Review

Do biological-based strategies hold promise to

biofouling control in MBRs?

Lilian Malaeb a, Pierre Le-Clech b, Johannes S. Vrouwenvelder a,c,d,George M. Ayoub e, Pascal E. Saikaly a,*aWater Desalination and Reuse Research Center and Division of Biological and Environmental Sciences and

Engineering, King Abdullah University of Science and Technology, Thuwal 23955-6900, Saudi ArabiabUNESCO Centre for Membrane Science and Technology, School of Chemical Engineering, The University of New

South Wales, Sydney, NSW, AustraliacDepartment of Biotechnology, Faculty of Applied Sciences, Delft University of Technology, Julianalaan 67, 2628 BC

Delft, The NetherlandsdWetsus, Centre of Excellence for Sustainable Water Technology, Agora 1, 8900 CC Leeuwarden, The NetherlandseDepartment of Civil and Environmental Engineering, American University of Beirut, Beirut, Lebanon

a r t i c l e i n f o

Article history:

Received 30 March 2013

Received in revised form

21 May 2013

Accepted 15 June 2013

Available online 27 June 2013

Keywords:

Anti-biofouling

Biofilm

Biological-based

Holistic control

Management

Membrane bioreactors

a b s t r a c t

Biofouling in membrane bioreactors (MBRs) remains a primary challenge for their wider

application, despite the growing acceptance of MBRs worldwide. Research studies on

membrane fouling are extensive in the literature, with more than 200 publications on MBR

fouling in the last 3 years; yet, improvements in practice on biofouling control and man-

agement have been remarkably slow. Commonly applied cleaning methods are only

partially effective and membrane replacement often becomes frequent. The reason for the

slow advancement in successful control of biofouling is largely attributed to the complex

interactions of involved biological compounds and the lack of representative-for-practice

experimental approaches to evaluate potential effective control strategies. Biofouling is

driven by microorganisms and their associated extra-cellular polymeric substances (EPS)

and microbial products. Microorganisms and their products convene together to form

matrices that are commonly treated as a black box in conventional control approaches.

Biological-based antifouling strategies seem to be a promising constituent of an effective

integrated control approach since they target the essence of biofouling problems. However,

Abbreviations: AHL, acyl-homoserine lactone; AFM, atomic force microscopy; AIP, autoinducer peptide; ARDRA, amplified ribosomaldeoxyribonucleic acid restriction analysis; ATP, adenosine triphosphate; CCCP, carbonyl cyanide chlorophenylhydrazone; CLSM,confocal laser scanning microscopy; COD, chemical oxygen demand; DNP, 2,4-dinitrophenol; DOC, dissolved organic carbon; DGGE,denaturing gradient gel electrophoresis; EPBR, enhanced biological phosphorous removal; ED, enzymatic disruption; EEM, excitationemission matrix; EPS, extracellular polymers; EU, energy uncoupling; F/M, food to microorganisms ratio; FISH, fluorescent in situ hy-bridization; FTIR, Fourier transform infrared; MALDI-TOF, matrix-assisted laser desorption/ionization -time of flight; MBR, membranebioreactor; MF, microfiltration; MLSS, mixed liquor suspended solids; MS, mass spectrometry; NF, nanofiltration; NMR, nuclear magneticresonance; NO, nitric oxide; NSM, natural small molecules; PAOs, polyphosphate accumulating organisms; PFU, plaque forming units;QQ, quorum quenching; QS, quorum sensing; RO, reverse osmosis; SEM, scanning electron microscopy; SIMS, secondary-ion massspectrometry; SMP, soluble microbial products; SRT, sludge retention time; STXM, scanning transmission X-ray microscopy; CS, 3,30,40,5-tetrachlorosalicylanilide; TEM, transmission electron microscopy; TOC, total organic carbon; T-RFLP, terminal restriction fragmentlength polymorphism; UF, ultrafiltration; 2D-PAGE, two-dimensional polyacrylamide gel electrophoresis.* Corresponding author. Tel.: þ966 5 44700129.E-mail address: pascal.saikaly@kaust.edu.sa (P.E. Saikaly).

Available online at www.sciencedirect.com

journal homepage: www.elsevier .com/locate/watres

wat e r r e s e a r c h 4 7 ( 2 0 1 3 ) 5 4 4 7e5 4 6 3

0043-1354/$ e see front matter ª 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.watres.2013.06.033

Membrane performance

Microbial community

biological-based strategies are in their developmental phase and several questions should

be addressed to set a roadmap for translating existing and new information into sustain-

able and effective control techniques. This paper investigates membrane biofouling in

MBRs from the microbiological perspective to evaluate the potential of biological-based

strategies in offering viable control alternatives. Limitations of available control methods

highlight the importance of an integrated anti-fouling approach including biological stra-

tegies. Successful development of these strategies requires detailed characterization of

microorganisms and EPS through the proper selection of analytical tools and assembly of

results. Existing microbiological/EPS studies reveal a number of implications as well as

knowledge gaps, warranting future targeted research. Systematic and representative

microbiological studies, complementary utilization of molecular and biofilm character-

ization tools, standardized experimental methods and validation of successful biological-

based antifouling strategies for MBR applications are needed. Specifically, in addition,

linking these studies to relevant operational conditions in MBRs is an essential step to

ultimately develop a better understanding and more effective and directed control strategy

for biofouling.

ª 2013 Elsevier Ltd. All rights reserved.

Contents

1. Biofouling problem in MBRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54482. Current status of antifouling strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5450

2.1. Commonly applied control strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54502.2. The potential of biological-based antifouling strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5450

2.2.1. Biological-based biofilm control strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54502.2.2. Application to MBRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5451

3. Implications from existing EPS/microbial studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54523.1. EPS studies in MBRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5452

3.1.1. EPS extraction and characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54523.1.2. Implications from EPS studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5453

3.2. Advanced microbial analysis of MBR fouling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54533.2.1. Analytical tools for microbial community characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54533.2.2. Biofouling microbial communities in MBRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54553.2.3. Implications in MBRs and other membrane applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5455

4. Current gaps in knowledge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54564.1. Microbiological studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54564.2. Biological strategies for MBR antifouling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5456

4.2.1. Challenges in biological-based strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54564.2.2. The importance of an integrated control approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5457

4.3. Linking operation to microbiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54575. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5458

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5459References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5459

1. Biofouling problem in MBRs

Membrane bioreactors (MBRs) are now broadly applied as

wastewater treatment technology that combines membrane

processes and suspended growth bioreactors. Compared to

other technologies e.g. activated sludge processes (Kim et al.,

2010; Judd, 2008; Visvanathan et al., 2000), MBRs allow

smaller footprints, higher-quality effluents, less sludge and

complete solideliquid separation; however, membrane

fouling remains in most cases a persistent problem

(increasing operating costs and reducing thewater quality and

quantity) (Wang et al., 2009; Kimura et al., 2009). Fouling re-

sults in a lower permeate flux, higher trans-membrane pres-

sure (TMP), frequent membrane cleaning or replacement and

overall degraded membrane performance (Mahendran et al.,

2011; Liao et al., 2004). Fouling can be organic, inorganic or

biological, although the boundary between these classifica-

tions is not rigid and the definitions of different fouling types

may overlap. For example, inorganic deposition can be a direct

consequence of biologically-induced mineralization between

biopolymers and salts (Wei et al., 2007; Herrera-Robledo et al.,

2011) and internal fouling caused by the adsorption of

wat e r r e s e a r c h 4 7 ( 2 0 1 3 ) 5 4 4 7e5 4 6 35448

dissolved organic and inorganic matter into membrane pores

in MBRs is known to occur simultaneously with biofouling

(Yamato et al., 2006; Rosenberger et al., 2006; Miura et al.,

2007).

