching ........................................................................ 12
ASSESSMENT OF NEW ENVIRONMENTAL QUORUM QUENCHING
BACTERIA AS A SOLUTION FOR MEMBRANE BIOFOULING
................................................................................................................ 15
Introduction ...................................................................................................... 15
Materials and Methods ..................................................................................... 17
3.2.1 Bacterial strains, growth media, culture condition ................................... 17
3.2.2 Isolation of QQ consortia from environmental samples ........................... 17
3.2.3 Strain identification and phylogenetic analysis ........................................ 18
3.2.4 Characterization of the quorum quenching activity of isolates................. 19
3.2.4.1 Measurement of quorum quenching activity...................................... 19
3.2.4.2 Detection of lactonase activity ........................................................... 19
3.2.4.3 Preparation of QQ-fibers .................................................................... 20
3.2.4.4 MBR operation ................................................................................... 20
Results and Discussion ..................................................................................... 21
3.3.1 Novel AHL degrading bacteria ................................................................. 21
3.3.2 Detection of QQ capacity .......................................................................... 25
3.3.3 Detection of exo-endo enzyme ................................................................. 27
3.3.4 Detecting the presence of lactonase using an acidification test ................ 28
3.3.5 Effect of QQ-fiber with T5 on C8-HSL biodegradation ........................... 30
3.3.6 Control of membrane biofouling by new antibiofouling agent Bacillus sp.
T5 ....................................................................................................................... 31
Conclusions ...................................................................................................... 34
Acknowledgments ............................................................................................ 35
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EVALUATION OF A NOVEL ANTI-BIOFOULING MICROORGANISM
(Bacillus sp. T5) FOR CONTROL OF MEMBRANE BIOFOULING
AND ITS EFFECT ON BACTERIAL COMMUNITY STRUCTURE
IN MEMBRANE BIOREACTORS .................................................... 37
Introduction ...................................................................................................... 37
Methods and Materials ..................................................................................... 39
4.2.1 QQ bacteria: Bacillus sp. T5 ..................................................................... 39
4.2.2 Preparation of sodium alginate beads........................................................ 40
4.2.3 Measurement of AHL degrading activity of the free T5 and T5 beads .... 41
4.2.4 Operation of MBR system ........................................................................ 41
4.2.5 Molecular analysis..................................................................................... 41
4.2.6 Analytical methods.................................................................................... 42
4.2.7 Statistical analysis ..................................................................................... 42
Results and Discussion ..................................................................................... 43
4.3.1 Molecular characteristics of Bacillus sp. T5 ............................................. 43
4.3.2 Immobilization of Bacillus sp. T5 and its anti-biofouling effect on MBR
performance ........................................................................................................ 45
4.3.3 Quorum quenching effects on microbial community structure ................. 48
Conclusion ........................................................................................................ 51
SELECTION OF THE QUORUM QUENCHING (QQ) BACTERIA FOR
MEMBRANE BIOFOULING CONTROL: EFFECT OF
DIFFERENT GRAM-STAINING QQ BACTERIA, Bacillus sp. T5
AND Delftia sp.T6, ON MICROBIAL POPULATION IN MBR..... 53
Introduction ...................................................................................................... 53
Material and Method ........................................................................................ 54
5.2.1 MBR systems ............................................................................................ 54
5.2.2 QQ bacteria: Bacillus sp. T5 and Delftia sp. T6 and preparation of the
immobilization media......................................................................................... 56
5.2.3 Measurement of AHL degrading activity via bioassay for free and
ımmobilised QQ bacteria ................................................................................... 57
5.2.4 Molecular analysis/ DNA extraction, PCR amplification and HiSeq
platform sequencing ........................................................................................... 57
5.2.5 Analytical methods.................................................................................... 58
5.2.6 Statistical analysis ..................................................................................... 58
Results .............................................................................................................. 58
5.3.1 QQ bacteria, activity of the beads ............................................................. 58
5.3.2 Anti-biofouling effect of QQ bacteria on MBR ........................................ 60
5.3.3 Differentiation of bacterial composition in the mixed liquor .................... 62
Discussions ....................................................................................................... 64
Compliance with Ethical Standards.................................................................. 65
5.6.1 Conflict of interest..................................................................................... 65
5.6.2 Ethical statement ....................................................................................... 65
CONCLUSIONS AND RECOMMENDATIONS ............................................. 67
REFERENCES ......................................................................................................... 71
CURRICULUM VITAE .......................................................................................... 83
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ABBREVIATIONS
QS : Quorum Sensing
QQ : Quorum Quenching
AHL : Acyl-homoserine Lactone
AIP : Autoinducing Peptides
AI-2 : Autoinducer-2
MBR : Membrane Bioreactor
EPS : Extracellular Polymeric Substances
SMP : Soluble Microbial Product
TMP :Trans-membrane Pressure
C8-HSL : N-octanoyl-homoserine Lactone
LB : Luria–Bertani Medium
COD : Chemical Oxygen Demand
gDNA : Genomic DNA
HRT : Hydraulic Retention Time
MLVSS : Mixed Liquor Volatile Suspended Solid
PCR : Polymerase Chain Reaction
SRT : Solids Retention Time
CEB : Cell Entraping Bead
PVDF : Polyvinylidene Fluoride
SCADA : Supervisory Control and Data Acquisition
TKN : Total Kjeldahl Nitrogen
X-GAL : 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside
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LIST OF TABLES
Page
QQ bacteria and their AHL degradation genes and enzymes. .................. 7

Quorum quenching strategies adopted to control of membrane biofouling.
................................................................................................................ 13
Table 3.1 : Identification of 19 AHL-degrading strains on the basis of 16SrRNA gene
sequence and their AHL degradation and lactonase profile. .................. 24
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LIST OF FIGURES
Page
Figure 3.1 : Preparation of QQ-fibers. Centrifuged QQ bacteria were injected into the
one side sealed PVDF hollow fiber microfiltration membranes from the
open side. Empty fibers not containing QQ bacteria. ............................. 20
Figure 3.2.:.Neighbour joining tree based on the 16S rRNA gene, showing
phylogenetic relationships among quorum quenching bacteria isolated
from pond, estuary and saltern. Bootstrap coefficients below 50% were
not shown. Scale bar, 0,05 substitutions per nucleotide position. .......... 22
Figure 3.3 : Quorum quenching activity of whole cell (C8-HSL degradation in in vitro
assay) of isolates. (a) ITU pond, (b) Haliç Estuary and (c) Çamaltı Saltern.
................................................................................................................ 26
Figure 3.4 : Comparison of the intracellular and extracellular QQ activities of the
isolated strains. The data are shown as means ± SD. Remaining AHL
levels in the sterile supernatant and cell extract of the isolates were
determined after incubation of the 2 mM AHL with sterile culture
supernatant and cell extract in Tris-HCL buffer (n=3). .......................... 28
Figure 3.5 : Lactonase activity of the supernatant and cell extract of the isolates. Data
shown are the averages (± SEM) of the restorated QS signal after
acidification test (n=3). ........................................................................... 29
Figure 3.6 : Bioassay for the determination of C8-HSL degradation in 60 min by
Bacillus sp. T5. ....................................................................................... 30
Figure 3.7 : Quorum quenching activity of QQ-fiber containing Bacillus sp. T5. The
C8-HSL degradation of QQ-fiber containing 8.2 mg Bacillus sp. T5/cm3
cubbyhole was determined in Tris−HCL solution. Control (vacant fiber)
was also tested to check the adsorption of C8-HSL on the fiber. Error bar:
standard deviation (n =3). ....................................................................... 31
Figure 3.8 : Application of the quorum quenching bacteria isolated from saltern for
wastewater treatment. TMP profile in the MBRs. Empty fibers and QQ-
fibers with Bacillus sp. T5 were inserted in the control and the QQ
reactors, respectively. ............................................................................. 32
Figure 3.9 : The CLSM images of biofilm formed on the membrane surface. (a1)
Control MBR, a plan view, (a2) Control MBR, cross-section view, (b1)
QQ MBR, a plan view, (b2) QQ MBR, cross-section view at the end of
the 10 days of MBR operation, stained with SYTO9. Magnification: X40.
Image size 501.76 × 501.76 μm. ............................................................ 33
Figure 3.10 : COD and TKN removal efficiencies of control and QQ MBR during the
operations................................................................................................ 34
Figure 4.1 : Standard curve for the calculation of residual amounts of C8-HSL. .... 39
Figure 4.2 : Pumping system for the preparation of sodium alginate beads. T5- 4%
sodium alginate suspension was dropped in 3% CaCl2 solution through
this system and forming beads left in CaCl2 solution for 8 hours .......... 40
Figure 4.3 : Comparison of the growth rate of Bacillus sp. T5 in the LB medium and
the synthetic wastewater. ........................................................................ 43
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Figure 4.4 : Gram staining of Bacillus sp. T5 after 24 h of incubation. ................... 44
Figure 4.5 : C8-HSL degradation of the free and immobilized Bacillus sp. T5. AHL
amounts in the samples were determined after incubation of the 200 nM
AHL with free cell and QQ-beads in Tris-HCL buffer. Vacant beads were
used as a control. Error bar: standard deviation (n=3) ............................ 45
Figure 4.6 : SEM image of (a) the surface and (b) the cross-section of the sodium
alginate beads. Immobilized rod-shaped Bacillus sp. T5 can be seen in the
figures. .................................................................................................... 46
Figure 4.7 : Transmembrane pressure (TMP) profiles during the MBR operation.
Empty beads and QQ-beads with Bacillus sp. T5 were applied to the
control and the QQ reactors. ................................................................... 47
Figure 4.8 : The CLSM images of biofilm on the membrane surface. (a) Control MBR,
cross-section view, (b) QQ MBR, cross-section view at the end of the
MBR operation, stained with Live/dead BacLight staining. Magnification:
X40. Image size 501.76 × 501.76 μm. .................................................... 48
Figure 4.9 : Dominant bacterial phyla in the MBRs and changes in the relative
abundances of the dominant phyla during quorum quenching process...49
Figure 4.10 : Relative abundance of dominant bacterial Genus in control and QQ
reactor. .................................................................................................... 51
Figure 5.1 : Schematic diagram of lab-scale MBR operation and preparation of the
beads. ...................................................................................................... 55
Figure 5.2 : Growth rate of Delftia sp. T6 in the LB medium and the synthetic
wastewater. ............................................................................................. 59
Figure 5.3 : Degradation of C8-HSL via QQ-beads with Bacillus sp. T5 and Delftia
sp. T6. C8-HSL concentration in the samples were determined using
bioassay. Vacant beads were used as a control. Error bar: standard
deviation (n=3)........................................................................................ 60
Figure 5.4 : Transmembrane pressure (TMP) profiles of the control and the QQ
reactors during the MBR operation with vacant beads and QQ-beads with
Bacillus sp. T5 (a) and Delftia sp. T6(b). ............................................... 61
abundances of the dominant phyla during quorum quenching process with
T5 (a) and T6 (b). (C: Control, Q:QQ reactor) ....................................... 62
Figure 5.6 : Relative abundance of dominant bacterial classes in control and QQ
reactor for operation with T6 (a) and T5(b). ........................................... 63
xviii
SUMMARY
The outcomes of the increase in the World population and industrialization, waste
discharging to non-receipt bodies or an insufficient treatment cause accumulation and
rapid decrease in natural resources day by day. The shrinkage of these resources
results in significant environmental issues. To overcome the water scarcity, there is a
global effort to expansion on water resources and reuse of wastewater. Membrane
Bioreactor (MBR) technology is an advanced biological wastewater treatment
technology. Nevertheless, biofouling on membrane surface remains one of the main
obstacles of profitable MBR operation.
Quorum Quenching (QQ) is an innovative approach attempting to solve the biofouling

problem in MBRs. QQ applications prevents the group behavior of microorganisms
therefore ceases/retards the formation of biofilm. Researchers have demonstrated that
Quorum sensing and biofilm formation are strongly linked and act to regulate the
biofilm formation on the membrane surface in MBR. C6-HSL and C8-HSL, 3-oxo-
C8-HSL C4-HSL, C6-HSL, C8-HSL, 3-oxo-C8-HSL, C10- HSL, C12-HSL, 3-oxo-
C12-HSL and C14-HSL have been detected from biofilm on the membrane surfaces
and in the mix liquor of. Because biofilm formation on membranes can be controlled
by QS, several studies have turned their attention to blocking AHL signals to either
reduce membrane biofouling or improve reactor performance. In the last 10 years,
research on QQ mediated strategies for mitigating membrane biofouling in MBRs has
intensified and notable success has been achieved in this regard.
Current thesis aims to isolate new and highly effective QQ bacteria from our national
resources and to understand what are the Achyl-homoserine lactone (AHL)
degradation pathways of the isolated new bacterial strains found in marine, pond and
saltern environments that are capable of interfering with AHL-mediated Quorum
Sensing. This study also evaluates applying the selected new QQ bacteria to the MBRs
with QQ-fiber/beads for the purpose of controlling biofilm formation. Finally assess
the interaction between the indigenous microbial community and two different QQ
bacteria, Bacillus sp. T5 (Gram positive) and Delftia sp. T6 (Gram negative), in order
to develop a comprehensive understanding of occurance of QQ mechanisms in MBR
step by step.
A BLAST search of GenBank was performed to identify if there was any taxonomical
relationship between the sequences available in the database and those of the 16S
rRNA gene of the 25 new isolates. New izolates belonged predominantly to
Gammaproteobacteria (G2, G4, T21, T22, T4, H1, H2, H3, H4, H7, H8, H9, H10),
xix
Betaproteobacteria (G6, T6, H5), Firmicutes (G5, T5), and Actinobacteria (G7). The
assessment of the partial nucleotide sequence of the amplified 16S rRNA gene
revealed a strong homology of 97-100 between this sequence and that of the bacteria
from the following genera: Shewanella, Acinetobacter, Klebsiella, Bacillus, Deftia,
Vibrio, Comamonas, Microbacterium, and Pseudomonas, Brevundimonas, Bosea,
Stenotrofomonas and Tistrella.
Minimal medium method was used which employs C8-HSL as the sole carbon source.
AHL degradation activity was measured via Biotest. The activity test results indicated
that Tistrella exhibited QQ activities for the first time, and strains T21, T22, T4, G2,
G4 G5, G6, H1, H3, H7, H8, H10 presented lactonase activity which have not been
recorded in the previous research. Strain T5 and T6 from saltern and strain S5 and S10
from leachate degraded more than 90% of the AHL, thus each of them exhibits
significant AHL activity. Results point that QQ is the strategy that may typically
adopted in the different environmental sources to overcome stress conditions and
achieve competitive advantages.
After isolation of the QQ bacteria, a new immobilization medium named QQ-fiber was
produced by hollow fiber membranes. MBRs was operated with QQ-fiber immobilized
with Bacillus sp. T5 and biofilm formation was successfully mitigated with 25 %
efficiency. To compare the immobilizations media and two different QQ bacteria,
sodium alginate beads entrapped with chosen strains Bacillus sp. T5 and Delftia sp.
T6. Total anti-biofouling effects of T5 and T6 beads were calculated as 85% and 75%,
respectively.
The one of the major finding of the thesis was bacterial community changes during the
QQ-MBR runs with QQ bacteria which investigated using metagenomic analysis. The
effect of QQ on the bacterial community was examined in detail on the basis of
phylum, class and genus. The gap in the understanding bacterial community dynamics
in MBRs was addressed by investigating the behavior of two types of QQ bacteria,
Bacillus sp. T5 and Delftia sp. T6, within control and QQ MBRs via Illumina's HiSeq
method using 16S rRNA genes. The analysis of the results and comparison of relative
abundances of taxa in MBR operations were revealed that different QQ bacteria have
effect on different taxa. Proteobacteria, Bacteroidetes, Chloroflexi, Firmicutes,
Acidobacteria and Actinobacteria were most QQ affected phyla in the microcosms.
Furthermore, when the QQ MBR was operated with gram-positive T5, a reduction in
the abundance of gram-positive classes and an increase in the abundance of gram-
negative classes was observed. On the contrary, when the QQ MBR was operated with
gram-negative T6, a reduction in gram-positive classes and an increase in gram-
negative classes was observed. As such, the outputs of the Illumina Hi-seq analysis
were demonstrated that use of gram-negative QQ bacteria in the reactor induced a
gram-positive microbial community, and vice versa. This indicates a close interaction
occurs between indigenous gram-negative and positive bacterial classes and Bacillus
sp. T5/Delftia sp. T6 is fundamental to the performance of MBRs. To the best of the
researchers’ knowledge, this is the first study of its kind to demonstrate such a
relationship. Molecular data of operation with mixed T5 and T6 was support this
finding.
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To summarize, within the the scope of this thesis, new and active species were isolated
from different sources, used in MBR as an antibiofouling agent and successfully
reduced biofilm and proven to be a highly effective biotechnological solution method
for biofouling problem. Moreover, three different immobilisation media (QQ-Fiber,
SA Beads and PVA Beads) were applied with selected QQ bacteria. Finally, according
to the results of molecular analysis, different QQ bacteria were found to act by
interacting with different microbial groups.
The results of this thesis reveal valuable information about the QQ technologies that
is indispensable application for mitigating biofouling. It is anticipated that the findings
of this thesis will lead to an enhanced knowledge of the relationships that exist between
QQ bacteria and the bacterial community structures in MBRs. Additionally, it
assistances to the researchs to select QQ bacteria/strategy in a different way from the
past selection method (like only consideration of the QQ activity) for MBR operations
and different biotechnological applications. Consequently, the results support the idea
that molecular and metagenomic analysis in MBR could be essential for membrane
biofouling control.
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MEMBRAN BİYOTIKANMASININ KONTROLÜ AMACIYLA YENİ
QUORUM QUENCHİNG BAKTERİLERİNİN İZOLASYONU VE Bacillus
sp./T5 Delftia sp. T6 TÜRLERİNİN MEMBRAN BİYOREAKTÖRDEKİ
MİKROBİYAL KOMÜNİTEYE ETKİSİNİN BELİRLENMESİ
ÖZET
Nüfusun artışı ve endüstrileşmenin bir sonucu olarak, atıkların arıtılmadan ya da