Among all types of fouling, biofouling is a dynamic, com-

plex and relatively slow process that involves interactions not

yet thoroughly understood (Pasmore et al., 2001; Baker and

Dudley, 1998). While it is possible to conduct controlled ex-

periments on other foulants, the enormous diversity of bac-

terial communities and their close association with excreted

extracellular polymers (EPS) and soluble microbial products

(SMP) complicate experimental approaches that would help

explicate this challenging phenomenon. Biofouling occurs as

a result of two mechanisms: (i) colonization of membrane

surfaces with microorganisms and (ii) production of mem-

brane foulants by microorganisms in the mixed-liquor. Both

the microbes and their products contribute to membrane

fouling, as evidenced by studies that show enhanced mem-

brane permeability due to quorum quenching, which targets

the microbes (e.g. Yeon et al., 2009a,b; Oh et al., 2012; Jiang

et al., 2013), and studies that correlate fouling to EPS/SMP

(e.g. Hwang et al., 2008; Nataraj et al., 2008; Wang et al., 2009;

Kimura et al., 2009). With the currently used analytical ap-

proaches, it is still challenging to determine which of the two

mechanisms is dominant and under which conditions. How-

ever, with advances in analytical characterization tools, it

becomes possible to conduct systematic studies that elucidate

the key players in the biofouling process and quantitatively

correlate the characteristics of both the microbial commu-

nities and their products to biofouling rates.

The importance of biofouling relative to other fouling

mechanisms is also ambiguous. Some researchers report that

sludge-cake resistance is much higher than pore blocking

resistance (Lee et al., 2001; Eusebio et al., 2010; Pendashteha

et al., 2011) and others consider the cake layer to be a

removable type of fouling while pore blocking as irremovable

by physical cleaning and hence requiring chemical methods

(Meng et al., 2009). Wu et al. (2011) assumed cake resistance to

be due to suspended solids whereas pore constriction and

blocking to be caused by solutes and colloids, respectively.

The accuracy of these divisions and of hypothetical biofouling

models, however, is questionable due to the complex in-

teractions among foulants as well as to differences in process

configuration, operating conditions and extraction methods.

The effects of operational and wastewater characteristics

have also been considered but discrepancies are commonly

noted among different studies. Research findings on the effect

of sludge retention time (SRT) are largely inconsistent (Meng

et al., 2009; Sweity et al., 2011). Increasing the SRT generally

leads to a better filterability (Nuengjamnong et al., 2005;

Trussell et al., 2006; Liang et al., 2007); however, a further in-

crease can intensify fouling (Han et al., 2005). Rosenberger and

Kraume (2002) found that specific SMP concentration

increased with sludge loading rate and decreased with sludge

age. Although shear stresses can alleviate fouling, shear

imposed on microbial flocs could result in the release of EPS

(Wang et al., 2009) and high shear conditions associated with

crossflow configuration can induce release and accumulation

of soluble organic matter in themixed liquor, which increases

its fouling propensity (Stricot et al., 2010). Fouling is also

reported to increase with higher food to microorganisms ratio

(F/M) (Trussell et al., 2006) yet themechanisms of these effects

remain to be clarified particularly in relation to floc charac-

teristics, which are in turn related to EPS (Jang et al., 2006).

Zhang et al. (2010) noted that during the start-up period, MBR

fouling is higher for variable than for constant organic loading

but after 2 SRTs, less fouling occurs with variable loading. A

high ratio ofmonovalent over polyvalent cations also worsens

filtration characteristics due to sludge defloculation (Van den

Broeck et al., 2010). Constant flux can prevent excessive

fouling (Defrance and Jaffrin, 1999) butmay lead to irreversible

fouling (Ye et al., 2005; Le-Clech et al., 2006). Moreover, low

fluxes cause much less homogenous live/dead cell profiles,

presumably due to starvation and oxygen stress, which can

increase cell membrane porosity, death and SMP release

(Hwang et al., 2008; Drews, 2010).

The biofouling process, led by various microorganism

types and their excreted products, develops into a complex

and difficult to control problem, so that conventional physical

cleaning processes such as back-washing and back-pulsing

are no longer effective (Chu and Li, 2005; Le-Clech et al.,

2006). Chemical cleaning also has limited success and micro-

organisms can adapt to cleaning procedures by changing their

physiological responses through gene regulation and meta-

bolism (Ma et al., 2009; Mahendran et al., 2011; Calderon et al.,

2011). Other control approaches focus on membrane modifi-

cation, so as to generate surfaces with lower fouling potential.

Yet, the stability of most proposed polymers and functional-

ized surfaces is often not evaluated for long-term filtration

where severe convective forces and cyclical cleaning protocols

are applied (Mansouri et al., 2010).

Control strategies commonly used in practice tend to treat

biofouling as a black box by ignoring its intrinsic biological

nature. Recently, biological-based antifouling strategies star-

ted to prove their potential as a more viable solution than

conventional control methods, based on engineering, mate-

rial, and chemical principles (Yeon et al., 2009b). As a result, an

integrated control approach that takes into account biological

methods is potentially more effective for understanding and

ultimately controlling this persistent problem that continues

to defy MBR operators. In order to define further needed

research efforts, an evaluation ofMBRmembrane biofouling is

presented from the microbiological perspective. This critical

review is divided into three distinct sections describing:

i. Limited effectiveness of conventional control methods

and potential of biological-based strategies in offering

better antifouling alternatives;

ii. Analytical tools presently used and insights from studies

onmicrobial communities and their EPS for an improved

understanding and management of the biofouling

problem;

iii. Existing knowledge gaps and future research needs in

terms of both microbiological investigations and

biological-based control applications developed specif-

ically for MBRs.

Finally, it is expected that an integrated approach that

combines various successful strategies is more likely to be

effective than any single antifouling method. Linking

wat e r r e s e a r c h 4 7 ( 2 0 1 3 ) 5 4 4 7e5 4 6 3 5449

microbiological/EPS studies to design and operational pa-

rameters that are representative of real-scale MBRs is crucial

for the development of such an approach with the ultimate

aim of effectively controlling MBR biofouling.

2. Current status of antifouling strategies

2.1. Commonly applied control strategies

Conventional physical (e.g. back-washing, back-pulsing, air

sparging) and chemical (e.g. acids, bases, oxidants, chelating

agents, polymeric coagulants, surfactants) cleaning methods

often fail to adequately control biofouling. In the membrane-

coupled upflow sludge blanket bioreactor analyzed by

Calderon et al. (2011), backflushing and chemical cleaning

using NaClO were found ineffective in completely removing

the biofouled layers from MBR membranes and remaining

populations supported rapid biofilm re-growth, leading to the

regeneration of a similar microbial community structure. In

fact, a biofilm formed under natural conditions may prove

easier to control in the long-term than one formed where

biocides are in use (Baker andDudley, 1998). Bacteria surviving

after a biocide application can develop defense mechanisms,

which render them even more resistant to the biocide when

reapplied later. Frequent chemical cleaningmoreover reduces

membrane lifetime (Judd, 2008), can impair permeate quality

(Tao et al., 2005), and is associated with undesirable waste

streams that have to be properly disposed of (Brepols et al.,

2008). It was further found that declogging the membrane to

remove particulatematter that fills void spaces inmembranes

can recover permeability for a short time but is followed by a

rapid permeability decline due to aggravated fouling over the

course of a few hours of time (Zsirai et al., 2012). Other control

strategies to reduce fouling and improve flux consistency

involve membrane modification (Table 1) but this also has

limited applicability as it is difficult to modify membrane

structures without occluding membrane pores or impacting

the separation performance (Mansouri et al., 2010). In addi-

tion, many coatings do not show long-term mechanical or

chemical stability. Membrane properties such as hydropho-

bicity, charge and roughness only affect the rate of early

bacterial attachment and under the specific conditions tested

and even the surfaces most resistant to biofilm formation

have been reported to show a 40% surface coverage within 2

days (Pasmore et al., 2001).

It was suggested that alternative antifouling strategies

directed towardsmicrobial groups that have shown resistance

to standard cleaning methods (e.g. members of the family

Sphingomonadaceae andmethanogenic Archaea) might be more

promising (Calderon et al., 2011). In this regard, biological-

based antifouling strategies might prove to be valuable and

may potentially enhance the effectiveness of currently

applied cleaning protocols. These strategies refer to the use of

certain chemicals that target a specific molecule or mecha-

nism that is responsible for the attachment, communication,

motility or growth of microbial cells. In this paper, the defi-

nition of biological-based antifouling strategies is also

expanded to include phages and predators,which respectively

infect and consume microorganisms.

2.2. The potential of biological-based antifouling

strategies

2.2.1. Biological-based biofilm control strategies

Among the commonly tested biological strategies for biofilm

control are quorum quenching (QQ), enzymatic disruption

(ED), energy uncoupling (EU), cell wall hydrolysis and the use

of microbial predation and bacteriophages. Biological-based

strategies are reported to offer the advantages of higher effi-

ciency, lower toxicity, more sustainability and less bacterial

resistance over other control approaches (Yeon et al., 2009b;

Xiong and Liu, 2010). QQ refers to the inhibition of the cell-

to-cell communication known as quorum sensing (QS) that

is used by microorganisms to coordinate their behavior e.g.

biofilm formation, motility, EPS production and detachment.

Table 1 e Specific membrane modifications to control biofouling in MBRs.

Membrane modifications and observations Reference

Original wastewater biofilms are highly tolerant to AgeNPa treatment but susceptibility is different for each

microorganism e.g. Thiotrichales is more sensitive to AgeNP than other bacteria.

Sheng and Liu (2011)

Modifying membranes to become hydrophilic or electrically charged only partially reduces biofouling. Liu et al. (2010)

Acrylic acid grafted membrane showed better antifouling than acrylamide modified membranes and surface

carboxyl-containing membranes were better than surface amido-containing membranes.

Li et al. (2010)

Amphiphilic comb copolymer additives in membranes prevent irreversible adhesion of bacteria. Adout et al. (2010)

Surface group functionalization, combining non-leachable biocidal moieties & anti-adhesion properties. Mansouri et al. (2010)

Ceramic membranes coated with fullerene C60b reduced microbial attachment & inhibited microbial respiratory

activity. One concern is that EPS & SMP may hinder contact between bacteria & C60.

Chae et al. (2009)

Surface modification of polypropylene hollow fiber MBR microporous membranes in MBR showed better

antifouling efficiency as follows: graft polymerization of acrylic acid > graft polymerization of acrylamide

acid > CO2 plasma treatment > NH3 plasma treatment.

Yu et al. (2008)

Synthesized amphiphilic copolymers (P123-b-PEG) decrease protein adsorption and increase flux recovery ratio. Wang et al. (2006)

Amphiphilic comb polymer (methyl methacrylate-based coating material with short non-fouling oligoethylene

glycol side chains) reduces nonspecific adsorption of biomolecules.

Hyun et al. (2006)

a AgeNP: silver nanoparticles.

b C60: fullerene.

wat e r r e s e a r c h 4 7 ( 2 0 1 3 ) 5 4 4 7e5 4 6 35450

Several signaling pathways have been identified for QS among

microorganisms including acyl-homoserine lactone (AHL)

signal genes, amino acid peptides, natural small molecules

(NSM) that can affect EPS synthesis and biofilm formation

(Lopez et al., 2009) and auto-inducers such as AI-2 signal.

Another widespread signal for biofilm disassembly is a

mixture of D-amino acids, which could disassemble biofilms

in Bacillus subtilis, Pseudomonas aeruginosa and Staphylococcus

aureus by releasing amyloid fibers that link the cells together

(Kolodkin-Gal et al., 2010). This could be a strategy used by

biofilm bacteria to create local channels and facilitate mass

transport within biofilms (Flemming and Wingender, 2010)

and may also prove to be an effective biofilm control solution.

EPS-degrading enzymes that have been successfully

applied for biofilm detachment include proteolytic enzymes

for protein hydrolysis (e.g. proteinase K, trypsin and subtili-

sin), polysaccharases for the hydrolysis of polysaccharides

(e.g. Dispersin B, Mutanase and dextranase) as well as DNases

(Petersen et al., 2005; Chaignon et al., 2007; Leroy et al., 2008;

Guezennec et al., 2012). Hydrolytic enzymes of cell walls

such as the antimycotic protein lysozyme have been used to

prevent microbial attachment and could act more specifically

than traditional biocides (Xiong and Liu, 2010). Energy

uncoupling via the addition of chemicals (e.g. 2,4-

dinitrophenol DNP, carbonyl cyanide chlorophenylhy-

drazone CCCP, and 3,3’,4’,5-tetrachlorosalicylanilide TCS) can

transport protons through the cellular membrane thus dissi-

pating the proton gradient and inhibiting adenosine triphos-

phate (ATP) synthesis. Xu and Liu (2011) showed evidence of

suppressed ATP synthesis and AI-2 production using DNP to

remove biofilm bacteria from nylon membrane surfaces.