yetersiz arıtılarak alıcı ortamlara deşarj edilmesi, doğal kaynakların günden güne
tükenmesine neden olmaktadır. Bu kaynaklardaki daralma iklim değişikliği vb. çok
mühim çevresel sorunlara neden olmaktadır. Bu nedenle, su kıtlığının üstesinden
gelmek amacıyla, su kaynaklarının genişletilmesi ve atık suların yeniden kullanılması
konusunda küresel bir çaba sarfedilmektedir. Membran Biyoreaktör (MBR)
teknolojisi, ileri düzey, biyolojik atıksu arıtma teknolojisidir. Bu teknoloji vasıtasıyla
atıksulardan yüksek kalitede, kullanılabilir su elde edilebilmektedir. Ancak membran
yüzeylerinde oluşan biyotıkanma hala MBR proseslerinin önündeki en büyük
engellerden biridir.
Quorum Quenching (QQ), MBR sistemlerindeki biyotıkanma sorununun çözümü için

geliştirilmişen en yenilikçi yaklaşımlardan biridir. QQ mikroorganizmaların grup
davranışlarını engellemekte böylece biyofilm oluşumunu en az seviyeye
indirgemektedir.
Gerçekleştirilen tezde, yeni ve çok etkili QQ bakterilerini milli kaynaklarımızdan
izole edilmesi, deniz, göl ve tuzla ortamlarından izole edilen türlerin AHL
degredasyon yollarının anlaşılması, seçilen QQ bakterilerinin biyofilm oluşumunu
engellemek amacıyla, değişik tutturma ortamları kullanılarak MBR sistemlerinde
uygulanması ve nihayetinde QQ bakterileri Bacillus sp.T5 ve Delftia sp. T6 ile
MBRdaki mikrobiyal kommünite arasındaki bağlantının incelenmesi neticesinde QQ
mekanizmasının genel işleyişi konusunda kapsayıcı bir yorum geliştirebilmektir.
Elde edilen sonuçlara göre, 13 genusa ait 25 QQ bakteri türü, göl, deniz, tuzla ve sızıntı
suyu ortamlarından izole edilmiş ve genetik olarak tanılanmıştır. Bu genuslar
Shewanella, Acinetobacter, Klebsiella, Bacillus, Deftia, Vibrio, Comamonas,
Microbacterium, and Pseudomonas, Brevundimonas, Bosea, Stenotrofomonas and
Tistrella’dır. İzolasyonlar, C8-HSL’nin tek karbon kaynağı olduğu minimal medium
metodu kullanılarak yapılmıştır. AHL degredasyon aktivitesi Biotest metodu ile
ölçülmüştür. İzolatların hücre ekstraktlarındaki AHL degredasyonunun, supernatanta
oranla daha yüksek olduğu gösterilmiştir. Bu durum izole edilen türlerden büyük
çoğunluğunun intraselüler aktivitelerinin daha güçlü olduğunu ortaya koymaktadır.
Aktivite testi sonuçlarına göre Tistrella’nın QQ aktivitesine sahip olduğu ayrıca T21,
T22, T4, G2, G4, G5, G6, H1, H3, H7, H8, H10 türlerinin ise laktonaz aktivitesi
gösterdiği ilk kez ortaya konulmuştur.
xxiii
İzolatlardan T5, T6, S5, S10 % 90 üzerinde AHL degredasyon aktivitesi göstererek en
aktif QQ türleri olmuştur. Bu sonuçlar göstermektedir ki, değişik yaşam ortamlarında
yaşayan bakteriler tarafından kendi türleri adına avantaj sağlamak için edinilmiş tipik
bir strateji olabilir.
İzolasyonları takiben, hallow fiber membranlar kullanılarak, yeni bir immobilizasyon
ortamı olan QQ-Fiber üretilmiştir. Bacillus sp. T5 tutturulmuş QQ-fiberler ile
gerçekleştirilen MBR operasyonu sonucu biyofilm oluşumu % 25 oranında
engellenmiştir. İki farklı tutunma ortamının ve QQ bakterilerinin karşılaştırılması
amacıyla seçilen QQ türleri olan Bacillus sp. T5 ve Delftia sp. T6. sodium aljinat
boncuklara tutturulmuş ve MBRa uygulanmıştır. Toplam anti-biyofoling etkisi T5 ve
T6 için %85 ve %75 olarak hesaplanmıştır.
MBRdaki bakteriyel kommünite dinamikleri konusunun incelenmesi alanındaki

boşluğu doldurmak için iki farklı QQ bakteri tipi Bacillus sp. T5 and Delftia sp. T6’nın
QQ ve kontrol reaktörlerine etkileri İllumina Hi-Seq platformu kullanılarak 16 S rRNA
gen temelli araştırılmıştır. Illumina Hi-Seq analizi sonuçları detaylı olarak phylum,
class ve genus bazında incelenmiştir ve neticesinde taksonların dağılım profili
incelendiğinde, farklı QQ bakterilerinin MBR’da farklı mikrobiyal grupları etkilediği
görülmüştür ve etkilerinin birbirinin tam zıttı yönde işlediği ortaya konulmuştur
Mikrokosmosdaki en çok etkilenen taxonlar Proteobacteria, Bacteroidetes,
Chloroflexi, Firmicutes, Acidobacteria and Actinobacteria olmuştur. QQ-MBR Gram
(+) Bacillus sp. T5 ile işletildiğinde gram negatif classların artmasına neden olurken
gram pozitif classların sayısını azaltmıştır. Tam tersine Gram (-) Delftia sp. T6 gram
negatif classları azaltırken gram pozitiflerin sayısının artmasına neden olmuştur.
Karışık işletme moleküler dataları da bu bulguyu destekler niteliktedir. Bu bulgular
MBR’daki mevcut bakterial komünitenin dağılımı ile QQ bakterileri arasında cereyan
eden sıkı ilişkinin MBR performansı için çok önemli olduğunu belirtmektedir.
Literatür araştırmasına göre, bu çalışma böyle bir ilişkiyi ilk kez ortaya koymaktadır.
Bu tez kapsamında, elde edilen tüm çıktılar ışığında, biyofilm kontrolü konusunda
vazgeçilmez olan QQ teknolojisi hakkında çok kıymetli sonuçlara ulaşılmıştır. Ayrıca
QQ bakterileri ile MBR içerisindeki mirobiyal kommünite arasındaki ilişki hakkındaki
mevcut kısıtlı bilginin genişletilmesine katkı sağlanmıştır. Bu sonuçlar, başta MBR
operasyonları olmak üzere pek çok farklı biyoteknolojik uygulama için QQ
bakterisi/stratejisi seçiminde araştırmacıların eski seçim metotlarından farklı yeni bir
yol izlemesinde yardımcı olacaktır. Mikrobiyal kommünite üzerindeki moleküler
analizlerin, membran sistemlerinde biyotıkanmanın önlenmesi ve böylece bu
sistemlerin iyileştirilmesine yönelik çok gerekli analizler olduğu ortaya konulmuştur.
xxiv
INTRODUCTION
Membrane bioreactor (MBR) is an advanced wastewater treatment technology that

combines the biological treatment processes and membrane processes. Though,
biofilm formation on the membrane surface cause biofouling during the MBR
process. Thus far, a comprehensive method have not be found for mitigating biofilm
formation complately. Quorum sensing (QS) is cell-to-cell communication using
signals that play a regulatory role in controlling the membrane surface biofilm in
MBR. Interfering with QS signals through Quorum Quenching (QQ) methods can
effectively reduce biofouling.
This thesis comprises new QQ bacteria/media as effective method of preventing

biofouling as an output of the membrane process. QS and QQ mechanisms are
relatively new concepts and, as such, there is a distinct lack of detailed studies on
these technologies. This study describes the new QQ agents were employed to apply
biological concepts to MBRs with the intention of controlling biofilm formation and
metagenomic interaction that was occurred with indigenous microorganism.
Purpose of Thesis
In this thesis, it was aimed that the use of newly isolated QQ bacteria to inhibit /
control the formation of biological film layer, to perform MBR screening and
biofouling studies in different hydraulic conditions, and to compare newly isolated
QQ bacteria. Additionally, to prepare a new immobilisation media, which could be
alternative to the alginate beads and other applications, is one of the aims. For this
purpose, the following objectives were planned:
 Isolation and genetically identification of new bacteria has QQ activity from

samples that taken from different source such as marine, saltern, leachate and
pond and use of biodegradation control in different MBR systems
 Comparison of isolated QQ bacteria with other bacteria in the literature in

terms of antibiofouling profile in MBR operation using standard methods
1
 To prepare a new immobilisation media design for new bacteria
 Within the framework of the targeted specificities, to evaluate the interaction

between bacterial population in MBR and new QQ bacteria.
Unique Aspect
The novel side of the thesis are isolation of new QQ bacteria, understanding the effect
of selected new QQ bacteria (Bacillus sp. T5, Delftia sp. T6) on microbial
community and examination the ways in which this novel QQ bacterium is critical
to the performance of MBRs. Microbial community analysis with high throughput
methods are very limited on the QQ-MBRs. Also this is the first study that made use
of the Illumina sequencing method for QQ-MBRs.The results of the molecular
investigation revealed that certain microbial taxa are very sensitive to QQ bacteria.
Because of the evaluated anti-biofouling effect of QQ bacteria was reached up to
85% it can be concluded that QQ process of T5/T6 was ultimately reflected in the
performance of the membrane bioreactors process.
Organization of the Thesis
The thesis contains an introduction followed by three data chapters (Chapters 2-4),
and thesis conclusions and recommendations (Chapter 5). Each data chapter
comprises of a combination of the overall research objectives and results.
Chapter 2 evaluated quorum quenching (QQ), which exists among bacteria that were
isolated from saltern, pond and marine habitats, and applied selected QQ bacterium
to inhibit biofilm formation in the membrane bioreactor (MBR). It was identified
nine genera belonging to 19 N-octanoyl-homoserine lactone (C8-HSL)-degrading
bacteria, namely Shewanella, Acinetobacter, Klebsiella, Bacillus, Deftia, Vibrio,
Comamonas, Microbacterium, and Pseudomonas. Both Bacillus sp. T5 and Delftia
lacustris T6 were isolated from a saltern and degraded all the C8-HSL; thus,
demonstrating the highest QQ capability. T5 was immobilized in a new
immobilization medium named QQ-fiber, which was composed of floating 20-cm
hollow fibers. To enlighten the antibiofouling potential of newly isolated Bacillus
sp. T5, it was applied to an MBR and the QQ effect was monitored during 12 days
of operation. According to transmembrane pressure (TMP) values, QQ bacterium led
2
to less biofouling than the control reactor. In MBRs operated with T5, TMP was
decreased with 25 % efficiency. The decrease in biofouling resulted from QQ-fiber
activity, and it was seen that T5 could be used successfully to inhibit biofilm
formation in MBR.
Chapter 3 examines the effects of a newly isolated quorum quenching (QQ) bacteria
(Bacillus sp. T5) on the microbial community has been evaluated via the Illumina
sequencing method. Membrane bioreactors (MBRs) operated with this novel QQ
bacterium to evaluate the improvement in the performance of MBR. Anti-biofouling
effect of T5 was enhanced as 71% compared to control reactor. Also, QQ bacteria
did not have any negative effect on the removal of organics during the process.
Gram-negative bacteria were found to be dominant than gram-positive bacteria.
Proteobacteria, Actinobacteria, Bacteroidetes, Acidobacteria, Firmicutes, and
Chloroflexi were dominant phyla in the control and QQ reactors. The proportion of
Alphaproteobacteria was most significant among Proteobacteria. The relative
abundances of Actinobacteria, Acidobacteria, and Firmicutes were significantly
affected by Quorum quenching mechanism. On the other hand, QQ activity of
Bacillus sp. T5 significantly influenced the relative abundance of Proteobacteria,
Bacteroidetes, and Chloroflexi. The QQ process appeared to generate variations in
the structure of the microbial community. According to the results of the molecular
analyses, the syntrophic interaction of Bacillus sp. T5 and indigenous gram-negative
and positive bacterial community is critical to the performance of MBRs.
Chapter 4 presents data on how the microbial community is present within QQ-
MBRs changes during the process of operation with different QQ bacteria. While an
advanced knowledge of the microbial community is required to develop and
implement effective QQ strategies, there remains a lack of understanding of this
process. This study aimed to evaluate that process by investigating the behavior of
two types of QQ bacteria, Bacillus sp. T5 and Delftia sp. T6. The anti-biofouling
effects of T5 and T6 in the QQ-MBR were 85 % and 76 % respectively. According
to the Illumina Hi-Seq results, gram-positive T5 triggered a reduction in the
abundance of gram-positive classes and an increase in the abundance of gram-
negative classes in the mixed liquor. On the contrary, operation with gram-negative
T6 caused, a reduction in gram-negative classes and an increase in gram-positive
classes. The results revealed that shifting in the indigenous microbial sturucture that
3
occurred by Bacillus sp. T5 and Delftia sp. T6 is vital for the performance of MBRs.
This is the first study which establish this relationship.
4
LITERATURE REVIEW1
Quorum Quenching Application in Membrane Bioreactors
Membrane bioreactors (MBRs) are frequently employed in advanced wastewater

treatment processes and have been verified to provide a better performance than
conventional activated sludge procedures (Drews, 2010; Meng et al., 2010).
However, unwanted accumulation of microorganisms on the membrane surface in
MBRs eventually results in biofouling (Chen et al., 2004). In spite of the research
and innovations in this area, it is thus far not possible to completely eliminate the
biofilm and this represents an important challenge for the sustainability of MBRs
(Malaeb et al., 2013).
Researchers have demonstrated that QS and biofilm formation are strongly linked
and act to regulate the biofilm formation on the membrane surface in MBR (Davies
et al., 1998; Waheed et al., 2017). C6-HSL and C8-HSL, 3-oxo-C8-HSL C4-HSL,
C6-HSL, C8-HSL, 3-oxo-C8-HSL, C10- HSL, C12-HSL, 3-oxo-C12-HSL and C14-
HSL have been detected from biofilm on the membrane surfaces and in the mix
liquor of MBR (S. R. Kim et al., 2013; Lade et al., 2014; Yeon et al., 2009a). Because
biofilm formation on membranes can be controlled by QS, several studies have
turned their attention to blocking AHL signals to either reduce membrane biofouling
or improve reactor performance (Ergön-Can et al., 2017; Köse-Mutlu et al., 2015;
Lee et al., 2016a; Oh et al., 2013; Yavuztürk Gül and Koyuncu, 2017). In the last 10
years, research on QQ mediated strategies for mitigating membrane biofouling in
MBRs has intensified and notable success has been achieved in this regard.
2.1.1 Enzymatic quorum quenching
1
This chapter is based on the paper Bahar Yavuztürk Gül, Börte Köse, Pyungkyu
Park, Chung-Hak Lee and Ismail Koyuncu, 2018. “Quorum Quenching for Fouling
Control in Membrane Bioreactors” Encyclopedia of Water : Science, Technology,
and Society.
5
A range of different types of AHL signals exist such as: lactonase, acylase,
oxidoreductase, decarboxylase and deaminase (Dong et al., 2000). Researches have
provided two core groups of AHL degradation enzyme; (i) lactonases that hydrolyze
the lactone ring of AHL, (Park et al., 2003; Zhang et al., 2002), (ii) acylases that
cleave the AHL amide bond and produce the free fatty acid and lactone ring (Park et
al., 2007; Romero et al., 2008)
The first study on QQ enzymes in MBR was conducted by Yeon et al. (Yeon et al.,
2009a). The authors directly introduced acylase I to a lab-scale MBR and achieved
mitigation of biofilm formation which caused a decrease in trans-membrane pressure
(TMP) of acylase treated MBR compared to the control case. In control reactor, it
took approximately 22 h for the TMP to reach maximum value 70 kPa and for QQ
reactor a maximum TMP of approximately 50 kPa was recorded after ∼37 h
operation. These results indicated that adding acylase I considerably improved
reactor performance due to the degradation of AHL. Because of the enzyme stability
problems and the necessity of continuous addition of enzyme, researchers try to find
more convenient ways for using enzymes, especially in large-scale long-term
operations to control membrane biofouling. has the attraction that it should have a
low risk for bacterial resistance as it does not affect the survival of the bacteria.
However, the short life time of the free enzyme and the cost of continuous treatment
are possible obstacles to adoption of this technique for large-scale applications. After
adoption of QQ in MBR through the degradation enzyme, researchers developed new
approaches to lower the operational cost of enzymes. To enhance the retention time
of the enzyme in the reactor Yeon et. al. (Yeon et al., 2009b) studied magnetic
immobilized enzyme carriers (MECs) in the MBR. Recorded TMP values were 76–
79 kPa and 36–39 kPa for the control and MEC MBR with the total recycled mode.
However, in continuous MBR, TMP exceeded 30 kPa after 48 h of operation for
control MBR but TMP of 10 kPa was recorded for the MEC MBR. This approach
obviously allows the reduction of biofouling in MBR. Although MEC can consist of
reusable immobilization media, studies have indicated disadvantages of the
approach, such as high operational cost. To overcome this obstacle, Jiang et al. (Jiang
et al., 2013) encapsulated the acylase enzyme with a low cost organic polymer
sodium alginate and observed enhanced reactor performance due to the reduced
biofilm formation. It took approximately 45 h to reach maximum TMP of 14 kPa for
6
QQ MBR, but control MBR reached 40 kPa within 55 h. Furthermore, the study
revealed that QQ has no harsh effect on removal of organics in MBR. Later, Lee et
al. (Lee et al., 2014) revealed an effective and sustainable strategy for preventing
biofilm development on membrane surfaces and developed magnetically separable
mesoporous silica immobilized with acylase. However, all these enzyme approaches
(free or immobilized), result in considerably increased cost due to enzyme
purification and stability problems which need to be overcome.
2.1.2 Bacterial quorum quenching
Difficulties and obstacles encountered in the use of direct enzymes for antibiofouling
have been the driving force to find more effective methods. Several groups of
bacteria are reported as producers of quorum quenching enzymes, which are able to
degrade AHL signals and regulate biofilm formation on the membrane surface. A
summary of the known QQ bacteria, along with their AHL degradation genes and
enzymes is provided in Table 2.1.
QQ bacteria and their AHL degradation genes and enzymes.