The use of bacteriophages to control biofilm growth is

another promising strategy due to the specificity of these

acellular microorganims, their rapid multiplication and

limited infectivity to prokaryotic organisms (Doolittle et al.,

1996; Lu and Collins, 2007). Polyvalent phages to prevent

concomitant adhesion and biofilm formation have proved

their effectiveness for multi-consortia (Jensen et al., 1998).

Phage resistance through reversion, however, can be a chal-

lenge. On the other hand, some phages (e.g. polyvalent phage

K) have shown that continuous propagation through bacterial

strains yields modified phages with enhanced lytic properties

toward the strains (O’Flaherty et al., 2005). Other studies

proved the ability of genetic engineered phages containing

biofilm-degrading enzymes to degrade EPS (Lu and Collins,

2007). Predators, which reduce biomass concentration and

alter its morphology due to grazing, motility or sloughing,

have also been used. Derlon et al. (2012) investigated the

impact of predation by eukaryotes on the development of

biofilm structures in gravity-driven dead-end UF systems

operated under ultra-low pressure conditions. They found

that an open and heterogeneous biofilm structure developed

in systems with predation, resulting in higher permeate

fluxes.

2.2.2. Application to MBRs

To date, a limited amount of biological control strategies has

been applied to membranes for water/wastewater treatment

in general, or to MBRs in particular. Poele and van der Graaf

(2005) used enzymatic cleaning by protease to remove

protein biofouling in UF membranes, which resulted in a

much higher efficiency compared to traditional alkaline

cleaning. The first studies in MBRs were conducted by Yeon

et al. (2009a,b), who demonstrated the potential of porcine

kidney acylase I and AHL-acylase enzymes to prevent MBR

biofouling by quenching AHL autoinducers. AHL-acylase

directly immobilized onto a nanofiltration (NF) membrane

was shown to prohibit the formation of mushroom-shaped

mature biofilm due to reduced EPS secretion (Kim et al.,

2011). To avoid practical issues of cost and stability of en-

zymes, Oh et al. (2012) proposed that QQ might be more

feasible, has a longer life span and does not require enzyme

purification. They encapsulated Recombinant Escherichia coli

producing N-acyl homoserine lactonase or Rhodococcus sp.

isolated from a real MBR inside microporous hollow fiber

membrane and could efficiently control biofouling (Jahangir

et al., 2012; Oh et al., 2012). The microbial-vessel maintained

its QQ activity over 100 days of MBR operation due to contin-

uous regeneration of living quorum, but the QQ effect was

largely dependent on the recirculation rate of themixed liquor

between the bioreactor and the membrane tank (Jahangir

et al., 2012). Higher recirculation rates facilitated transport of

signal molecules from the biofilm into the bulk mixed liquor

and then to the microbial-vessel (Jahangir et al., 2012). Jiang

et al. (2013) showed that using porcine kidney acylase I

enzyme inhibited biofilm formation and enhancedmembrane

permeability with no apparent effects on effluent quality of

the lab-scale MBR that was fed with synthetic wastewater. In

this study, QQ was found to increase settleability, reduce the

production of polysaccharides and proteins and reduced vis-

cosity and relative sludge hydrophobicity (Jiang et al., 2013).

Ngo and Guo (2009) found that a green bioflocculant from a

natural starch-based cation reduces fouling in an aeratedMBR

but noted that further study on its sustainability in biological

treatment is required. It was also suggested that selection

pressure to enhance populations of Bacillus and Pseudomonas

species for their enzymes that digest carbohydrates and pro-

teins might be effective in reducing membrane fouling (Ng

and Ng, 2010). As for energy uncoupling, no application has

been conducted to MBR membranes. The study by Xu and Liu

(2011), which used DNP to remove biofilms from nylon

membrane surfaces is promising but was conducted for hy-

drophilic flat-sheet nylon membranes used in dead-end

microfiltration (MF) filtration of batch-fed synthetic

wastewater.

In terms of the use of bacteriophages, Goldman et al. (2009)

reported that the addition of bacteriophages reduced by

40e60% the microbial attachment to UF membrane in MBR

treating effluents containing three bacterial species. However,

few phages (1e10 PFU/100 mL) were detected in the permeate,

which can pose a problem in real applications. Moreover, a

combination of several phages would be required to combat

the multiple bacterial species encountered in real-scale

systems.

As to biofouling control using predation, available studies

are very limited. A metazoa population, primarily composed

of rotifiers and oligochaete worms has been shown to play an

effective role in MBR biofouling control and reducing cake

formation on the membrane (Luxmy et al., 2001); yet it was

mentioned in this study that high mixed liquor suspended

wat e r r e s e a r c h 4 7 ( 2 0 1 3 ) 5 4 4 7e5 4 6 3 5451

solids (MLSS) in MBR might suppress the activity of these

metazoa. Derlon et al. (2012) noted that predation is especially

useful in systems operated without cross-flow that may pro-

vide suitable conditions for the larger eukaryotic organisms to

develop (Derlon et al., 2012). Table 2 summarizes success

stories of biological-based antifouling strategies as applied to

MBRs in particular. The enhancement in permeability as re-

ported in the corresponding studies is given in terms of trans-

membrane pressure or permeability.

On the other hand, studies discussed in this section on the

successful application of biological-based methods to reduce

biofouling in membranes other than MBRs (Poele and van der

Graaf, 2005; Goldman et al., 2009; Kim et al., 2011; Xu and Liu,

2011; Derlon et al., 2012) are also encouraging although the

expansion toMBR conditions requires accounting for themore

complex environment and parameters involved. To enhance

thedevelopment and application of the above biological-based

antifouling strategies inMBRs requiresfirst understanding and

characterizing the main elements responsible for biofouling.

These include both the microorganisms and their associated

EPS as discussed in the next section.

3. Implications from existing EPS/microbialstudies

3.1. EPS studies in MBRs

Membrane modules and materials, hydrodynamic parameters,

process and environmental conditions, and physicochemical

properties of the treated water are among the dominant factors

determining membrane biofouling (Liao et al., 2004). These fac-

torsmaybedivided into twocategories:biomassandoperational

parameters. A summary of the impact of these parameters has

beenpresentedinseveral reviews (e.g. Le-Clechetal., 2006;Meng

et al., 2009; Drews, 2010). Among the most important and com-

plex biomass parameters is related to EPS, which is often

differentiated into loosely bound, tightly bound and soluble EPS,

also known as slime polymers (SMP). EPS consist of macromol-

ecules of microbial origin (polysaccharides, proteins, glycopro-

teins, lipoproteins) (Flemming, 1997) and insoluble material

(sheaths, capsular polymers, condensed gel, loosely bound

polymers and attached organic material) produced by active

secretion, shedding of cell surface material or cell lysis

(Jang et al., 2005).

3.1.1. EPS extraction and characterization

EPS nature and quantity vary greatly among biofilms,

depending on the microorganisms present, shear forces,

temperature and nutrients while EPS identification largely

depends on the isolation method (Flemming and Wingender,

2010). Extraction and characterization methods for EPS have

been extensively reviewed in the literature (e.g. Zhang et al.,

1999; Drews, 2010). The determination of EPS concentration

mostly relies on measuring carbohydrates and proteins while

different characterization methods can give different results.

Some authorsmeasure total or dissolved organic carbon (TOC/

DOC) or chemical oxygen demand (COD) instead of measuring

individual carbohydrate and protein fractions (Haberkamp

et al., 2007; Wang et al., 2009); however, with both

Table 2 e Biological control methods applied to MBRs.

Methoda Description of biological-based antifouling strategy

used in MBR

Type of MBR [flux] Enhancement of permeability interms of TMPb [compared

to control MBR]

Reference

ED Bioassay with Agrobacterium

tumefaciens A136 supplemented

with spectinomycin and

tetracycline to maintain two

plasmids that provide the AHL

response system

Batch MBR with total

recycle mode [15 Lmh]

w32 h to reach 40 kPa [w20 h in

control]; maximum TMP 48 kPa at

w40 h [70 kPa at w23 h for control]

Yeon et al. (2009a)

ED Magnetic enzyme carriers

prepared by immobilizing the

QQ Porcine kidney acylase I on

a magnetic carrier and recycled

back from the redrawn sludge

Batch MBR with total

recycle mode [15 Lmh]

Maximum TMP 36e39 kPa [76e79 kPa

in control] in 3 operation cycles

(15e20 h)

Yeon et al. (2009b)

Continuous MBR

[15 Lmh]

TMP 10 kPa throughout the

experiment [30 kPa in 48 h for the

control]

QQ QQ bacteria encapsulated

inside a porous vessel

(microbial-vessel)

Continuous MBR

[30 Lmh], filtration

(60 min), relaxation

(1 min), MLSS

recirculated at

7.5e30 mL/min

Maximum TMP 30 kPa at 68 h

[48 h in control]

Jahangir et al. (2012)

QQ Microbial vessel containing

recombinant E. coli

Continuous MBR

[20 Lmh]

39 h to reach 25 kPa [28 h in control] Oh et al. (2012)

Continuous MBR

[20 Lmh]

Maximum TMP 11 kPa

[45 kPa in control]

ED Porcine kidney acylase I Continuous MBR

[15 Lmh]

Maximum TMP 14 kPa at w45 h

[40 kPa at 55 h in control]

Jiang et al. (2013)

a QQ: Quorum quenching; ED: Enzymatic disruption.

b TMP: trans-membrane pressure.

wat e r r e s e a r c h 4 7 ( 2 0 1 3 ) 5 4 4 7e5 4 6 35452

photometric methods or TOC measurements, only surrogate

concentrations are determined and no information on indi-

vidual constituents or synergistic effects is obtained so that

two samples with equal net carbohydrate, protein or TOC

concentration can exhibit largely different behaviors (Drews,

2010). Measurement of mechanical properties in EPS such as

elastic and shear modulus, adhesive strength or tensile

strength can be challenging but a range of values for these

properties is reported in the literature (Yun et al., 2006; Gao

et al., 2011a; Sweity et al., 2011).

3.1.2. Implications from EPS studies

Contradictory results in the literature are commonly found

when addressing the influence of a specific parameter on

membrane biofouling, especially those related to EPS. For

example, in some MBRs, protein was identified as the main

foulant (e.g. Ng and Ng, 2010; Gao et al., 2011b; Wang et al.,

2011) and thought to be more involved than sugars in gener-

ating electrostatic bonds with multivalent cations, a principal

factor in stabilizing aggregate structures (Dignac et al., 1998).

Carbohydrates were nonetheless found to show a better cor-

relation to membrane permeability in other studies (Germain

et al., 2005; Kimura et al., 2005). Using NMR and FTIR, Kimura

et al. (2005) found that with higher food-microorganisms ratio

(F/M), membrane foulants became more proteinaceous.

Metzger et al. (2007) fractionated MBR fouling layers by

different removalmechanisms and identified an upper loosely

bound layer with a similar composition to the biomass flocs,

an intermediate fraction with a higher concentration of car-

bohydrates and a lower layer featuring a relatively higher

concentration of strongly bound proteins. The generation of

EPS in the lower fraction of the bio-cake layer over long pe-

riods of operation was also found to be responsible for the

rapid increase in TMP under subcritical flux (Hwang et al.,

2008). Loosely-bound EPS was found to show more

significant positive correlation with fouling than tightly-

bound EPS during long term operation of a pilot-scale MBR

(Wang et al., 2009).

It is the general current consensus that proteins and

carbohydrates are assumed to be the major fractions

contributing to fouling, although other components may

also play a significant role. For example, Subhi et al. (2013)

found that biopolymers and low-molecular-weight neu-

trals were the major contributors to irreversible fouling.

During studies of alginate biosynthesis in P. aeruginosa,

Whitchurch et al. (2002) discovered that the majority of the

extracellular material was not exopolysaccharides but DNA

and found that addition of DNase I to the culture medium

strongly inhibited biofilm formation. Lyko et al. (2007) found

an important influence on MBR membrane fouling by both

soluble humic substances and carbohydrates in complexes

with metal cations. Three-dimensional excitation-emission

matrix (EEM) fluorescence spectra analysis also revealed

that membrane fouling was associated with fulvic acid-like

substances (Wang et al., 2011). These findings highlight the

critical role of the method of analysis chosen and that can

only measure a limited amount of compounds (Subhi et al.,

2011). The above contradictions also emphasize the need for

standardization in measurement methods and analytical

tools and for well-controlled experiments. Proper

measurement of trans-membrane pressure and early

biofouling detection are also important in MBRs as has been

shown in other types of membranes (Vrouwenvelder et al.,

2009a, 2011). Future studies employing more of the

advanced techniques can reveal information on EPS fin-

gerprints and origins beyond structure and composition.

However, protocol optimization and adequate sample pu-

rification for new techniques such as MALDI/TOF/MS are

important (Kimura et al., 2012). Furthermore, in lab-scale

MBRs, operating and environmental conditions are subject

to higher fluctuations than in large plants and time scales

often differ significantly, which stresses the importance of

conducting more studies on real-scale MBRs in order to

validate laboratory research findings.