Bacterial strains Gene Enzyme Reference
AHL (Wang et al.,
Microbacterium testaceum AiiM
Lactonase 2010)
AHL (Dong et al.,
Bacillus sp. AiiA
Lactonase 2002)
AHL (Dong et al.,
Bacillus thuringiensis AiiA
Lactonase 2002)
AHL (Park et al.,
Arthrobacter sp. AhlD
Lactonase 2003)
(Carlier et al.,
AHL
Agrobacterium tumefaciens AttM, AiiB 2003; Zhang et
Lactonase
al., 2002)
(Uroz and
AHL
Rhodococcus erythropolis QsdA Heinonsalo,
Lactonase
2008)
(Carlier et al.,
Variovorax paradoxus ND* AHL Acylase 2003; Zhang et
al., 2002)
7
Table 2.1 (continued) : QQ bacteria and their AHL degradation genes and
enzymes.

(Morohoshi et
Shewanella sp. Aac AHL Acylase
al., 2008)
(Abed et al.,
Marinobacter sp. ND ND
2013)
(Abed et al.,
Halomonas gomseomensis ND ND
2013)
AHL (Romero et al.,
Salinicola salaries ND
Lactonase 2012)
(Romero et al.,
Olleya marilimosa ND ND
2012)
(Romero et al.,
Maribacter sp. ND ND
2012)
(Lee et al.,
Pseudomonas sp. 1A1 ND AHL Acylase
2013)
AHL (Tang et al.,
Muricauda oleria MomL
Lactonase 2015)
AHL (Wong et al.,
Chryseobacterium sp. AidC
Lactonase 2012)
(Romero et al.,
Anabaena sp. Aiic AHL Acylase
2008)
(Lin et al.,
Ralstonia eutropha AiiA AHL Acylase
2003)
PvdQ, (Huang et al.,
Pseudomonas aeruginosa AHL Acylase
QuiP 2003)
(Park et al.,
Streptomyces sp. AhlM AHL Acylase
2005)
(Uroz et al.,
Comamonas sp. ND AHL Acylase
2007)
Oxido- (Uroz et al.,
R. erythropolis W2 ND
reductase 2005)
AHL (Dong et al.,
Erwinia carotovara AiiA
Lactonase 2000)
AHL (Chan et al.,
Acinetobacter sp. ND
Lactonase 2011)
(Jafra et al.,
Delftia sp. ND ND
2006)
AHL (Gao et al.,
Ochrobactrum sp. AidH
Lactonase 2013)
Pseudoalteromonas AHL (Huang et al.,
QsdH
byunsanensis Lactonase 2012)
8
Table 2.1 (continued) : QQ bacteria and their AHL degradation genes and
enzymes
(Lin et al.,
Ralstonia eutropha AiiA AHL Acylase
2003)
(Yavuztürk Gül
AHL
Acinetobacter baylyi ND and Koyuncu,
Lactonase
2017)
(Yavuztürk Gül
AHL
Bacillus infantis ND and Koyuncu,
Lactonase
2017)
(Yavuztürk Gül
AHL
Delftia litopenaei ND and Koyuncu,
Lactonase
2017)
(Yavuztürk Gül
Pseudomonas AHL
ND and Koyuncu,
nitroreducens Lactonase
2017)
(Yavuztürk Gül
AHL
Vibrio azureus ND and Koyuncu,
Lactonase
2017)
(Yavuztürk Gül
Acinetobacter AHL
ND and Koyuncu,
calcoaseticus Lactonase
2017)
(Yavuztürk Gül
AHL
Shewanella decolorationis ND and Koyuncu,
Lactonase
2017)
(Yavuztürk Gül
AHL
Shewanella putrefaciens ND and Koyuncu,
Lactonase
2017)
*ND: Not defined.
Because of the high extraction and purification cost and stability problems of
enzymes, it could be more logical to make use of the bacteria themselves, which
already produce QQ enzyme, to maintain efficient and economical QQ applications
in MBR (Lee et al., 2013; Oh et al., 2013; Yavuztürk Gül and Koyuncu, 2017).
Therefore, QQ bacteria have been isolated from nature or MBR by many researchers
and their QQ activities screened (Table 1). The most commonly studied QQ
bacterium for biofilm control in MBR has been Rhodococcus sp. BH4 (Köse-Mutlu
et al., 2015; Lade et al., 2014; Oh et al., 2013; Weerasekara et al., 2016). In addition,
researchers have used QQ bacteria in MBR operations by immobilizing or
encapsulating them in very different media. Recently, Yavuztürk Gül and Koyuncu
(Yavuztürk Gül and Koyuncu, 2017) have successfully prevented biofilm formation
with new antibiofouling bacteria Bacillus sp. T5, which has 91% QQ activity and is
9
isolated from the saltern. Oh et al. (H.-S. Oh et al., 2012) applied the recombinant
Escherichia coli (producing lactonase enzyme) to the MBR. Although they
demonstrated effective inhibition of biofouling in lab-scale MBRs, this effect may
not survive in the pilot/real-scale MBR. Therefore, the strategy shifted to isolate the
indigenous QQ bacteria for wastewater treatment. Oh et al. (Oh et al., 2013) isolated
a QQ bacterial strain Rhodococcus sp. BH4 and encapsulated to Rhodococcus sp.
BH4 with a microbial vessel. The microbial vessel was simply produced from a
hollow fiber ultrafiltration membrane and the BH4 was filled in. This apparatus was
not free to move within the MBR but allowed the bacteria to be physically restrained.
The authors observed that MBR with the microbial vessel was postponed for 11
hours to reach the same TMP value (25 kPa) as the control. Jahangir et al. (Jahangir
et al., 2012a) applied the microbial vessel in the external submerged MBR. They
placed the vessel near the membrane and obtained more noticeable results for
mitigating biofilm formation. Reaching the same TMP values (30 kPa) was
postponed by 20 hours for MBR with the microbial vessel. Furthermore, they
revealed that the specific allocation of the microbial vessel is highly critical because
of the non-mobile characteristic of the vessels. Similarly, Cheong et al. (Cheong et
al., 2014) developed a ceramic microbial vessel (CMV) and immobilized
Pseudomonas sp. 1A1. It was observed that MBR with recombinant E. coli was
postponed for 10 hours to reach the same TMP value (25 kPa) as the control.
Furthermore, it was found the amount of extracellular polymeric substances was
noticeably reduced and biofilm formation was efficiently decreased with CMV. It
took around 10 days in the control reactor to reach the TMP of ∼60 kPa, but after 10
days the QQ reactor reached the TMP of ∼10 kPa. Recently, to cope with the fixed
properties of the vessels, Yavuztürk-Gül and Koyuncu (Yavuztürk Gül and
Koyuncu, 2017) focused on developing a new free moving immobilization medium
named QQ-fiber, which consisted of floating 20 cm hollow fibers. They
immobilized newly isolated Bacillus sp. T5 in the QQ fibers to detect the
antibiofouling potential of T5 during 12 days of operation. According to the
transmembrane pressure (TMP) values, QQ bacterium led to less biofouling than the
control reactor. It took around 10 days in the Control reactor to reach the TMP of
200 mbar, whereas after 10 days the QQ reactor with QQ-fibers reached the TMP of
120 mbar. In MBRs operated with T5, TMP was decreased with 25 % efficiency. To
overcome the disadvantages (limited space to grow for bacteria, diffusion of only
10
soluble and small molecules from the membrane, nonmobile features etc.) of using
hollow fibers as an encapsulation apparatus, Kim et al. (S. R. Kim et al., 2013)
developed a new immobilization media called cell entrapping beads (CEBs). CEBs
were more economical and efficient than both the microbial vessel method and the
enzymatic quorum quenching method. CEBs are free moving beads that were
prepared by dropping sodium alginate-Rhodococcus sp. BH4 suspension into CaCl2
solution. They revealed remarkable results in terms of inhibition of biofilm formation
which originates from both the QQ effect of BH4 and the physical washing effect of
CEBs. According to their results it took 1.8 days for the TMP to reach 70 kPa in the
control reactor, whereas it took 18.8 days for the reactor with CEBs. Thus, CEBs
delayed the time required to reach the same TMP of 70 kPa by a factor of 10,
compared with the control MBR. This is very significant because the reduction in
the TMP rise caused energy savings in the MBR process. Despite the fact that it is
not accurate to compare the results of the microbial vessel and CEBs directly, it can
be said that the antibiofouling performance of CEBs is better than the microbial
vessels. Kim et al. (Kim et al., 2014) coated Rhodococcus sp. BH4 entrapping
microcapsules by a polymeric membrane layer i.e. PSf, PES and PVDF, managed to
prevent leakage of QQ bacteria from macrocapsules in harsh environmental
conditions. They applied macrocapsules to MBR fed with real waste water and the
results showed a notable anti-biofouling capacity in continuous MBR. TMP was
monitored and it took 3–10 days to reach TMP of 40 kPa for the control MBR. On
the other hand, it took 23 days to reach the same TMP for the QQ MBR.
Because of the short lifetime of the sodium alginate CEBs, and bacterial loss incurred
through removal of excess sludge from the MBR, Ergön-Can et al. (Ergön-Can et al.,
2017) developed a novel bacterial immobilization apparatus named a rotary
microbial carrier frame (RMCF) that provides mobility, stability, and reusability. QQ
bacteria (Rhodococcus sp. BH4) were filled onto a rotating polycarbonate frame
covered with a microfiltration membrane. They showed that transmembrane pressure
(TMP) was decreased with 65% efficiency. Köse-Mutlu et al. (Köse-Mutlu et al.,
2015) compared three different immobilization media (CEBs, microbial vessel, and
RMCF) under the same operational conditions. They also showed the effect of
different Rhodococcus sp. BH4 amounts and fluxes on the QQ mechanism. The flux
was found to be more effective than the amount of bacteria. While the effect of CEB
11
on the inhibition of biofouling was the most significant, the vessel had the minimum
influence on the TMP reduction. On the other hand, the authors found that RMCF is
more feasible than other methods according to the cost analysis results.
Although, sodium alginate beads have been developed and verified for their excellent
anti-biofouling potential in a pilot-scale MBR with flat-sheet membrane modules,
QQ-beads can hardly penetrate into the structure of dense hollow fiber (HF) bundles.
Nahm et al. (Nahm et al., 2017) developed QQ bacteria entrapping sheets (QQ-
sheets) as new floating QQ-media to enhance the reactor performance in a QQ-MBR
with an HF module. QQ-sheets showed 2.5 times greater QQ activity than the QQ-
beads, because beads did not contact the inner part of the modules. The results
indicate that the advantages of the QQ-sheets over the QQ-beads were more notable
in the MBR with the multi-layer-HF module.
These bacteria immobilization strategies and types of QQ bacteria, used for

mitigation of biofouling, are remarkably important because they allow the expansion
of the QQ effect.
2.1.3 Fungal quorum quenching
Until now, the decomposition of signal molecules in MBRs, both enzymatic QQ and
bacterial QQ have been reported. Lee et al. (Lee et al., 2016a) reported for the first
time fungal-to-bacterial QQ for biofouling control in MBRs used for wastewater
treatment. Farnesol was the first known fungal QS signal molecule and was produced
by Candida albicans (Hornby et al., 2001). Farnesol was also revealed to mitigate
biofilm formation of some bacteria and fungi via affecting their metabolite pathways
(Jabra-Rizk et al., 2006; Koo et al., 2003).
Lee et al. (Lee et al., 2016b) demonstrated the QQ effect of C. albicans on MBR by
its entrapping beads and revealed that fungal QQ greatly inhibited biofilm formation
in the QQ-MBR. They calculated the ratio of the TMP values between the vacant-
MBR and the conventional-MBR (physical washing effect) as 6,9. On the other hand,
the ratio between QQ-MBR and conventional MBR was found to be 24,4. They
concluded that the joint effect of the QQ activity and the physical washing effect
could reduce biofouling in an MBR more significantly. Table 2.2 shows a summary
of the different strategies to control membrane biofouling based on the quorum
quenching phenomenon.
12
Quorum quenching strategies adopted to control of membrane biofouling.
Enzyme/Bacteria/Fungus
Type of QQ Immobilization Media
Free Enzyme
Enzymatic QQ Magnetic Enzyme carrier Acylase
Encapsulated with sodium alginate
Immobilized in mesoporous silica
Microbial vessel Recombinant Escherichia coli
Microbial vessel Rhodococcus sp. BH4
Ceramic microbial vessel (CMV) Pseudomonas sp. 1A1
QQ-Fiber Bacillus sp. T5
Microbial beads Rhodococcus sp. BH4
Bacterial QQ
Macroencapsulation of quorum quenching bacteria by polymeric
Rhodococcus sp. BH4
membrane layer
Rotary microbial carrier frame (RMCF) Rhodococcus sp. BH4
Bacteria entrapping sheets (QQ-sheets) Rhodococcus sp. BH4
Fungal QQ Microbial beads Candida albicans
13
Immobilization media that microorganisms attached varies according to the type of
MBR, module and etc. If summarize the up-to-date immobilization strategies and
compare their efficiency, it can generally be concluded that the bacterial QQ results
are more effective than the enzymatic QQ. Furthermore, mobile immobilization
environments are found to be more successful in reducing TMP than fixed ones. The
performance of the beads is much higher than the immobilization media produced
by the membranes. The removal of endurance problems of beads via production of
polymer-coated beads is seen as a highly effective application to prevent biofouling.
14
ASSESSMENT OF NEW ENVIRONMENTAL QUORUM QUENCHING
BACTERIA AS A SOLUTION FOR MEMBRANE BIOFOULING2
Introduction
Quorum sensing (QS) is a phenomenon, where bacteria communicate through

chemical signals and organize their group behavior by sensing their population
density. QS is mediated by the production and emission of autoinducers.
Autoinducers are small signaling molecules that amass in the extracellular
environment. Bacteria release, identify and react to the accumulation of these
molecules in order to control the gene expression of a community (Fuqua and
Greenberg, 2002). When bacteria sense that their population density has reached a
threshold level, autoinducers are released. These trigger the expression of multiple
genes in the population which, in turn, serve to regulate a range of significant
biological functions such as aggregation, antibiotic biosynthesis, secondary
metabolite production, biofilm maintenance and differentiation, luminescence,
motility, plasmid conjugal transfer, siderophore production, swarming, symbiosis or
virulence (Davies et al., 1998; Fuqua and Greenberg, 2002; Raina et al., 2012;
Williams, 2007). A range of different classes of signals exist within a QS system
such as: (1) N-Acyl-homoserine Lactones (AHLs), (2) Furanosyl borate
(Autoinducer-2), (3) Oligopeptides (5–10 amino acid cyclic thiolactone), (4)
Hydroxyl-palmitic acid methylester, and (5) Methyl dodecanoic acid. Of these, the
best characterized is AHLs. (Dong and Zhang, 2005; García-Contreras et al., 2013).
The term quorum quenching (QQ) describes the mechanisms that are responsible for
deactivating the QS communication systems by interrupting the signaling process.
The QQ mechanism uses competitive binding to inactivate the autoinducer
2
This chapter is based on the paper Yavuztürk Gül B, Koyuncu I (2017) Assessment
of new environmental quorum quenching bacteria as a solution for membrane
biofouling. Process Biochem 61:137–146. doi: 10.1016/j.procbio.2017.05.030.
15
synthases/receptors or switches off signal transmission through the degradation of
the signaling molecules. Existing research to date has identified QQ mechanisms in
bacteria (Dong et al., 2002; Park et al., 2007; Uroz, 2003), marine algae (Manefield,
2000), root associated fungi (Uroz and Heinonsalo, 2008), plants (Gao et al., 2003;
Teplitski et al., 2000), and mammalian cells (Draganov et al., 2005). Many studies
have focused in depth on the enzymatic degradation of AHL. Research in this area
has developed three main categories of AHL degradation enzyme that correlate with
their enzymatic mechanisms; (i) AHL lactonases: these hydrolyze the lactone ring of
AHL, generating the corresponding N-acyl-homoserine (Park et al., 2003; Zhang et
al., 2002). The process of hydrolysis can also occur spontaneously in the presence of
alkaline pH and can be reversed at lower pH levels (Yates et al., 2002), (ii) AHL
acylases: these cleave the AHL amide bond and generate the corresponding free fatty
acid and lactone ring (Park et al., 2007; Romero et al., 2008), (iii) AHL oxidases and
reductases (oxidoreduction) (Uroz et al., 2005).
Existing studies have proven the presence of QQ activities in a range of different

environments, including soil (Chan et al., 2011; Chong et al., 2012; Christiaen et al.,
2011), and aquatic environments (Romero et al., 2012, 2011; Tang et al., 2015), and
a number of researchers have successfully isolated several strains and genes that are
capable of degrading AHLs. However, knowledge as to how QQ mechanisms
suppress QS in saline environments remains relatively limited, although some
existing studies do indicate that the QQ mechanisms in this environment inhibit QS
through the compounds produced by Marinobacter, Halomonas and archaean strains
(Abed et al., 2013).
Given the fact that QS is known to have the ability to control a variety of different
functions, as outlined above, there is the potential that the compounds that interfere
with bacterial QS can be employed for alternative purposes; for example, to produce
of commercially desirable bacterial pigments (Kulandaisamy Venil et al., 2013), to
eliminate pathogenic activity in aquacultural and agricultural environments (Chan et
al., 2011; Jafra et al., 2006; Romero et al., 2014), or to operate as antifouling agents
(Jahangir et al., 2012b; Lee et al., 2013; Yeon et al., 2009b). Recent research
explored the potential that quorum quenching has as an innovative method of
controlling biofouling in a membrane bioreactor (MBR) for the advanced treatment
of wastewater (S. R. Kim et al., 2013; Köse-Mutlu et al., 2015; H. S. Oh et al., 2012).
16
The goals of this study were: (i) To isolate and identify those bacteria found in
estuary, pond and saltern environments that are capable of interfering with AHL-
mediated QS, (ii) To determine the extent to which these forms of bacteria have
quenching capabilities with using bacterial biosensor Agrobacterium tumefaciens,
and (iii) To evaluate the inhibition of membrane biofilm that resulted from QQ
activity during the MBR operation.
Materials and Methods
3.2.1 Bacterial strains, growth media, culture condition
The AHL degrading bacteria were screened on a minimal medium containing sterile
distilled water and 2,5 mM N-octanoyl-l-homoserine lactone (C8-HSL) (Sigma–
Aldrich, USA) as a sole carbon source, which is the most abundant autoinducers in
the membrane bioreactors (Leadbetter and Greenberg, 2000; H. S. Oh et al., 2012;
Uroz, 2003). By sodium requirements of halophilic marine and saltern QQ bacteria
a modified minimal medium containing C8-HSL (2,5 mM) and NaCl (1,9%-5%
w/vol) was used (Poli et al., 2013). Ringer solution was used to dilute the soil
samples. All of the isolated bacteria were cultivated in a Luria–Bertani (LB) medium
at 30°C. Agrobacterium tumefaciens A136 (Ti)- (pCF218)(pCF372) (Fuqua and
Winans, 1996) the reporter strains for the bioassay, were cultured in LB at 30 ̊C and
supplemented with spectinomycin and tetracycline when necessary (McLean et al.,

2004).
3.2.2 Isolation of QQ consortia from environmental samples
Samples were taken from three different environmental origins for bacterial isolation
and screening for QQ activity against C8-HSL. Sample locations were chosen for its
different microbial diversity. One of the samples was collected in coastal
environment in Haliç, Marmara Sea, one from the highly polluted pond in Istanbul
Technical University, Istanbul and the third sample was obtained from the Çamaltı
Saltern Area, İzmir, Turkey.
Marine, pond and soil samples were taken using a 50-mL sterile laboratory material
and hence large amount of water and soil samples were taken. In order to achieve
homogeneous suspension for serial dilutions, samples were vigorously vortexed then
17
1 ml of the water sample was inoculated into 100 ml of modified minimal medium
for marine samples and minimal medium for pond samples. Soil samples of saltern
were suspended and serially diluted with ringer solution. After samples were serially
diluted to 1/104 of their original strength, the specimens were seeded in a modified
minimal medium supplemented with 2.5 mM C8-HSL as a sole carbon source. The
inoculation mixtures were incubated at 30°C for 48 hours while shaking 180 rpm.
These suspensions were subinoculated into a medium containing C8-HSL at 2 days
intervals (1% v/v). Through this procedure, it was possible to isolate the bacteria that
were capable of surviving with an C8-HSL as a sole carbon source. After the third
enrichment cycle, 200 μl cell suspensions were spread on Luria−Bertani (LB) agar
to obtain pure colonies. After incubation for 48 hours at 30 °C, 33 colonies were able
to grown on LB agar. A total of 19 colonies were selected with different color and
morphology and subcultured on LB in several passages. Single colonies stored at –
80 °C and used for QQ screening (Jahangir et al., 2012b).
3.2.3 Strain identification and phylogenetic analysis
The identification of the isolated bacteria was carried out by amplifying and partial
sequencing of the 16S rRNA gene using the universal primers 27F(5′-
AGAGTTTGATCCTGGCTCAG-39) and 1492R(5′-
GGTTACCTTGTTACGACTT-39) (Weisburg et al., 1991). The PCR cycling
conditions were as follows: 94°C for 5 min, followed by 30 cycles at 94°C for 30 s,
45°C for 30 s, and 72°C for 60 s. The amplified products were purified and sequenced
using an automated ABI 3100 Genetic Analyzer (Applied Biosystems, USA) and a
Bigdye Terminator cycle sequencing kit. Gene sequences were compared with
GenBank databases using the BLASTN program. A phylogenetic tree was
constructed using the neighbour-joining method with CLUSTAL W and MEGA
Version 5 (Tamura et al., 2011). The 16S rRNA nucleotide sequences determined in
the present study were deposited in the GenBank database under accession numbers
KR705931- KR705949, respectively.
18
3.2.4 Characterization of the quorum quenching activity of isolates
The QQ activity of the newly isolated 19 strains was measured by degradation of a

signal molecule, N-octanoyl-l- homoserine lactone (C8-HSL) in a Tris–HCl buffer
solution (50 mM, pH 7.0). To identify if the enzymes of the isolates were intracellular
or extracellular, both the QQ behaviors of the culture supernatants and the cell
extracts were separately examined each strain. The strains were incubated in an LB
medium for 24 hours (30°C, 180 rpm), then a Tris–HCl buffer solution (50 mM, pH
7.0) was used to dilute the suspensions (OD600 value of 1.0). Centrifugation (12,000
g, 10 min) and filtration were then used to separate the supernatant from the cells,
and the cell pellets were resuspended with the Tris–HCl buffer solution. This resulted
in the suspension of the whole cell. The cell extracts were acquired by sonicating the
whole cell suspension on ice then C8-HSL was added to the buffer to form a final
concentration of 200 nM. Finally, the QQ activities of the whole cells, supernatants
and the cell extracts were identified by assessing the extent to which C8-HSL was
degraded over different periods of time. A bioassay was used to measure the residual
concentrations of C8-HSL (Jahangir et al., 2012b; H. S. Oh et al., 2012). Using the
bioassay method with Agrobacterium tumefaciens A136 as the reporter strain, the
blue color that appeared on the X-gal (5-bromo-4-chloro-3-indolyl-beta-D-
galactopyranoside)-coated agar plate was attributed to exogenous AHL molecule
degradation (Fuqua and Winans, 1996; H. S. Oh et al., 2012). Briefly, A. tumefaciens
A136 was cultivated overnight in a 20 mL LB medium with spectinomycin and
tetracycline, then added to an LB agar containing 40 mg X-Gal. Then, 10 ml of the
mixture was overlaid on the surface of a 10 ml LB agar plate. After preparation of
the bioassay plate, samples of the whole cell, supernatant and cell extract were then
loaded into the well of the indicating agar plate. The residual amounts of C8-HSL
were calculated based on the calibration curve obtained by the sizes of the colored
zones, corresponding to each standard concentration of the C8-HSL.
A method described by Yates and colleagues (Yates et al., 2002) was employed to
determine the extent to which the AHLs were inactivated by lactonolysis, through
the formation of the corresponding N-acylhomoserine compound. This approach
19
uses acidification of the reaction mixture to pH 2 using HCl to stimulate
reconstruction of the lactone ring. The recovery of C8-HSL was analyzed via
bioassay.
A QQ-fiber was designed to immobilize QQ bacteria using hydrophilic

polyvinylidene fluoride (PVDF) hollow fiber microfiltration membranes (Philos Co.
Ltd MegaFlux I, Korea) that have 0.2 μm nominal pore size. The bottoms of every
20-cm length hollow fibers were sealed with silicon adhesive, 8.2 mg QQ bacteria
were injected into the hollow fiber from the open side, and then the open side was
sealed (Figure 3.1). Empty fibers not containing QQ bacteria were also prepared the
same way. The C8-HSL degradation potential of QQ-fiber was determined also by
using bioassay.
Preparation of QQ-fibers. Centrifuged QQ bacteria were injected into

the one side sealed PVDF hollow fiber microfiltration membranes from the open
side. Empty fibers not containing QQ bacteria.
Activated sludge was taken from a wastewater treatment plant (İstanbul, Turkey) and
acclimated to the synthetic wastewater before MBR operation. The composition of
20
the synthetic wastewater was as follows (mg/L): glucose, 500; urea, 100; (NH4)2SO4,
50; KH2PO4, 50; MgSO4 7H2O, 50; NaCl, 50; CaCl2 2H2O, 10; and NaCO3, 100.
Two parallel membrane bioreactors, the control reactor with 25 empty fibers and the
QQ reactor with a 25 QQ-fibers, were designed and operated under a constant flux of
18.5 L/m2/h. The working volume and the mixed liquor suspended solid (MLSS)
concentration of each reactor were 4,5 L and 12,000-12,700 mg/L, respectively.
PVDF hollow fiber membrane modules with an effective area of 90 cm2 were used
in both reactors. Hydraulic retention time (HRT) and sludge retention time (SRT)
were set to 27 hours and 30 days, respectively. MLSS, TKN and chemical oxygen
demand (COD) were determined according to standard methods. Biofilm layers of
the membrane surfaces were observed using confocal laser scanning microscopy
(CLSM, C1 plus, Nikon, Japan).
Results and Discussion
3.3.1 Novel AHL degrading bacteria
A total of 33 isolates were obtained from the various environments. 19 new isolates
were selected, based on their distinct colony morphology and color. These consisted
of 5 strains (T21, T22, T4, T5, and T6) from the Çamaltı Saltern; 5 strains (G2, G4,
G5, G6, and G7) from the ITU pond; and 9 strains (H1, H2, H3, H4, H5, H7, H8, H9
and H10) from the Haliç estuarine. There is a possibility that the number of isolated
QQ strains is underestimated because the strains were first selected based on their
capacity to interfere with C8-HSL only. Further analysis may show that new isolates
have the capacity to interfere with other AHL molecules.
A BLAST search of GenBank was performed to identify if there was any

taxonomical relationship between the sequences available in the database and those
of the 16S rRNA gene of the 19 new isolates. Of the 19 isolates, 13 belonged
predominantly to Gammaproteobacteria (G2, G4, T21, T22, T4, H1, H2, H3, H4,
H7, H8, H9, H10), three to Betaproteobacteria (G6, T6, H5), two to Firmicutes (G5,
T5), and one to Actinobacteria (G7). New isolates were characterized and classified
in nine genera and 14 species (Figure 3.2). The assessment of the partial nucleotide
sequence of the amplified 16S rRNA gene revealed a strong homology of 97-100
21
between this sequence and that of the bacteria from the following genera: Shewanella
(5), Klebsiella (2), Acinetobacter (3), Bacillus (2), Delftia (2), Vibrio (2),
Comamonas (1), Microbacterium (1), and Pseudomonas (1).
Neighbour-joining tree based on the 16S rRNA gene, showing

phylogenetic relationships among quorum quenching bacteria isolated from pond,
estuary and saltern. Bootstrap coefficients below 50% were not shown. Scale bar,
0,05 substitutions per nucleotide position.
22
The newly isolated strains belonged to genera that had previously been proven to
exhibit QQ activity (Table 3.1). The strains H7, H8, and H10 were identified as
Shewanella putrefaciens, which is found mainly in marine environments as a fish
pathogen. The H2 strain identified as Shewanella xiamenensis along with the H3
strain (Shewanella decolorationis), belonged to the same genera as the marine
bacterium. Morohoshi at al. (Morohoshi et al., 2008) reported that the Shewanella
sp. strain MIB015 degrades AHL and reduced biofilm formation. Among the isolated
QQ bacteria obtained from the Haliç estuary, strains H4 and H9 presented a 99%
identity match with Klebsiella pneuomoniae. Klebsiella pneumoniae is a very
common gram-negative bacterium that causes pneumonia. This bacterium also
presents AHL degradation activity and it was found that ahlK, an ahlD homologue,
encodes an AHL-lactonase in K. pneumoniae (Park et al., 2003). Strain H1 is a
member of the Pseudomonas nitroreducens (97%). It has been categorized as
belonging to the P. aeruginosa species and is typically found in soil, coastal habitats,
plant and animal tissue. Lee et al. (Lee et al., 2013) isolated and used Pseudomonas
sp. 1A1 in an MBR system for biofouling inhibition
23
Table 3.1 : Identification of 19 AHL-degrading strains on the basis of 16SrRNA gene sequence and their AHL degradation and lactonase
profile.
Isolate
Source Accession no. Remaining C8- Extracellular Intracellular
Code Identity of closest bacteria
HSL Cons. (nmol)* Lactonase Lactonase
G2 Acinetobacter baylyi (99%) KR705931 50 +++ ++
ITU Pond,
Istanbul
G4 Acinetobacter baylyi (99%) KR705932 66,7 +++ +

G5 Bacillus infantis (99%) KR705933 83,3 ++ ++
G6 Delftia litopenaei (98%) KR705934 57,4 + +
G7 Microbacterium kitamiense (98%) KR705935 61 ND ND
T21 Vibrio azureus (99%) KR705936 63,7 + +
Çamaltı
Saltern,
T22 Vibrio sp. (99%) KR705937 54,7 + ++

İzmir
T4 Acinetobacter calcoaseticus (98%) KR705938 63,5 +++ +

T5 Bacillus cereus (99%) KR705939 18 +++ +
T6 Delftia lacustris (99%) KR705940 18,4 _ _
H1 Pseudomonas nitroreducens (97%) KR705941 83,3 +++ _
Haliç Estuary, İstanbul
H2 Shewanella xiamenensis (99%) KR705942 55,3 ++ +