3.2. Advanced microbial analysis of MBR fouling

In addition to EPS studies, it is important to properly charac-

terize the microorganisms that are responsible for biofouling.

This provides the basis for endeavors directed at developing

biological-based antifouling strategies.With the advancement

of molecular biology techniques, more studies have been

conducted to characterize microbial communities responsible

for biofouling in MBRs. Table 3 gives a brief description of the

analytical tools used for characterizing both the microbial

communities (structure and function) and biofilms (microor-

ganisms with associated EPS matrix) on membrane surfaces.

3.2.1. Analytical tools for microbial community

characterization

Culture-based microbial characterization approaches intro-

duce well-known biases such as the potential to exclude the

detection of important microbial species (Herzberg et al.,

2010). These approaches tend to select for the fittest and

least fastidious microorganisms, whereas non-culture-based

molecular biology techniques can provide more reliable and

useful information (Table 3). Pre-genomic tools refer to DNAor

RNA-based molecular techniques that target a single gene

(commonly the 16S rRNA gene) or RNA molecule. These tools

are important for assessing microbial community structure

and identifying functionally important members of unchar-

acterized communities (Rittmann et al., 2008). However, the

obtained information on community structure and diversity is

only weakly linked to metabolic and functional capabilities

(Tringe and Hugenholtz, 2008).

With the advent of genomic (metagenomic) and post-

genomics (metatranscriptomic and metaproteomic) ap-

proaches, as described in Table 3, methodological studies of

expressed subsets of genes or proteins can be conducted.

Metagenomics can unveil a large number of sequence reads

with unidentified origin, offering information on microbial

diversity, gene content and metabolic potential (Taylor et al.,

2007; Warneckea, and Hess, 2009; Rademacher et al., 2012).

Post-genomic approaches such as metatranscriptomics and

metaproteomics give information on community function by

identifying themessenger RNA (expressed gene transcripts) or

expressed proteins and can reveal the link between commu-

nity structure and function (Wilmes and Bond, 2006; Helbling

et al., 2011). These high throughput next-generation

sequencing techniques surpass conventional methods in

wat e r r e s e a r c h 4 7 ( 2 0 1 3 ) 5 4 4 7e5 4 6 3 5453

Table 3 e Available analytical tools for membrane biofouling characterization.

Techniquea Principles Information gained & limitations

Molecular biology techniques

Pre-genomic DGGE Amplified gene fragments are separated on a

denaturing gel based on their different

melting behavior

Provides information on the community

structure and dynamics of predominant

members in a community; DGGE bands can be

sequenced

Clone library PCR amplified gene fragments are inserted

into a plasmid and sequenced using Sanger

sequencing

Phylogenetic information of predominant

members; may not reflect true diversity

T-RFLP PCR amplified fluorescently labeled terminal

restriction fragments (T-RFs) are separated

based on size or capillary electrophoresis &

identified with automatic DNA sequencer

Community structure and dynamics of

predominant members; underestimates diversity

(limited number of

T-RFs can be resolved); not possible to combine

with sequencing

16S rRNA gene

pyro-sequencing

A high-throughput sequencing technology

capable of obtaining few thousand sequences

of partial 16S rRNA gene; using barcoded

primers, hundreds of PCR amplified samples

(amplicons) can be sequenced simultaneously

Information on community structure and

diversity of dominant and rare members; short

sequence read lengths are generated with the

Roche 454 platform; cannot accurately resolve

sequences to the genus & species level

FISH Fluorescently labeled DNA oligonucleotide

probes hybridize to RNA molecules

(rRNA or mRNA)

Visualization of spatial interactions &

quantification of target microbial cells;

identification is reliant on probe specificity

Genomic Meta-genomics Relies on shotgun sequencing, isolated DNA

are fragmented, cloned into a suitable vector

and sequenced; with massive parallel

sequencing platforms (Roche 454, Illumina,

ABI SOLiD) generates large number of short

sequence reads

Structure and metabolic potential; doesn’t show

if the predicted genes are expressed or not and if

the DNA comes from viable or dead cells; relies

on available reference genome or functional

databases & on bioinformatic tools

Post-genomic Meta-transcriptomics Isolated RNA are sequenced using

pyrosequencing technology (Roche 454),

which is capable of obtaining ten to hundreds

of thousands sequences from the RNA pool

Links community function to phylogenetic

structure; isolation of mRNA is still a challenge

because of its very short half-life; requires

powerful bioinformatic tools

Meta-Proteomics Extracted proteins are separated on a gel

followed by mass spectrometry or are

digested into peptides followed by separation

and identification using liquid

chromatography tandem mass spectrometry

(LC-MS/MS)

True ability of microbial community to perform

reactions; ability to link community structure to

function; extraction of proteins from complex

environments is still a challenge; depends on the

availability of metagenomic sequence data

Biofilm characterization methods

Ultrasonic

reflectometry

The reflected time and amplitude of sound

waves are compiled into frequency

distributions

Data on earliest interactions between foulants

and membrane materials; real-time detection

CLSM Uses white lasers for providing 3D

information of the biofouling layers and

studying the overall biofilm morphology with

high resolution

With fluorescent labels, in situ characterization

of biofilms (e.g. EPS; cell viability; target microbial

cells); biofilm thickness & 3D images of biofilms

MRI Non-invasive characterization using

magnetic field gradients to obtain nuclear

spin density images

Gives a bulk analysis of biofilm and information

on hydrodynamic parameters

STXM Provides differentiation of classes of

biomolecules without the use of fluorescent

labels; allows chemical speciation &

compositional mapping based on bonding

structure

Limited by absorption saturation; mount

between 2 fragile silicon nitride windows;

inaccuracies in reference spectra; distortion for

thick biofilms; needs sample pretreatment

SIMS þ FISH FISH probes are imaged using SIMS so that

several elements can be observed

simultaneously

Microbial identity and elemental composition at

cell level; links bacterial identity with function

AFM Mapping of the distribution of

macromolecules

Morphological images; needs sample

pretreatment

SEM/TEM Magnified imaging of biofouling layers using

electron beam

Pre-treatment may lead to artifacts & loss of

morphology

a DGGE, denaturing gradient gel electrophoresis; T-RFLP, terminal restriction fragment length polymorphism; FISH, fluorescent in situ hy-

bridization; rRNA, ribosomal RNA; mRNA, messenger RNA; CLSM; confocal laser scanning microscopy; MRI, magnetic resonance imaging;

STXM, scanning transmission X-ray microscopy; SIMS, secondary-ion mass spectromety; AFM, atomic force microscopy; TEM; transmission

electron microscopy; SEM, scanning electron microscopy.

wat e r r e s e a r c h 4 7 ( 2 0 1 3 ) 5 4 4 7e5 4 6 35454

profiling complex bacterial communities (Kwon et al., 2010; Ye

and Zhang, 2011; Guo and Zhang, 2012); however, the enor-

mous amount of generated data requires proper bioinformatic

analysis, involving the assembly, mapping and interpretation

of large quantities of short sequence reads (Simon and Daniel,

2009; Horner et al., 2009; Vakhlu et al., 2012). Another chal-

lenge is associated with the difficulty to convert this data into

practical solutions and/or better understanding of the

biofouling phenomenon. The application of genomic and

post-genomics methods are still limited in the field of

biofouling in membranes for water/wastewater treatment in

general, or in MBRs in particular.