H3 Shewanella decolorationis (99%) KR705943 83,7 ++ ++
H4 Klebsiella pneumoniae (99%) KR705944 48 + ++
H5 Comamonas testosterone (99%) KR705945 90 ++ +
H7 Shewanella putrefaciens (99%) KR705946 74 +++ _
H8 Shewanella putrefaciens (99%) KR705947 69,7 +++ +
H9 Klebsiella pneumoniae (99%) KR705948 76,4 ++ +
H10 Shewanella putrefaciens (99%) KR705949 59 +++ +
*The amount of degraded C8-HSL was measured using bioassay after cultivation of whole cell in a minimal medium containing 2 nM C8-HSL for 1 hour. The restoration of the QS signal after acidification is presented
by +. – indicates that absence of lactonase. The number of plus symbol indicates the amount of C8-HSL recovered: +, 20-50% recovered of initial C8-HSL; ++, 50-75%; +++, 75-100. ND, not determined.
24
3.3.2 Detection of QQ capacity
The AHL-degrading capacity of 19 new strains was determined using a whole cell
bioassay. A bioassay was performed using the reporter strain Agrobacterium
tumefaciens A136 that served as a biosensor for the detection of the inhibition of C8-
HSL activity. From Figure 3.3, all the isolated strains exhibited effective utilization
ability through the degradation of more than 50% of 200 nM C8-HSL. Among them,
37% of the strains degraded more than 70% of the C8-HSL within one hour.
The genus Bacillus, which was widely distributed, has previously been reported to
exhibit QQ activity (Dong et al., 2002). Two isolated strains, T5 and G5, were
identified as Bacillus cereus (99%) and Bacillus infantis (99%), respectively. Strain
T5 exhibited remarkable C8-HSL-degrading activity potential; however, G5 did not.
A similar finding was observed for the G6 and T6 strains. Strain G6 was identified
as Delftia litopenaei (98%), and strain T6 as Delftia lacustris (99%), which belong
to the same genus; however, only the latter has remarkable C8-HSL-degradation
potential. Interestingly, even though the strains shared more than 99% sequence
identity with the 16S rRNA gene sequence and were from the same genus, such as
Delftia (T6-G6) and Bacillus (T5-G5), or from the same species, such as
Acinetobacter baylyi (G2-G4) and Klebsiella pneumoniae (H4-H9), their C8-HSL
degradation capabilities were different.
Strain G7 was identified as Microbacterium kitamiense (98%). It is reported that

some other Microbacterium members are able to degrade AHLs (Wang et al., 2012).
Three isolates obtained from different sources, namely strain T4 (Acinetobacter
calcoaceticus) from Çamaltı saltern, strain G2 and strain G4 (Acinetobacter baylyi)
from ITU pond, belonged to the same genus as opportunistic pathogens in the
Proteobacteria group. The genus Acinetobacter is widely distributed in aquatic and
terrestrial habitats, and several members of this genus are capable of utilizing AHL
(Table 3.1). The isolates (T21-T22, T4, T5, T6), identified as Vibrio azureus,
Acinetobacter calcoaceticus, Bacillus cereus and Delftia lacustris, respectively,
which were obtained from the Çamaltı saltern, were capable of degrading AHL
effectively.
25
a
Quorum quenching activity of whole cell (C8-HSL degradation in in

vitro assay) of isolates. (a) ITU pond, (b) Haliç Estuary and (c) Çamaltı Saltern.
26
3.3.3 Detection of exo-endo enzyme
Being capable of producing intracellular (endoenzyme) or extracellular (exoenzyme)

enzymes, that depend on where the enzymes function, is important for sustaining the
enzyme stability. To determine the degradation pathway of the AHL, the activity test
was performed for supernatant and cell extracts of the isolates separately, except for
Micobacterium kitamiense G7 because of its limited growth capacity in the LB broth.
The C8-HSL degradation results obtained from the supernatant and cell extracts are
presented in Figure 3.4. The remaining C8-HSL amount reveals that AHL
degradation was much higher in the cell extracts than in the supernatant. Strain T6
degraded 60% of the C8-HSL and had the most extracellular activity. Except for
strains G2 and G6, all other isolates exhibited stronger intracellular activity. Strains
H7, H8, T4, T5, and T6 could intracellularly degrade over 80% of the C8-HSL, even
though the amount of the remaining C8-HSL concentrations were negligible for the
T5 and T6 strains. Interestingly, although strains T6 and G6 belong to the same
genus, they have different degradation pathways. Both exhibit intracellular and
extracellular activity; however, strain T6 can achieve much higher levels of
intracellular activity than strain G6. Of the isolated strains, members of the genera
Acinetobacter G2, G4, and T4 have different C8-HSL degradation pathways. While
strains T4 and G4 presented high QQ activity in the cell extract, strain G2 did not,
and the extracellular enzyme activity of strain G2 was lower than that of strain T4
(Fig 3.4).
27
Comparison of the intracellular and extracellular QQ activities of the
isolated strains. The data are shown as means ± SD. Remaining AHL levels in the
sterile supernatant and cell extract of the isolates were determined after incubation
of the 2 mM AHL with sterile culture supernatant and cell extract in Tris-HCL
buffer (n=3).
3.3.4 Detecting the presence of lactonase using an acidification test
Lactonase activity causes AHLs to transform to their cognate N-acyl homoserine

(AHS) derivative. This ring-open form is not an active QS signal molecule. The
procedure suggested by Yates et al. (Yates et al., 2002) was used to test the isolates
in order to identify the existence of putative lactonase activity. Once the bioassay
had been completed, HCl was applied to the AHL degradation media to reduce the
pH to 2.0. This triggered the ring closure of the AHS and restored the AHL
molecules.
In this study, the lactonase activity of the supernatants and cell extracts of the isolates
were investigated separately to determine the localization of the lactonase enzyme.
The results presented the presence of lactonase activity in these isolates except for
Delftia lacustris T6 (Figure 3.5).
28
Lactonase activity of the supernatant and cell extract of the isolates.
Data shown are the averages (± SEM) of the restorated QS signal after acidification
test (n=3).
The recovery of C8-HSL from the cell extract was not observed for: the H1 strain
identified as Pseudomonas nitroreducens, the H7 strain identified as Shewanella
putrefaciens, and the T6 strain identified as Delftia lacustris. Furthermore, the
Delftia lacustris strain T6 almost completely degraded the C8-HSL so that it could
not be significantly retrieved from the cell extract or supernatant by acidification.
This indicated that the enzymatic activity of the T6 strain was very different to that
of lactonase. As such, whether oxidoreductase and/or acylase was responsible for the
QQ capacity of the Delftia lacustris T6 is yet to be confirmed. According to the
results, all the acquired isolates, except for strains H1, H7, and T6, exhibited
intracellular lactonase activity due to the recovery of the lactone ring (Table 3.1).
The amount of C8-HSL recovered following the acidification of the spent culture
media indicated that the other strains that belong to the genera Acinetobacter,
Bacillus, Vibrio, Shewanella, Klebsiella, Pseudomonas, and Comamonas appeared
to exhibit a wide-spectrum of extracellular or intracellular lactonase activity.
Acidification allowed the recovery of almost all the C8-HSL for B. cereus T5 and
50% for B. infantis G5. This supports the findings related to the enzymatic activity
of lactonase described thus far for this genus.
29
Existing research in this area has previously reported AHL-lactonase activity, as
shown in Table 3.1. For the first time, this study demonstrated C8-HSL -lactonase
activities in the following strains: T21, T22, T4, G2, G4 G5, G6, H1, H2, H3, H5,
H7, H8, and H10. Strains T5 and T6 utilized all the C8-HSL tested and exhibited
higher degrading activity within one hour than the alternative strains. Specifically,
Bacillus cereus strain T5, eradicated most of the C8-HSL within just 30 min. In
addition, it was revealed that the saltern isolates T6, has a different QQ enzyme than
lactonase within the tasted strains (Fig.3.5). This indicates that the stress conditions
in the environment, such as high salinity, may trigger the QQ mechanism of some of
the microorganisms that are present in the consortia or may cause overexpression of
the different QQ enzymes. Also, most of isolated strains exhibit much higher levels
of intracellular activity than extracellular activity. It can be said, intracellular enzyme
activity may adopt by environmental bacteria because of to achieve enzyme stability.
3.3.5 Effect of QQ-fiber with T5 on C8-HSL biodegradation
A new QQ strain Bacillus sp. T5 was chosen for MBR application because of its
remarkable enzyme activity. QQ activity of T5 was tested by degradation of C8-HSL
in Tris−HCL solution, and 200 nM C8-HSL completely degraded with the free cells
in the reaction time of about 30 min (Fig. 3.6).
Bioassay for the determination of C8-HSL degradation in 60 min by

Bacillus sp. T5.
30
T5 immobilized to QQ-fiber to investigate the potential of inhibiting biofilm
formation on membrane surface. The C8-HSL degradation potential of QQ-fiber
containing 8.2 mg T5/cm3 cubbyhole was also determined by using C8-HSL as a
signal molecule. It was chosen as 8,2 mg T5/cm3 because in our previous study
(Köse-Mutlu et al., 2015) we got good results with approximately the same amount.
As shown in Figure 3.7, the degradation efficiency of C8-HSL with the QQ-fiber
was measured to be 55% in the reaction time of 120 min when the QQ-fiber
contained 8.2 mg of the T5. While the adsorption of C8-HSL on the vacant QQ-fiber
was negligible, the decrease in the concentration of C8-HSL was attributed mainly
to its decomposition by the QQ-fiber inside.
250,0 QQ-
fiber
Control
200,0
C8HSL Conc. (nM)
150,0
100,0
50,0
0,0
0 50 100 150
Time (min)
Quorum quenching activity of QQ-fiber containing Bacillus sp. T5.

The C8-HSL degradation of QQ-fiber containing 8.2 mg Bacillus sp. T5/cm3
cubbyhole was determined in Tris−HCL solution. Control (vacant fiber) was also
tested to check the adsorption of C8-HSL on the fiber. Error bar: standard deviation
(n =3).
3.3.6 Control of membrane biofouling by new antibiofouling agent Bacillus sp.

T5
QQ-fiber with Bacillus sp. T5 was applied to submerged MBR. To evaluate the
potential of biofouling inhibition on membrane surface, TMP profiles of the control
and QQ MBR were compared by comparison of the areas under the TMP curve. As
shown in Figure 3.8, it took around 10 days in the Control reactor to reach the TMP
31
of 200 mbar, whereas it took 10 days the QQ reactor with QQ-fibers to reach the
TMP of 120 mbar.
250
200
TMP (mbar)
150
100
50
0
0 2 4 6 8 10 12
Time (Day)
QQTMP CONTROL TMP
Application of the quorum quenching bacteria isolated from saltern for

wastewater treatment. TMP profile in the MBRs. Empty fibers and QQ-fibers with
Bacillus sp. T5 were inserted in the control and the QQ reactors, respectively.
This shows that T5 immobilized QQ-fibers, inhibiting biofilm formation and

decreasing the TMP. Initial fouling is high for this MBR operation. As expected,
high flow and high MLSS concentration caused the rapid increase in TMP profile.
In addition, the reinforced properties of the membrane may have prompted this
sudden increase. According to the area under the TMP graphs; it can be said that
Bacillus sp. T5 metabolism resulted in 25% decreasing on TMP values compared
with the control. After 10 days of operation, inhibition of biofilm formation on
membrane surface was confirmed visually via CLSM. From the CLSM image (Fig
3.9), it can be seen that the biofilm formation on the membrane which from the QQ
MBR was thinner than that from the control MBR. These data support the evidence
of quorum quenching effect on the inhibition of biofouling.
32
The CLSM images of biofilm formed on the membrane surface. (a1)
Control MBR, a plan view, (a2) Control MBR, cross-section view, (b1) QQ MBR,
a plan view, (b2) QQ MBR, cross-section view at the end of the 10 days of MBR
operation, stained with SYTO9. Magnification: X40. Image size 501.76 × 501.76
μm.
The effect of the QQ mechanism on COD and TKN removal efficiencies of control
and QQ MBR during the microfiltration process were also determined. As shown in
Figure 3.10, the differences in COD and TKN profiles were negligible.
33
100
COD Removal (%)

80
60
40
20
0
1 3 5 7 9 10
Time (Day)
QQ MBR Control MBR
100
TKN Removal (%)
80
60
40
20
0
1 2
Time (day)
QQ MBR Control MBR
COD and TKN removal efficiencies of control and QQ MBR during

the operations.
As a result, these findings suggesting that quorum quenching did not have any
negative effect on biodegradation of the organics. In the literature, there is one
bacteria Rhodococcus sp BH4, have been widely studied for controlling membrane
biofouling (Kim et al., 2015; Oh et al., 2013). T5 has greater potential like BH4
because it degrades 91% of the C8-HSL in 15 min and all of the C8-HSL in 30 min
in aqueous solution. Oh et al. (H. S. Oh et al., 2012) used microbial vessel as an
immobilization medium for Rhodococcus sp. BH4 and applied microbial vessel to a
submerged MBR. The biggest disadvantage of a microbial vessel is its nonmobile
characteristic, so we developed floating hollow fibers as an immobilization medium
in activated sludge. The newly isolated QQ bacterium T5 with the new
immobilization medium QQ-fiber is a novel approach to inhibit membrane fouling.
Conclusions
Although plenty of studies have been conducted on the isolation of QQ bacteria from
marine and soil environments, very few have reported on saltern and estuarine. This
34
is the significant report of isolation and identification of bacteria that are capable of
utilizing C8-HSL from very different environments as a sole carbon source. 19 new
strains were isolated from marine, pond and estuarine. All of the isolated strains
exhibited effective utilization ability. According to the results, all the acquired
isolates, with the exception of H1, H7, and T6, exhibited intracellular lactonase
activity due to the recovery of the lactone ring. The chosen strain of Bacillus sp. T5,
with great potential for C8-HSL degradation, was immobilized successfully with the
newly developed immobilization medium QQ-fibers and then applied to MBR.
Mitigation of biofilm formation rate of QQ MBR is 25% more than the control MBR
and the formation of biofilm was suppressed for 10 days. Consequently Bacillus sp.
T5 is an effective QQ bacterium for controlling biofouling.
Acknowledgments
This work supported by The Scientific and Technological Research Council of

Turkey (TUBITAK) (Project No: 114Y706)
35
36
EVALUATION OF A NOVEL ANTI-BIOFOULING MICROORGANISM
(Bacillus sp. T5) FOR CONTROL OF MEMBRANE BIOFOULING AND ITS
EFFECT ON BACTERIAL COMMUNITY STRUCTURE IN MEMBRANE
BIOREACTORS3
Introduction
MBRs are commonly employed in advanced wastewater treatment processes that are
designed to separate the solid and liquid stages, and they have been proven to deliver
a better performance than conventional activated sludge processes (Drews 2010;
Meng, Liao, et al. 2010). However, biofilm forms on the membrane surface in MBRs,
resulting in biofouling. Despite the efforts of many researchers, it is not yet possible
to apply physicochemical treatment methods to completely remove the biofilm that
is generated by MBRs, and this continues to represent a significant challenge that
undermines the sustainability of this technology (Malaeb, Le-Clech, et al. 2013).
Researchers have demonstrated that cell-to-cell signals, which are referred to as

quorum sensing (QS) signals, finely regulate the biofilm that develops on the
membrane surface during the operation of an MBR (Davies, Parsek, et al. 1998). As
such, interfering with these signals through QQ can serve to reduce the amount of
biofouling that occurs in MBRs, and many researchers have turned their attentions
to blocking these signals as a means of controlling biofouling (Yeon, Lee, et al. 2009;
S. R. Kim, Oh, et al. 2013; Köse-Mutlu, Ergön-Can, et al. 2015; Ergön-Can, Köse-
Mutlu, et al. 2017). The main biological agents that are employed in biological
treatments are microorganisms. Some researchers have examined how QQ bacteria
3
This chapter is based on the paper Yavuztürk Gül B., Imer D. Y., Park P-K.,
Koyuncu I. (2018) Evaluation of a novel anti-biofouling microorganism (Bacillus sp.
T5) for control of membrane biofouling and its effect on bacterial community
structure in membrane bioreactors. Water Sci Technol wst2017592. doi:
10.2166/WST.2017.592).
37
generate QQ enzymes and have found that they can be employed to foster an
approach to managing biofouling that is stable, environmentally friendly, and
economically viable (Huang, Ki, et al. 2008). To this end, there is a distinct need for
ongoing research to focus specifically on QQ bacteria and their potential to reduce
biofouling in MBRs.
While a study has been conducted that analyzed microbial 16S rRNA sequences in
various MBR operating conditions, the effect that the QQ had on the MBR
communities was not specifically examined in details. Only very few studies exist in
the literature on this field with high-throughput sequencing technologies (Lim, Kim,
et al. 2012; H. W. Kim, Oh, et al. 2013). In light of the fact that the effect of QQ
mechanisms have on the composition of a microbial community and the subsequent
implications for membrane fouling remains relatively unknown, there is a need to
examine the dynamics of the microbial community, assess the associated metabolic
products, and consider the influence of the operation parameters during the QQ
process. In addition to improving existing knowledge and understanding of
biofouling, in-depth research could also potentially identify the role QQ bacteria
plays during the process of membrane fouling. Unfortunately, the molecular methods
that are commonly used to analyze microbial communities, such as terminal
restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel
electrophoresis (DGGE), are incapable of generating comprehensive data about the
microorganisms that are present. However, one method that does hold significant
potential in this area in that of Illumina sequencing. Illumina is a high-throughput
sequencing technology that generates comprehensive 16S RNA data for a given site
and covers diverse microbial populations, which has been found to enhance
sensitivity and reduce the cost of sequencing (Logares, Sunagawa, et al. 2014). In
addition, it has the ability to deal with all DNA materials. As such, it represents a
very powerful tool that can be employed to develop detailed insights into the
occurrence of microbial and functional genes in MBRs.
The purpose of this study was to examine the anti-biofouling effect of a new QQ
bacterium, Bacillus sp. T5, in an MBR.
The aims of the research were as follows:
(1) characterize the highly effective QQ bacterium Bacillus sp. T5;
38
(2) assess the anti-biofouling effect of T5 in immobilization media in MBR; and
(3) use the Illumina sequencing method to determine how QQ impacts the relative
abundance of the microbial community during the QQ-MBR processes as a means
of better understanding membrane biofouling. This approach may be helpful for
understanding the microbial relationships that exist between QQ bacterium Bacillus
sp. T5 and bacterial community structures in MBRs and how these hinder the
improvement of both the stability and efficiency of the membrane bioreactors.
Methods and Materials
4.2.1 QQ bacteria: Bacillus sp. T5
Quorum Quenching bacterium Bacillus sp. T5 (Accession no: KR705939), was

obtained from the Çamaltı Saltern (İzmir, Turkey) via an enrichment culture method
(Yavuztürk Gül and Koyuncu 2017). Gram staining was carried out according to the
procedure described by Beveridge (2001). The growth of strain T5 was examined
both in autoclaved Luria-Bertani (Miller, US) (LB) medium and synthetic
wastewater. The growth curve of the T5 was plotted by measuring the optical density
at a wavelength of 660 nm (OD660). The specific growth rate of Bacillus sp. T5 was
determined based on the growth curve (Figure 4.1) .
Figure 4.1 : Standard curve for the calculation of residual amounts of C8-
HSL.
39
4.2.2 Preparation of sodium alginate beads
As a cell immobilization media sodium alginate (Sigma-Aldrich) beads were used.

The isolated Bacillus sp. T5 was cultured in LB broth at 31 ºC at 180 rpm for
overnight. Then, T5 culture was centrifuged 9000 rpm for 15 min, washed and
resuspended in 8 ml sterile distilled water (S. R. Kim, Oh, et al. 2013). To make 4%
sodium alginate-T5 suspension 8,2 mg/ml T5 was mixed with 192 ml sterile sodium
alginate suspension. T5-sodium alginate suspension was dropped in 3% CaCl2
solution through a pumping system (Figure 4.2). Formed beads were left in CaCl2
solution for 8 hours to increase the stiffness of the beads and beads were washed with
sterile distilled water. The average diameter of the beads was 3 mm. Empty beads
were prepared with the same procedure without the addition of T5 for the control
reactor.
Figure 4.2 : Pumping system for the preparation of sodium alginate beads.
T5- 4% sodium alginate suspension was dropped in 3% CaCl2 solution through this
system and forming beads left in CaCl2 solution for 8 hours
40
4.2.3 Measurement of AHL degrading activity of the free T5 and T5 beads
The N-Acyl-Homoserine Lactone (C8HSL) degrading activity of T5 was measured

according to bioassay method described in elsewhere (McLean, Pierson, et al. 2004;
Lee, Cheong, et al. 2013). Agrobacterium tumefaciens was used as reporter strain.
By the degradation rate of the C8-HSL, the potential quorum quenching activity of
Bacillus sp. T5 and T5 immobilized sodium alginate beads were determined. Briefly,
C8HSL was added to 30 ml 50 mM Tris-HCL (pH=7) buffer to obtain a C8HSL final
concentration of 200 nM. 50 pieces of T5 immobilized beads were added to C8HSL-
Tris-HCL mixed solution to evaluate the beads activity. Free T5 activity were
measured after centrifuging of T5 and centrifuged T5 was added to C8HSL-Tris-HCl
solution. Samples were taken at certain periods of time in an hour. The bioassay was
used to measure the residual concentrations of C8HSL. Bioassay plates were
prepared according to described in previous studies. The residual amounts of C8HSL
were calculated based on the calibration curve obtained by color zone sizes,
corresponding to each standard concentration of the C8HSL.
4.2.4 Operation of MBR system
Activated sludge was taken from a wastewater treatment plant (İstanbul, Turkey) and
acclimated to the synthetic wastewater before MBR operation. Two laboratory-scale
MBRs were operated in parallel under a constant flux of 48 L/(m2h). Control reactor
had empty beads whereas QQ reactor had T5 immobilized beads. Reactor volumes
and mixed liquor suspended solid (MLSS) concentrations were 4.5 L and 13,300-
13,600 mg/L, respectively. The submerged PVDF hollow fiber membrane modules
with an effective area of 88 cm2 were used in both reactors. Hydraulic retention time
(HRT) and sludge retention time (SRT) were set to 10 h and 30 d, respectively. The
composition of the synthetic wastewater was as follows: glucose, 500 mg/L; urea,
100 mg/L; (NH4)2SO4, 50 mg/L; KH2PO4, 50 mg/L; MgSO4 7H2O, 50 mg/L; NaCl,
50 mg/L; CaCl2 2H2O, 10 mg/L; and NaCO3, 100 mg/L.
4.2.5 Molecular analysis
DNA was extracted from 500 mg of collected samples using a PowerSoil DNA
isolation kit (Mo Bio Laboratories, USA) following the manufacturer’s procedure.
Concentrations of extracted DNA were determined via a NanoDrop UV–vis
spectrophotometer (Thermo Scientifics, USA). 515F (50-
41
GTGCCAGCMGCCGCGGTAA-30) and 806R (50-
GGACTACVSGGGTATCTAAT-30) primers specific for V4 region (length, ca. 250
bp) of the rRNA gene were selected, required Illumina adapters and barcode
sequences were added to the primers. Extracted DNA was amplified using PCR
following the amplification protocol, which is as follows: initial denaturation for 3
min at 94 °C, followed by 20 cycles of 45 s at 94 °C, 30 s at 53 °C, 90 s at 65 °C,
and a final elongation step of 10 min at 65 °C (Shahi et al., 2016). All DNA samples
were further purified using the Wizard DNA Clean-Up System (Promega) in
accordance with manufacture’s protocol. The samples were then quantified using
Qubit 2.0 Fluorometer (Invitrogen, NY, USA). 16S rRNA genes were sequenced
following the Illumina method (Illumina, Inc., CA, USA) with paired-end read
cycles. Sequence analysis and the identification of operational taxonomic units
(OTUs) were obtained using the methods suggested by Giongo et al. (2010) and
Fagen et al. (2012). At least 80% of sequence similarity was considered as the
domain and phylum. OTUs abundance matrices for each taxonomic rank were
created using the total number of reads, which showed 16S rRNA sequences
matching with the database, and matrices of each sample were divided by the total
number of pairs for normalizing varying sequencing depths.
4.2.6 Analytical methods
MLSS, chemical oxygen demand (COD) and total Kjeldahl nitrogen (TKN) were
determined according to Standard Methods (1998). The surface and cross section of
the empty and QQ beads were visualized by scanning electron microscopy (SEM).
Biofilm layers of the membrane surfaces were observed using confocal scanning
laser microscopy (CLSM, C1 plus, Nikon, Japan).
4.2.7 Statistical analysis
R 3.1.1 analysis was used to conduct Statistical analyses (www.r-project.org).

Histogram, q-q plots and the Shapiro-Wilk’s test were performed to examine data
normality. Variance homogeneity was also investigated by using The Levene’s test.
One-way analysis of variance (ANOVA) or independent-samples t-test was used to
check against the variations in QQ bacterium Bacillus sp. T5 and bacterial
community structures. To provide multiple comparisons, The Tukey’s test was used.
42
Values of tests were pointed out as mean and standard deviation. Important
difference was detected at the p< 0.05 level of importance.
Results and Discussion
4.3.1 Molecular characteristics of Bacillus sp. T5
Gram staining, QQ activity of free and immobilized T5 and growth rate in LB

medium and in synthetic wastewater (ww) of T5 were evaluated to observe microbial
characteristics. As shown in Figure 4.3, Bacillus sp. T5 growth rate was higher in the
LB medium than the synthetic ww.
T5 LB T5 SW
8
7
6
OD 660 nm
5
4
3
2
1
0
0 0,5 1 1,5 2 2,5 3 3,5
Time (day)
Figure 4.3 : Comparison of the growth rate of Bacillus sp. T5 in the LB

medium and the synthetic wastewater.
The specific growth rate of T5 during the exponential growth phase were 0.45 h−1
and 0.23 h−1 in LB medium and synthetic ww, respectively. As shown in Figure 4.4,
T5 was a gram-positive and rod-shaped bacterium.
43
Figure 4.4 : Gram staining of Bacillus sp. T5 after 24 h of incubation.
The AHL degradation results obtained from the free cell and T5 immobilized beads
were presented in Figure 4.5. Bacillus sp. T5 degraded all the C8-HSL tested and
exhibited great activity. The amount of the remaining C8-HSL concentration was
negligible for the free T5.
44
Free T5 Immobilised T5 Control
250
200
C8-HSL Cons. (nM)
150
100
50
0
0 10 20 30 40 50 60 70 80
Time (day)
Figure 4.5 : C8-HSL degradation of the free and immobilized Bacillus sp. T5.
AHL amounts in the samples were determined after incubation of the 200 nM AHL
with free cell and QQ-beads in Tris-HCL buffer. Vacant beads were used as a
control. Error bar: standard deviation (n=3)
4.3.2 Immobilization of Bacillus sp. T5 and its anti-biofouling effect on MBR

performance
The beads were measureed as 3 mm in diameter and had a spherical shape with a
smooth surface and uniform size. In Figure 4.6, the surface and the cross-sectional
SEM images showed the morphologies of the vacant beads and QQ-beads. Rod-
shaped T5 were spread on the bead surface.
45
a b
Figure 4.6 : SEM image of (a) the surface and (b) the cross-section of the
sodium alginate beads. Immobilized rod-shaped Bacillus sp. T5 can be seen in the
figures.
Before MBR operation, AHL degradation potential of the QQ-beads containing 8.2
mg T5/cm3 cubbyhole and the vacant beads were also measured. As shown in Figure
3.2, the degradation efficiency of C8HSL with the QQ-beads was measured to be
75% in the reaction time of 70 min. The adsorption of C8HSL on the vacant beads
was negligible.
The mitigate of biofouling during the MBR operation was analyzed via monitoring
transmembrane pressure (TMP). TMP rise was delayed substantially after applying
T5 immobilized sodium alginate beads in the QQ reactor (Figure. 4.7). Since TMP
rises due to biofouling formation during MBR operation, TMP values were
controlled to evaluate mitigation of biofouling in control and QQ reactors.
Prevention efficiency of T5 was calculated using the areas under the TMP curves.
As shown in Figure 3.4, during 12 days of MBR operation TMP was reached to 480
mbar in control reactor whereas TMP of QQ reactor was reached to 110 mbar. QQ
reactor decreased TMP rise at least four times regarding control reactor.
46
QQ TMP Control TMP
600
500
400
TMP (mbar)
300
200
100
0
0 2 4 6 8 10 12 14
Time (day)
Figure 4.7 : Transmembrane pressure (TMP) profiles during the MBR

operation. Empty beads and QQ-beads with Bacillus sp. T5 were applied to the
control and the QQ reactors.
This was the evidence of that T5 immobilized QQ beads, inhibited biofilm formation
and decreased TMP. According to the calculated area under the TMP curves; QQ
effect of Bacillus sp. T5 resulted in 71% decrease in TMP values compared with the
control. Inhibition of biofilm formation on membrane surface was confirmed visually
via CLSM after 12days of operation. In Figure 4.8, biofilm thickness formed was
compared. According to the CLSM image of from QQ reactor was thinner than that
from the control reactor. These data strongly supported the evidence of QQ effect on
the inhibition of biofouling.
47
a b
Figure 4.8 : The CLSM images of biofilm on the membrane surface. (a)
Control MBR, cross-section view, (b) QQ MBR, cross-section view at the end of
the MBR operation, stained with Live/dead BacLight staining. Magnification: X40.
Image size 501.76 × 501.76 μm.
Our previous study (Yavuztürk Gül and Koyuncu 2017) was conducted with
different immobilization media namely QQ-fiber through the encapsulation of the
very same QQ bacteria, Bacillus sp. T5. QQ-fiber was applied to a submerged MBR
and resulted in 25% decrease in biofilm formation. According to the results, sodium
alginate beads as an immobilization media increased the QQ effect of T5 25% to
71% on MBR. These results indicated that QQ bacterium, Bacillus sp. T5 could
effectively mitigate the biofouling in MBR and QQ beads with T5 have higher QQ
activity potential than the study based on QQ-fiber with same bacteria. These results
are consistent with previous studies reporting that sodium alginate beads were more
effective immobilization media with high QQ activity compared to the vessels (Oh,
Kim, et al. 2013; Köse-Mutlu, Ergön Can, et al. 2015).
To determine whether QQ mechanism has any side effect on COD and TKN removal
efficiencies, biodegreadation of organics of control and QQ reactors during the
microfiltration process was also monitored. The differences in COD and TKN
removal efficiencies of QQ and control reactors were negligible. As a result, these
findings suggested that QQ mechanism of T5 did not have any negative effect on the
removal of organics.
4.3.3 Quorum quenching effects on microbial community structure
Microbial composition of the mixed liquor in control and QQ reactor was analyzed
at the different taxonomic level during the MBR operation. Figure 4.9 showed the
48
relative abundances in phyla in the studied samples. Proteobacteria (35 ± 2.8%),
Actinobacteria (12± 2.3%), Bacteroidetes (11.7 ± 2.6%), Acidobacteria (4.2 ±
0.9%), Firmicutes (6.3 ± 1.5%), and Chloroflexi (5.2 ± 0.9%) were determined to be
the dominant phyla in the control and QQ reactors, respectively. Proteobacteria and
Actinobacteria were most dominant phylum in the activated sludge, which was in
accordance with with former researches that revealed Proteobacteria to be a
dominant phylum in activated sludge (Yuki Miura, Yoshimasa Watanabe, et al.
2006; Teplitski, Chen, et al. 2004; Lim, Kim, et al. 2012; H. W. Kim, Oh, et al. 2013).
Moreover, among Proteobacteria, the proportion of Alphaproteobacteria (38.2 ±
0.6%) and Betaproteobacteria (21.5± 0.4%) was much higher than
Deltaproteobacteria (12.3 ± 0.3%) and Gammaproteobacteria (8.2 ± 0.3%).
According to molecular analysis in MBR, most of the dominant phyla belonged to
gram-negative bacteria, which again confirmed the importance of these bacterial
groups in the biodegradation of signal molecules.
abundances of the dominant phyla during quorum quenching process.
Relative abundances of the microbial community were different in the control and
QQ reactor as expected. It was clear from the Figure 4.9 that the relative abundances
of Actinobacteria, Acidobacteria, and Firmicutes were significantly affected by
49
Quorum quenching mechanism in QQ reactor, whereas the same phyla in control
reactor were not affected (p < 0.05). On the other hand, QQ activity of Bacillus sp.
significantly influenced the relative abundance of Proteobacteria, Bacteroidetes, and
Chloroflexi. However, there are no significant differences in the relative abundances
of the same phyla in control reactor. This suggested that QQ bacterium Bacillus sp.
T5 has the ability to change the proportion of certain microbial groups in the mixed
liquor. Regarding the dominant bacterial strains in the MBR, Bacteroides sp.,
Acinetobacter sp., Defluvibacter sp., Desulfitobacterium sp., Streptococcus sp.,
Flavobacterium sp., Staphylococcus sp., Pseudomonas sp., and Bacillus sp. species
were in abundance (Figure 4.10). These species constitued more than 50% of the
dominant bacterial strains. Acinetobacter and Flavobacterium (Zhang, Choi, et al.
2006) have been reported as one of the pioneer genera in the membrane fouling in a
lab-scale MBR. Other genera such as Brevundimonas, Acinetobacter,
Sphingomonas, and Aquaspirillum have also been reported to be present in low
abundance in the mixed liquor but dominant on biofilm (Zhang, Choi, et al. 2004).
According to our results, relative abundance of Acinetobacter and Flavobacterium
much higher in the mixed liquor of the QQ reactor than the control reactor and
increased during the QQ process. These findings imply that QQ bacterium T5 may
prevent selectively to attach specific bacterial, especially Proteobacteria, groups to
the membrane surface.
50
Figure 4.10 : Relative abundance of dominant bacterial Genus in control and
QQ reactor.
During MBR operation, MLSS and COD removal rate were maintained at constant
levels. However, the relative abundance of microbial communities in mixed liquor
demonstrated high diversities for each reactor. Furthermore, we demonstrated that
QQ mechanism had a great effect on microbial taxa in QQ reactor compared to the
control one. This may be an indicator for a strong evidence of close association
between microbial community structure with QQ and biofouling characteristics.
Microbial diversities and their relative abundances in the community provide
significant information about the main phyla involved in the inhibition of biofilm
process in the QQ reactor. Consequently, the assessment of indigenous bacteria and
the probable effects of quorum quenching on the population dynamics of MBR are
necessary for understanding QQ process.
Conclusion
The findings of this study indicated that Bacillus sp. T5 had a strong impact on the
performance of MBR. Anti-biofouling effect of T5 immobilized beads was evaluated
as 71%. The results of the molecular investigation revealed that Actinobacteria,
Acidobacteria, Firmicutes, Proteobacteria, Bacteroidetes, and Chloroflexi were
very sensitive to QQ process of Bacillus sp. T5 and this was ultimately reflected in
51
the performance of the membrane bioreactors. Therefore, molecular analysis of
microbial community in MBR is strongly suggested for improving MBR systems
and membrane biofouling mitigation .
Acknowledgments
This work supported by The Scientific and Technological Research Council of
Turkey (TUBITAK) (Project No: 114Y706)
52
SELECTION OF THE QUORUM QUENCHING (QQ) BACTERIA FOR
MEMBRANE BIOFOULING CONTROL: EFFECT OF DIFFERENT
GRAM-STAINING QQ BACTERIA, Bacillus sp. T5 AND Delftia sp.T6, ON
MICROBIAL POPULATION IN MBR4
Introduction
MBRs are commonly employed within advanced wastewater treatment processes,