Biofilm characterization techniques and image-analysis

algorithms offer growing opportunities to better understand

the biofouling process. For example, atomic force microscopy

(AFM) can distinguish variations in cell deposition (Zaky et al.,

2012). CLSM images help determine time-dependent devel-

opment of microcolonies on membrane surfaces (Miura et al.,

2007; Zator et al., 2007). Other biofilm characterization

methods (Table 3) are also available but have not been used to

their full potential in MBR studies. In reverse osmosis (RO)

modules, techniques such as magnetic resonance imaging

(MRI) have been applied to capture water flow and subsequent

changes in biofilm distribution due to cleaning (Creber et al.,

2010). Vrouwenvelder et al. (2009b) employed in-situ non-

destructive MRI to support visual observations of pressure

drop measurements in spiral-wound NF and RO membranes.

In MBRs, the application of these advanced biofilm charac-

terization methods is still limited. The main limitation is the

higher complexity of MBR systems and the need to prepare

well-designed experimental setups with specific and

methodical objectives. Accordingly, a broad scope for exten-

sive research exists in this area. A systematic approach that

integrates emerging ‘omics’ technologies and combinations of

biofilm characterization methods offers a wealth of opportu-

nities to collect multiple pieces of information on biofilm

development on membrane surfaces. The proper selection of

these tools is vital for ultimately developing better biological

antifouling strategies that can also benefit the advancement

of physical, chemical as well as other non-biological control

methods.

3.2.2. Biofouling microbial communities in MBRs

Many studies reported the phylum Proteobacteria to be the

dominant phyla in biofouled MBR membranes (Lim et al.,

2004; Choi et al., 2006; Jinhua et al., 2006; Miura et al., 2007;

Zhang et al., 2006; Ahmed et al., 2007; Huang et al., 2008b;

Duan et al., 2009; Fontanos et al., 2010; Gao et al., 2011b;

Piasecka et al., 2012). In addition to Proteobacteria, other

phyla such as Firmicutes (Lim et al., 2004; Calderon et al.,

2011), Bacteriodetes (Fontanos et al., 2010; Huang et al.,

2008b), Actinobacteria (Lim et al., 2004) and Planctomycetes

(Piasecka et al., 2012) have also been reported to be pre-

dominant in MBR fouled membranes. Some researchers

argue that targeting pioneer bacteria that initially colonize

on membrane surfaces might help develop better control

strategies; yet, very few studies have addressed pioneer

bacteria (Zhang et al., 2006; Piasecka et al., 2012). Choi et al.

(2006) noted that Acinetobacter is important in the early

colonization of MF membrane in a lab-scale MBR. Miura et al.

(2007) noted that initially the biofilm in hollow-fiber MF

membrane of MBR treating municipal wastewater was

composed of Alpha-, Beta-, Gamma- and Delta-Proteobacteria

whereas in mature biofilm, Dechloromonas-related bacteria

became dominant. Zhang et al. (2006) reported a list of bac-

teria that might be the pioneer bacteria colonizing the sur-

face of flat-sheet MF membranes in an MBR treating

synthetic paper mill wastewater. Most of the sequences

belonged to Alpha-, Beta- and Gamma- Proteobacteria, the

Cytophaga-Flavobacterium-Bacteroides clade, and an uncultured

bacterium division TM7. Gao et al. (2011b) reported that

species belonging to the phylum Alphaproteobacteria were

prone to form initial biocake but as conditions become more

anoxic in thicker biocake, habitats were changed with a

succession from Betaproteobacteria to Deltaproteobacteria.

Piasecka et al. (2012) identified 25 pioneer operational taxo-

nomic units (OTUs) belonging to the phyla Firmicutes, Pro-

teobacteria, Bacteroidetes and Planctomycetes on membrane

surface after one day of filtration in a lab-scale MBR with

double-flat sheet membranes operated on molasses waste-

water. In an inclined plate MBR, Fontanos et al. (2010) re-

ported that although the same biofouling microbial groups

were found on polyethylene (PE) and polyvinylidene fluoride

(PVDF) hollow fiber membranes, the community dominance

exhibited different trends with the advent of fouling.

3.2.3. Implications in MBRs and other membrane applications

A common finding in several studies is that the microbial

community identified on biofouled MBR membranes differs

significantly from the mixed liquor community (Piasecka

et al., 2012; Gao et al., 2010; Huang et al., 2008b; Miura

et al., 2007; Zhang et al., 2004; Choi et al., 2006; Jinhua

et al., 2006). For example, Miura et al. (2007) noted that the

filamentous Chloroflexi, which accounted for nearly 20% of

total hybridized cells in the mixed liquor was not detected on

the membrane surfaces of pilot-scale MBRs. Similarly, Gao

et al. (2010) noted the absence of Bacteroidetes and other

bacteria that were abundant in suspended biomass. Choi

et al. (2006) and Zhang et al. (2004) further reported that

genera such as Brevundimonas, Acinetobacter, Sphingomonas,

and Aquaspirillum, which were present in low abundance in

the mixed liquor became dominant on fouled MF mem-

branes, implying that specific bacterial populations selec-

tively attached to the membrane surface. Piasecka et al.

(2012) observed that this difference between mixed liquor

and biofouling communities was more pronounced in the

initial phases of filtration.

Research in process water RO systems has been suc-

cessful in identifying a specialized bacterial community

responsible for initiating and dominating biofouling i.e.

Sphingomonas spp. (Bereschenko et al., 2010). A common

cosmopolitan group of biofouling bacteria was, moreover,

identified on five different seawater RO membranes from

different parts of the world, operated under different condi-

tions and collected at different times of the year (Zhang

et al., 2011). Whether specific initiating and dominating

species can be identified in MBRs and if so, how this infor-

mation can be linked to developing better control needs to be

determined by first filling specific knowledge gaps that still

remain in MBRs.

wat e r r e s e a r c h 4 7 ( 2 0 1 3 ) 5 4 4 7e5 4 6 3 5455

4. Current gaps in knowledge

4.1. Microbiological studies

Although the genetic basis of biofilm formation has been

investigated for several bacterial species, including E. coli

(Pratt and Kolter, 1998), P. aeruginosa (Heydorn et al., 2000;

O’Toole and Kolter, 1998) and Vibrio cholera (Watnick and

Kolter, 1999), similar studies targeting the specific microor-

ganisms responsible for membrane biofouling in MBRs are

very limited. Successful application of biological-based anti-

fouling strategies requires a deeper knowledge of the mi-

crobial ecology of the communities responsible for biofouling

and which may completely differ from the ones that have

been investigated in the literature on biological control in

biofilms. Despite the increasing number of studies, the mi-

crobial ecology of microorganisms responsible for biofouling

in MBRs has not been systematically addressed. Whereas

some researchers claim that targeting the pioneer bacteria is

important (Zhang et al., 2006; Piasecka et al., 2012), since

severe fouling is often observed in initial phases when the

biomass is not yet fully acclimated (Li et al., 2012), most

existing microbial studies in MBRs have focused on the

mature bacterial biofilm ecology (Lim et al., 2004; Jinhua

et al., 2006; Miura et al., 2007; Huang et al., 2008a; Duan

et al., 2009; Gao et al., 2010).