and previous research has concluded that their performance outperforms traditional
activated sludge approaches (Drews, 2010; Meng et al., 2017). However, during the
process of operation, microorganisms accumulate on the membrane surface in
MBRs, and this results in biofouling on a long-term basis. Despite the significant
developments that have emerged in this area, to date, scientists have yet to identify
a method by which the formation of biofilms can be prevented. As such, membrane
biofouling remains a significant issue that undermines the sustainability of MBR.
Previous studies have found that cell-to-cell signals, or quorum sensing (QS) signals
as they are also known, play a regulatory role in controlling the membrane surface
biofilm that develops during the operation of an MBR (Davies et al., 1998).
Researchers have found that interfering with QS signals through QQ methods can
effectively reduce biofouling and significant effort has been invested in identifying
approaches through which QS signals can be blocked (Yeon et al., 2009a, 2009b).
Bacterial QQ represents a promising approach to controlling biofouling in MBRs
because it enhances the efficiency of the treatment and is less energy intensive than
alternative approaches (Ergön-Can et al., 2017; Köse-Mutlu et al., 2015; Oh et al.,
4
This chapter is based on the paper Yavuztürk Gül, B., Imer, D.Y., Park, P.-K.,
Koyuncu, I., 2018. Selecting the Right Quorum Quenching (QQ) Bacteria for
Membrane Biofouling Control: Effect of Different Gram-staining QQ Bacteria,
Bacillus sp. T5 and Delftia sp. T6, on Microbial Population in MBR. Water Science
and Technology.
53
2013). In more recent studies, researchers have successfully isolated novel QQ
bacteria from both MBRs and nature (Yavuztürk Gül and Koyuncu, 2017). However,
to date, the decision as to which QQ bacteria to use as a means of impeding QS
signals has been based on the level of AHL degradation and no effort has been made
to assess the impact that the QQ bacteria has on the microbial community.
Although a solid understanding of the microbial community present within biofilm

is critical to the development of effective QQ strategies, knowledge in this area
remains extremely limited (Jo et al., 2016). As such, there is a requirement to
evaluate the impact that different QQ bacteria of varying properties have on the
microbial community that is present in an MBR.
In the current study, we assessed the relationship between the microbial community
and two different QQ bacteria, Bacillus sp. T5 (Gram positive) and Delftia sp. T6
(Gram negative), both of which exhibit significant Acyl-Homoserine Lactone (AHL)
activity. The objective of this research was to use the Illumina sequencing method to
identify how different QQ bacteria impact the relative abundance of the microbial
community during QQ-MBR processes. It is anticipated that the findings of this
research will lead to an enhanced knowledge of the relationships that exist between
QQ bacteria and the bacterial community structures present in MBRs and,
consequently, the development of effective methods of controlling biofouling and
enhancing the performance of membrane bioreactors.
Material and Method
5.2.1 MBR systems
Two laboratory-scale submerged MBRs comprising two identical aerobic tanks, the
control reactor with vacant beads without QQ bacteria and the QQ reactor with QQ
beads, were designed (Figure 5.1). Two sets of MBR operations for T5 and T6 were
run under the same conditions. Each reactor was operated at a filtration flux of 23
L/m2h to maintain the MBR operation. Activated sludge was obtained from a
wastewater treatment plant (İstanbul, Turkey) and acclimated with the synthetic
wastewater.
54
Figure 5.1 : Schematic diagram of lab-scale MBR operation and preparation of the beads.
55
The composition of the synthetic wastewater (ww) was as follows (mg/L): glucose,
500; urea, 100; (NH4)2SO4, 50; KH2PO4, 50; MgSO4 7H2O, 50; NaCl, 50; CaCl2
2H2O, 10; and NaCO3, 100. Dissolved oxygen concentration was maintained at
5.5−6 mg/L. The working volume and mixed liquor suspended solid (MLSS)
concentration of each reactor were maintained at 4.5 L and 11.000−11.200 mg/L for
operation with T5 and 11.200−11.700 mg/L for operation with T6, respectively. The
hydraulic retention time (HRT) and sludge retention time (SRT) were maintained at
19 h and 30 d. The two identical, submerged PVDF hollow fiber membrane modules
with an effective area of 88 cm2 were used in both reactors for two MBR operations.
In order to quantify the degree of membrane biofouling, changes of trans membrane
pressure (TMP) values which critical membrane performance parameter was
observed. Changes in TMP were monitored via fully automated system, SCADA, for
two operations. The efficiency of mitigating biofouling formations was estimated
through the proportion of calculated area under the TMP curves of the control and
QQ reactors for each run.
5.2.2 QQ bacteria: Bacillus sp. T5 and Delftia sp. T6 and preparation of the
immobilization media
QQ bacteria Bacillus sp. T5 (Accession no: KR705939) and Delftia sp. T6

(Accession no: KR705940) were used (Yavuztürk Gül and Koyuncu, 2017). Gram
staining was done according to the procedure described in the literature (Beveridge,
2001). The growth rate of T6 was evaluated in both Luria-Bertani (LB) medium and
synthetic ww by measuring the optical density at a wavelength of 660 nm (OD660).
The specific growth rate of QQ bacteria were calculated from the logarithmic scale
of the growth curve (Nair, 2007).
Sodium alginate (Sigma-Aldrich) beads were used as a cell immobilization media.

Bacillus sp. T5/Delftia sp. T6 were immobilised to the beads described in the
literature (S. R. Kim et al., 2013). Vacant beads were prepared without the addition
of T5/T6 for the control reactors (Figure 5.1). The concentration of Bacillus sp.
T5/Delftia sp. T6 was approximately 9 mg/mL in the beads. The average diameter
of the beads was approximately 3 mm.
56
5.2.3 Measurement of AHL degrading activity via bioassay for free and
ımmobilised QQ bacteria
The C8-HSL degrading activity of T5/T6 was qualified according to the bioassay
method described in previous studies (Lee et al., 2013; McLean et al., 2004). To
determine the C8-HSL degrading activity of the beads, a 30 mL Tris-HCL (50 mM,
pH=7) buffer was prepared and C8-HSL was added to make the 200 nM final
concentration. T5/T6 immobilized beads were incubated in an hour (31 °C, 180 rpm),
and samples were taken at certain intervals. The concentrations of the residual C8-
HSL molecules were measured on the basis of the calibration curve gained by color
zone sizes, corresponding to each standard concentration of the C8-HSL.
5.2.4 Molecular analysis/ DNA extraction, PCR amplification and HiSeq

platform sequencing
DNA was extracted from 1000 mg of samples via a PowerSoil DNA isolation kit
(Mo Bio Laboratories, USA) due to the manufacture’s procedure. The extracted
DNA was quantified using NanoDrop UV–vis spectrophotometer (Thermo
Scientifics, USA). The V4 regions of the 16S rRNA genes were amplified with the
primers (length, ca. 250 bp) 515F (50-GTGCCAGCMGCCGCGGTAA-30) and
806R (50-GGACTACVSGGGTATCTAAT-30) primers specific for V4 region
(length, ca. 250 bp). Illumina adapters and barcode sequences which are required
were added to the primers. Extracted DNA was amplified using PCR, amplification
protocol as follows: initial denaturation for 3 min at 94 °C, followed by 20 cycles of
45 s at 94 °C, 30 s at 53 °C, 90 s at 65 °C, and a final elongation step of 10 min at 65
°C (Shahi et al., 2016). Using the Wizard DNA Clean-Up System (Promega), all
DNA samples were purified. The samples were measured using Qubit 2.0
Fluorometer (Invitrogen, NY, USA). 16S rRNA genes were sequenced following the
Illumina method (Illumina, Inc., CA, USA) with paired-end read cycles. The
methods suggested by Giongo et al. (Giongo et al., 2010) and Fagen et al. (R. Fagen,
2012) used for sequence raw data analysis and the identification of operational
taxonomic units (OTUs). At least sequence similarity of 80 % of was sustained as
the domain and phylum. For each taxonomic rank OTUs abundance matrices were
created via the total number of reads, which showed 16S rRNA sequences matching
57
with the database, and matrices of each sample were divided by the total number of
pairs for normalizing varying sequencing depths.
5.2.5 Analytical methods
The concentration of the mixed-liquor suspended solids (MLSS), chemical oxygen

demand (COD) and total Kjeldahl nitrogen (TKN) were measured according to
Standard Methods (Standard Methods for the Examination of Water and Wastewater,
1998). The dissolved oxygen concentration was measured by a DO meter (YSI,
Model DO200, USA).
5.2.6 Statistical analysis
Statistical analysis was carried out using R 3.1.1 (www.r-project.org). To examine

data normality, histogram, q-q plots and the Shapiro-Wilk’s test were performed.
Variance homogeneity was also determined by The Levene’s test. One-way analysis
of variance (ANOVA) or independent-samples t-test was used to check against the
variations in QQ bacterium Bacillus sp. T5 and Delftia sp. T6 and bacterial
community structures. The Tukey’s test was used to provide multiple comparisons,.
Values of tests were pointed out as mean and standard deviation. Important
difference was detected at the p< 0.05 level of importance.
Results
5.3.1 QQ bacteria, activity of the beads
Growth of Bacillus sp. T5 was calculated in our previous study (Yavuztürk Gül et
al., 2018). The growth rate of Delftia sp. T6 was calculated in both LB medium and
synthetic ww and is shown in Figure 5.2. Each QQ bacteria grows faster in the LB
medium than the synthetic ww. The specific growth rates of T5 and T6 during the
exponential growth phase were 0.45 h−1 and 0.23 h−1 in LB medium and 0.44 h−1 and
0.28 h−1 in synthetic ww, respectively.
58
6
OD 660 nm
4
T6 LB
2 T6 SW
0
0 1 2 3 4
Time (day)
Figure 5.2 : Growth rate of Delftia sp. T6 in the LB medium and the synthetic
wastewater.
9 mg/cm3 cubbyhole QQ bacteria immobilized to the sodium alginate beads, which

were approximetly 3 mm in diameter and spherical with a smooth surface. AHL
degradation potential was measured for the QQ-beads and vacant beads. As shown
in Figure 5.3, the degradation efficiency of C8-HSL of the beads with T5 and T6 was
measured 77% and 71% in the reaction time of 120 min, respectiveley. The vacant-
bead without the QQ bacteria did not exhibit a considerable decrease in the C8-HSL
concentration.
59
200,0
150,0 T5
C8-HSL Conc. (nM)
T6
Control
100,0
50,0
0,0
0 20 40 60 80 100 120 140
Time (min)
Figure 5.3 : Degradation of C8-HSL via QQ-beads with Bacillus sp. T5 and
Delftia sp. T6. C8-HSL concentration in the samples were determined using
bioassay. Vacant beads were used as a control. Error bar: standard deviation (n=3)
5.3.2 Anti-biofouling effect of QQ bacteria on MBR
Two laboratory-scale aerobic reactors (control and QQ) were operated in parallel
under the same operating conditions. QQ beads and vacant beads were applied to the
QQ-MBR and control MBR to observe their inhibition potential of biofouling. TMP
increase during the biofouling occurence in time because of that inhibition of
biofouling was detected via monitoring the rise of the TMP profiles of the control
and QQ-MBR during the operations and compared areas under the TMP curves to
evaluate anti-biofouling efficiency of T5 and T6 through the QQ mechanism (Figure
5.4).
It is observed that rising of TMP is successfully controlled with QQ bacteria T5 and

T6. In Figure 5.4, the control reactor was reached the TMP of 300 mbar in 20 d,
whereas the QQ-reactor with QQ beads to reach the TMP of 100 mbar within the 20
d for operation with T5 and the control MBR took 11 d for the TMP to reach 420
mbar in the operation of T6 but the QQ-MBR took 11 d to reach 150 mbar. According
to the calculated area under the TMP curves, T5/T6 immobilized the QQ beads and
mitigated biofilm formation by 85−76%, compared with the control MBR.
60
a
400
TMP (mbar) QQ (T5) TMP

200 CONT.TMP
0
0 5 10 15 20 25
Time (day)
b
400
TMP (mbar)
200
0
0 2 4 6 8 10 12
Time (day)
Figure 5.4 : Transmembrane pressure (TMP) profiles of the control and the
QQ reactors during the MBR operation with vacant beads and QQ-beads with
Bacillus sp. T5 (a) and Delftia sp. T6(b).
The results are consistent with our previous studies reporting the QQ effect of the
same bacteria with different immobilization media (Yavuztürk Gül and Koyuncu
2017a; Yavuztürk Gül et al. 2017b).
During the microfiltration process, COD removal efficiencies of control and QQ

MBR were evaluated as 96-98% and the differences in COD profiles of QQ and
control reactors were negligible.
61
5.3.3 Differentiation of bacterial composition in the mixed liquor
Illumina analysis was performed to assess differentiation in the bacterial community.

Bacterial composition of the mixed liquor in the control and the QQ reactor was
determined (Figure 5.5).
a Proteobacteria
Bacteroidetes
Actinobacteria
Chloroflexi
Planctomycetes
Acidobacteria
Firmicutes
0 20 40 60 80 100
b Proteobacteria
Bacteroidetes
Actinobacteria
Chloroflexi
Planctomycetes
Acidobacteria
Firmicutes
Verrucomicrobia
Cyanobacteria
0 20 40 60 80 100
Relative
abundances of the dominant phyla during quorum quenching process with T5 (a)
and T6 (b). (C: Control, Q:QQ reactor)
The phyla Proteobacteria, Actinobacteria, and Bacteroidetes were most dominant in

the control and QQ reactors for each run. It was found that the average relative
abundance of Proteobacteria was 31 % in the control MBR, whereas it was 40 % in
the QQ-MBR in the first run (T5 operation), 34 % in the control, and 28 % in the QQ
62
reactors in the second run (T6 operations). The average relative abundances of the
dominant bacteria classes in T5 and T6 operations are shown in Figure 5.6.
a T6
100
80
60
40
20
0
C1 C2 C3 C4 Q1 Q2 Q3 Q4
Bacteroidia Acidimicrobiia Chloroflexia

Planctomycetacia Acidobacteriia Bacilli
Verrucomicrobiae Clostridia Others
Unclassified (at Class level)
b T5
100
80
60
40
20
0
C1 C2 C3 C4 Q1 Q2 Q3 Q4
Bacteroidia Acidimicrobiia Chloroflexia

Planctomycetacia Acidobacteriia Bacilli
Verrucomicrobiae Clostridia Others
Unclassified (at Class level)
Figure 5.6 : Relative abundance of dominant bacterial classes in control and

QQ reactor for operation with T6 (a) and T5(b).
In the first operation with the T5, abundances of Bacilli, Bacteroidia, and
Chloroflexia were increased over time in the QQ-MBR, whereas Acidimicrobiia and
Acidobacteria were decreased over time in the QQ-MBR. There are not significant
differences in the relative compositions of the same classes in the control reactor (P
< 0.05). In the second run with T6, Bacilli, Bacteroidia, and Chloroflexia decreased
over time in the QQ-MBR, whereas Acidimicrobiia and Acidobacteria increased
63
over time in the QQ-MBR. The abundances of the same classes were not
significantly changed in the control reactor (P < 0.05).
Discussions
According to the results of MBR operations, the anti-biofouling effect of T5 and T6

was evaluated as 85% and 71%. Delftia sp. T6 successfully controlled TMP rising
likewise the QQ bacterium T5. The QQ activity of T6 controlled the occurrence of
biofilm and stretched the time required to reach the same TMP. These findings
indicated that Bacillus sp. T5 and Delftia sp. T6 had a strong impact on the
performance of membrane bioreactors.
Although Bacillus sp. T5 has been already reported to mitigate biofouling with
different immobilization media in MBR (Yavuztürk Gül et al., 2018; Yavuztürk Gül
and Koyuncu, 2017), it still remains necessary to make clear the impact of the QQ
bacteria to microbial population of mixed liquor in MBR.
According to the findings of the Illumina Hi-seq sequences analysis, the phyla
Proteobacteria, Actinobacteria, and Bacteroidetes were most dominant in the
control and QQ reactors for each run, which is consistent with previous studies (H.
W. Kim et al., 2013; Lim et al., 2012; Teplitski et al., 2004; Yuki Miura et al., 2006).
The QQ effect of T5 caused a decrease in the abundance of the Actinobacteria but
an increase in the abundance of Proteobacteria and Bacteroidetes. The QQ effect of
T6 caused an increase in the amount of Actinobacteria but a decrease in
Proteobacteria and Bacteroidetes. When phylum results are examined, it is observed
that the QQ mechanisms of T5 and T6 have the opposite effect on microbial
community composition. In order to examine this opposite effect further, the results
of the class abundance have been compared (Figure 5.6). It was observed that gram-
negative QQ bacteria (T6) increased the relative abundances of gram-positive classes
and decreased gram negatives. Contrarily, gram-positive QQ bacterium (T5)
increased relative abundances of gram-negative classes and decreased gram
positives.
This said opposite effect of QQ bacteria may originated/caused two different

consequences: 1) The amount of certain microbial groups increased in mixed liquor
because they may be more likely formed to produce biofilm or 2) Dependent on the
64
QQ mechanism, they might be prevented from binding biofilm and survived on
mixed liquor, thus their population increased for this reason.
In this study, we demonstrated that different QQ bacteria (one gram positive and the
other gram negative) had an opposite effect on microbial taxa in the QQ reactor
compared with the control. This was reflected in the microbial population dynamics.
Findings of the study are significant and can inform ongoing research efforts to
identify and develop a new QQ strategy. The development of a comprehensive
molecular analysis of the microbial community that is present in MBRs could play a
critical role in the identification of membrane biofouling control methods and the
selection of the most appropriate QQ bacteria to achieve control objectives. It can be
concluded that the interaction between microbial community structure with quorum
quenching bacteria may lead to choose suitable bacteria/strategy for a different
purpose or may be used in studies that aim to control different bacterial groups via
QQ. Also, provide significant information for the future research may be conducted
on the effect of different QQ bacteria on biofilm composition in the QQ-MBR and
the propensity of the microbial taxa that are promoted in presence of QQ bacteria to
produce EPS/biofilm.
Acknowledgments
This study was funded by The Scientific and Technological Research Council of
Turkey (TUBITAK) (Project No: 114Y706).
Compliance with Ethical Standards
5.6.1 Conflict of interest
The authors declare that they have no conflict of interest.
5.6.2 Ethical statement
This article does not contain any studies with human participants or animals
performed by any of the authors.
65
66
CONCLUSIONS AND RECOMMENDATIONS
Attempts to reduce the formation of biofilm using the Quorum Quenching method in
MBR systems have emerged as an innovative approach. Quorum quenching be able to
reduce membrane biofouling efficiently thus improve the filtration process. For the
last decades, the development of antibiofouling strategies based on QQ in MBR is a
relatively hot topic of biofouling researches. Nevertheless, several questions need to
be adressed about biofilm formation mechanisms and detailed consequence of activity
of QQ bacteria in MBR. Despite, plenty of studies carried out on quorum quenching,
membrane and/or module modification in MBR, studies of QQ should continue deeper
and more specific about the mecanism of QQ. Majorly one bacterial strain
(Rhodococcus sp., BH4) has been reported to have been used for QQ in MBR until
now. In this thesis, new QQ bacteria were isolated from our national resources in order
to obtain new QQ bacteria. Bacteria with high AHL degradation activity were selected
from among those experiments and it was demonstrated that they were very suitable
for Biofouling Control. It has been determined that QQ bacteria effectively retard the
biofilm layer formed on the membrane surface and can be used as an antibiofouling
agent according to all data. Results of the thesis are briefly summarized below:
 Source of the QQ bacteria were pond, saltern, marine and leachate. 25 QQ

bacteria were obtained and the accession number of the isolates were deposited
in NCBI GenBank.
 All species have been determined as over 50% of QQ activities,
 For the first stage, Bacillus sp. T5 was selected and applied to the MBR.
Operation with QQ-Fiber and beads were performed with this species.
 According to results it was proven that strain T5 QQ was very convenient for
controlling biofilm and effectively mitigate the biofilm layer formed on the
membrane surface.
 It is stated that the QQ effect of the QQ-fiber is lower than the effect of the
beads applications in reactors. It may cause that the immobilised QQ bacteria
may not have enough area for growth in the QQ-fibers.
67
 For the second stage, MBR operation was performed with one of the most
active QQ bacteria, Delftia sp. T6. The TMP data and the operating parameters
are compared, it has been determined that the T6 strain effectively control the
biofilm layer formed on the membrane surface and can be used as a biofouling
agent such as T5.
 For the third stage, the joint effect of sodium alginate beads with T5 and T6
have been evaluated and observed the changes in the microbial population in
MBR. The retardation in the increasing of the TMP in mixed operation was
lower than that of using two types of QQ bacteria individually. The
antagonistic effect against each other could be trigger this situation.
 Since the sodium alginate beads was dispersed within 12-20 days during the
operation, reusable and stable immobilization media development studies have
been made and alginate beads with added PVA have been produced and
optimized for this purpose. MBR operations with S4 bacteria were carried out
in different hydraulic conditions and the QQ effects of newly produced durable
beads were revealed.
 The one of the major finding of the thesis was bacterial community changes
during the QQ-MBR runs with T5 and T6 which investigated using the Ilumina
Hi-Seq platform. The effect of QQ on the bacterial community was examined
in detail on the basis of phylum, class and genus. It has been demonstrated that
the microbial groups affected by the T5 and T6 were different and their shifting
effects of bacterial population dinamics were in the exact opposite direction.
Bacillus sp. T5 caused an increase in the number of gram negative classes while
the number of gram positive classes decreased. On the contrary, Delftia sp. T6
was reduce gram negative classes and led to an increase in the number of gram
positives. Molecular data of operation with mixed T5 and T6 was support this
finding.
To summarize, within the the scope of this thesis, new and active species were isolated
from different sources, used in MBR as an antibiofouling agent and successfully
reduced biofilm and proven to be a highly effective biotechnological solution method
for biofouling problem. Moreover, three different immobilisation media (QQ-Fiber,
SA Beads and PVA Beads) were applied with selected QQ bacteria and PVA beads
68
have been found most suitable for MBR operations. Finally, according to the results
of molecular analysis, different QQ bacteria were found to act by interacting with
different microbial groups.
Since QQ is promising, ecofriendly and natural process, studies on QQ in MBR stands

hopeful alternative for controlling biofouling for future applications. One of the
potential research area of the QQ concept could be the isolation of new pure cultures
that have a high ability on QQ. Controlling to the spesific microbial group as a target
by QQ bacteria could be also a new perspective for QS studies. In addition, during the
QQ process, active groups/species on the microbial community can be quantified via
targeted genes encoding AHL/lactonases. provide more deep understanding of the QQ
mecanism. It is obvious that devolopment of an innovative immobilization medium is
the other research point which would help commercialize bio-products of QQ. In the
light of the findings of the thesis, one of the significant research area could be
metagenomic analysis of the QQ-MBR. Molecular analysis of the interaction of the
QQ bacteria and indigenous microbial community is critical to the performance of
MBR. Comprehensive QQ studies should be continue to carried out with the
perspective of molecular and metagenomic analysis because of the significance
reflection of the QQ in the relative abundance of the microbial groups. Future QQ
studies should eventually be moved from the lab scale to the pilot scale and ultimately
to the real scale MBR in our country.
69
70
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82
CURRICULUM VITAE
Name Surname : Bahar YAVUZTÜRK GÜL
Place and Date of Birth : İstanbul, 18.03.1981
E-Mail : baharyavuzturk@yahoo.com
EDUCATION :
 B.Sc. : 2003, Marmara University Department of Biology

 M.Sc. : 2005, Marmara University Institute of Pure and Applied Sciences,
Biology
PROFESSIONAL EXPERIENCE AND REWARDS:
 (2014-2015) TÜBİTAK Project Scholarship “Yüksek performanslı ileri osmoz

membranı üretimi ve bu membranlar ile gerçek atıksu arıtım performanslarının
belirlenmesi” (Project Number: 113Y340).
 (2014-2017) İstanbul Technical University BAP Project Scholarship “Quorum
Quenching Bakterilerinin Çevresel Kaynaklardan İzolasyonu ve Biyofilm
Gelişiminin Önlenmesi Amacıyla Membran Biyoreaktörlerde Kullanılması”.
 (2015-2017) 2523 - TÜBİTAK-NRF Project Scholarship “Study on Hydraulic

Factors in Membrane Bioreactors Controlled by Quorum Quenching
Technology” (Project Number: 114Y706).
83
PUBLICATIONS, PRESENTATIONS AND PATENTS ON THE THESIS:
 Yavuztürk Gül, B., Imer, D.Y., Park, P.K., Koyuncu, I. (2018). Selecting the
Right Quorum Quenching (QQ) Bacteria for Membrane Biofouling Control:
Effect of Different Gram-staining QQ Bacteria, Bacillus sp. T5 and Delftia sp.
T6, on Microbial Population in MBR. Water Science and Technology, Under
Review.
 Yavuztürk Gül B, Imer DY, Park PK, Koyuncu I. (2018) Evaluation of a
novel anti-biofouling microorganism (Bacillus sp. T5) for control of membrane
biofouling and its effect on bacterial community structure in membrane
bioreactors. Water Sci. Technol. 77, 971–978. doi:10.2166/wst.2017.592.
 Yavuztürk Gül, B., Koyuncu, I. (2017). Assessment of new environmental
quorum quenching bacteria as a solution for membrane biofouling. Process
Biochem. 61, 137–146. doi:10.1016/j.procbio.2017.05.030.
 B. Yavuztürk Gül, B. Kose Mutlu, T. Ergon Can, I. Koyuncu, Isolation and
Identification of Quorum Quenching (QQ) Bacteria from Different Ecological
Environments, and QQ MBR Operation with New Isolate, International
Workshop on MBR/QQ-MBR, 19 November 2015, Soeul.
 B. Yavuztürk Gül, B. Kose Mutlu, T. Ergon Can, I. Koyuncu, Quorum
Quenching (QQ) Bakterilerinin Farklı Ekolojik Ortamlardan İzolasyonu,
Tanılanması, QQ Potansiyellerinin Belirlenmesi ve MBR’de Uygulanması, 4.
Ulusal Membran Teknolojileri ve Uygulamaları Sempozyumu, 7-8 Ekim 2015,
Isparta.
 B. Yavuztürk Gül, Pyung Kyu Park, Chung Hak Lee, I. Koyuncu,
Comparision of QQ Bacteria isolated from marine, pond, saltern and landfill
leachate for biofouling control in MBR, International Water Industry
Conference, IWIC 2016, Daegu city, S. Korea.
 B. Yavuztürk Gül, I Koyuncu, Evaluation of N-Acyl-Homoserine Lactone
degradation by bacteria isolated from marine, pond, saltern, leachate and
biofouling control with Bacillus sp. T5 in MBR, IMSTEC 9th International
Membrane Science and Technology Conference, 5-8 December 2016,
Adelaide, Australia.
 B. Yavuztürk Gül, Derya İmer, Pyung Kyu Park, I. Koyuncu, Farklı QQ
Bakterilerinin MBR’da Biyotıkanmaya ve Mkrobiyal Komüniteye Etkisi,
Membran Biyoreaktör Teknolojileri Çalıştayı, 2017, İstanbul.
 B. Yavuztürk Gül, Derya İmer, Pyung Kyu Park, I. Koyuncu, Evaluation of
QQ Bacteria isolated from marine, pond, saltern and landfill leachate for
biofouling control in MBR,3rd International Conference on Desalination Using
Membrane Technology, 2-5 April 2017, Spain.
 B. Yavuztürk Gül, Derya İmer, Pyung Kyu Park, I. Koyuncu, Comparison of
different Quorum Quenching Bacteria as a Solution for Membrane Fouling,
8th International Water Association Membrane Technology Conference, 5-9
Eylül 2017, Singapore.
 B. Yavuztürk Gül, Derya İmer, Pyung Kyu Park, I. Koyuncu, Değişik
Tutunma Ortamlarının ve QQ Bakterilerinin MBR’da Biyotıkanma Üzerindeki
84
Etkisinin Karşılaştırılması, 5. Ulusal Membran Teknolojileri ve Uygulamaları
Sempozyumu, 21-23 Eylül 2017, İstanbul.
 Yavuztürk Gül B., Koyuncu I. 2018. Biyofilm oluşumunu önleyen yeni
bakteri türlerinin izolasyonu ve biyoteknolojik kullanımı. File Number: 2018-
GE-214911, Application Number: 2018/06908
OTHER PUBLICATIONS, PRESENTATIONS AND PATENTS:

 M.Sc. Thesis, 2005 Isolation and definition of extremophiles from Çamaltı
saltern area in Turkey
 Poli A, Nicolaus B, Denizci AA, Yavuzturk B, Kazan D (2013) Halomonas
smyrnensis sp. nov., a moderately halophilic, exopolysaccharide-producing
bacterium. Int J Syst Evol Microbiol 63:10–8. doi: 10.1099/ijs.0.037036-0
 Yavuztürk B., Toksoy Öner, E., Denizci, A.A., Kazan, D, (2005),
Extremophiles From Camalti saltern area in Turkey, ECB12: 12th European
Congress on Biotechnology, August 21-24, Journal of Biotechnology, V 118,
Issue 1, Supplement 1, p 164-165.
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Bacillus Sp. T5 AND Delftia - T6 On Microbial Community

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ISTANBUL TECHNICAL UNIVERSITY  GRADUATE SCHOOL OF SCIENCE

ENGINEERING AND TECHNOLOGY

ISOLATION OF NOVEL QUORUM QUENCHING BACTERIA FOR THE

Bahar YAVUZTÜRK GÜL

Department of Environmental Engineering

Environmental Biotechnology Programme

ISOLATION OF NOVEL QUORUM QUENCHING BACTERIA FOR THE

Bahar YAVUZTÜRK GÜL

Department of Environmental Engineering

Environmental Biotechnology Programme

Thesis Advisor: Prof. Dr. İsmail KOYUNCU

MEMBRAN BİYOTIKANMASININ KONTROLÜ AMACIYLA YENİ

Bahar YAVUZTÜRK GÜL

Çevre Mühendisliği Anabilim Dalı

Çevre Biyoteknolojisi Programı

Tez Danışmanı: Prof. Dr. İsmail KOYUNCU

Thesis Advisor : Prof. Dr. İsmail KOYUNCU ..............................

Jury Members : Prof. Dr. Süleyman ÖVEZ .............................

Prof. Dr. Mehmet KİTİŞ ..............................

Prof. Dr. Bülent İNANÇ ..............................

Prof. Dr. Halil HASAR ..............................

Date of Submission : 21 March 2018

March, 2018 Bahar YAVUZTÜRK GÜL

QQ bacteria and their AHL degradation genes and enzymes. .................. 7

Quorum Quenching (QQ) is an innovative approach attempting to solve the biofouling

Nüfusun artışı ve endüstrileşmenin bir sonucu olarak, atıkların arıtılmadan ya da

Quorum Quenching (QQ), MBR sistemlerindeki biyotıkanma sorununun çözümü için

MBRdaki bakteriyel kommünite dinamikleri konusunun incelenmesi alanındaki

Membrane bioreactor (MBR) is an advanced wastewater treatment technology that

This thesis comprises new QQ bacteria/media as effective method of preventing

 Isolation and genetically identification of new bacteria has QQ activity from

 Comparison of isolated QQ bacteria with other bacteria in the literature in

 Within the framework of the targeted specificities, to evaluate the interaction

Organization of the Thesis

Quorum Quenching Application in Membrane Bioreactors

Membrane bioreactors (MBRs) are frequently employed in advanced wastewater

2.1.1 Enzymatic quorum quenching

2.1.2 Bacterial quorum quenching

QQ bacteria and their AHL degradation genes and enzymes.

Bacterial strains Gene Enzyme Reference

These bacteria immobilization strategies and types of QQ bacteria, used for

2.1.3 Fungal quorum quenching

Quorum sensing (QS) is a phenomenon, where bacteria communicate through

Existing studies have proven the presence of QQ activities in a range of different

Materials and Methods

3.2.1 Bacterial strains, growth media, culture condition

supplemented with spectinomycin and tetracycline when necessary (McLean et al.,

3.2.2 Isolation of QQ consortia from environmental samples

3.2.3 Strain identification and phylogenetic analysis

The QQ activity of the newly isolated 19 strains was measured by degradation of a

A QQ-fiber was designed to immobilize QQ bacteria using hydrophilic

Preparation of QQ-fibers. Centrifuged QQ bacteria were injected into

Results and Discussion

3.3.1 Novel AHL degrading bacteria

A BLAST search of GenBank was performed to identify if there was any

Neighbour-joining tree based on the 16S rRNA gene, showing

G4 Acinetobacter baylyi (99%) KR705932 66,7 +++ +

T22 Vibrio sp. (99%) KR705937 54,7 + ++

T4 Acinetobacter calcoaseticus (98%) KR705938 63,5 +++ +

H2 Shewanella xiamenensis (99%) KR705942 55,3 ++ +

Strain G7 was identified as Microbacterium kitamiense (98%). It is reported that

Quorum quenching activity of whole cell (C8-HSL degradation in in