On the other hand, only one study has characterized

archaea in biofouling layers where in addition to the dominant

bacterial phyla (Firmicutes and Alphaproteobacteria in this case),

archaeal clones e mostly affiliated to the order Meth-

anosarcinales and family Methanospirillaceae were identified

(Calderon et al., 2011). Few studies in the literature were based

on real wastewater (e.g. Huang et al., 2008b; Miura et al., 2007;

Jinhua et al., 2006; Lim et al., 2004) while most others used

synthetic wastewater and the majority of microbiological

studies were conducted on lab-scale MBRs with very few on

pilot-scale systems (Calderon et al., 2011; Jinhua et al., 2006)

and none on real-scale systems. Moreover, existing microbi-

ological studies in MBRs have focused only on the microbial

community structure (who is there?) but not function (i.e.

expressed genes and proteins). Studying both the structure

and function of the biofouling community is important in

future microbiological studies since a common function

maybe identified for different microbial communities. Com-

mon genes may be identified for important functions (e.g. EPS

formation or QS) even from different phylogenetic groups.

Advanced biofilm characterization and next-generation high

throughput sequencing tools (metagenomics, meta-

proteomics, metatranscriptomics) can be especially helpful in

this regard as illustrated in Table 3. So far, existing microbi-

ological studies inMBRs have utilized tools such as T-RFLP and

clone library analysis of 16S rRNA gene (Piasecka et al., 2012;

Gao et al., 2010), DGGE and clone library analysis of 16S

rRNA gene (Duan et al., 2009; Huang et al., 2008b; Jinhua et al.,

2006), quinone profile analysis (Ahmed et al., 2007; Lim et al.,

2004), amplified ribosomal deoxyribonucleic acid restriction

analysis (ARDRA) and clone library analysis of 16S rRNA

gene (Zhang et al., 2006) and FISH (Fontanos et al., 2010; Miura

et al., 2007).

Genomics and post-genomics techniques are expected to

offer new clues to better control, but how this information can

be channelized into devising effective, sustainable and cost-

effective control strategies still needs further research.

Although technical challenges such as DNA, RNA, and protein

extraction, mRNA instability and lack of reference genomes

may hamper the use of omics for some time, the growing use

of these techniques offers the opportunity for a more

comprehensive understanding of the structure and function

of biofouling communities. Post-genomic approaches have

been successfully applied to detect expression profiles (mRNA

and proteins) and provide functional insights for mixed cul-

ture environmental samples such as the gut microbiota

(Gosalbes et al., 2011) and activated sludge (Wilmes et al.,

2008). One successful story of applying these tools is in

enhanced biological phosphorous removal (EPBR) processes.

Combining new sequencing technologies from -omics ap-

proaches with single-cell studies and with process and phys-

ical/chemical studies allowed linking intracellular metabolite

flux models to comprehensive system-scale models for EPBR

(Nielsen et al., 2012). In the field of membrane biofouling,

-omic tools are still not widely applied. Miyoshi et al. (2012)

analyzed two-dimensional polyacrylamide gel electropho-

resis (2D-PAGE) spots by N-terminal amino acid sequencing

analysis and identified two well-characterized outer mem-

brane proteins originating from Pseudomonas genus, namely

OprF and OprD, contributing to membrane fouling in MBRs.

More studies employing these tools can play a vital role in

clarifying the functions and interactions among biofouling

microorganisms and could help improve our understanding of

critical aspects of the biofouling process. The practical success

with filamentous bacteria is a good demonstration of manip-

ulation of populations for better control of these microor-

ganisms. Molecular tools have been applied to study

filamentous bacteria, which can cause poor settling (bulking)

or foaming. Because of an increased understanding of their

identity and ecology, it has been possible to develop specific

control measures for several of these unwanted organisms,

which include changes in plant operation, process layout or

addition of chemicals (Nielsen et al., 2009). Thus, a better

understanding of the microbiology and proper linking with

operational parameters is expected to help in developing

more effective control strategies and cleaning methods.

4.2. Biological strategies for MBR antifouling

4.2.1. Challenges in biological-based strategies

Most of the biological-based antifouling strategies are still in

the developing phase andmany face their own challenges and

limitations. In QQ, for example, care should be taken so that

desirable biochemical reactions undertaken by the microbial

consortia would not be affected by a particular quorum-

quenching strategy (Choudhary and Schmidt-Dannert, 2010;

Jiang et al., 2013). Inhibition of ATP synthesis by chemical

uncouplers may be a feasible alternative for alleviating

biofouling, but it has the disadvantage that most ATP un-

couplers are classified as aromatic and can be toxic (Xiong and

Liu, 2010), thereby limiting their practical application.

Conversely, the enzymatic method is nontoxic and environ-

mentally friendly but enzymes are unstable and are highly

wat e r r e s e a r c h 4 7 ( 2 0 1 3 ) 5 4 4 7e5 4 6 35456

pH-, temperature-, and salt concentration- sensitive. For

bacteriophages, in addition to phage resistance challenges,

nutritional limitation is known to influence the stability of

bacteriaephage interactions where increasing the input of

nutrients can lead to a large increase in phage numbers, a

small increase in bacteria and a reduction in the dynamic

stability of both populations (Bohannan and Lenski, 1997).

Also, the presence of a non-susceptible bacterial population

could protect bacteriophage-susceptible strains, possibly by

creating ‘spatial refugees’ within the depths of the biofilm

(Tait et al., 2002).

Despite the success of recent studies described before (e.g.

QQ used by Oh et al., 2012), more research is needed to

examine the scale-up of these results to real MBRs and vali-

date their effectiveness using real wastewater. Much remains

to be learned about the role of QS in biofilm formation. For

instance, more than 100 species of Proteobacteria, which is a

common phylum of biofouling bacteria, are known to contain

AHL signal genes, which could be responsible for QS signaling

pathways; however, only few of the species identified in bio-

fouled membranes show QS-regulated control of EPS regula-

tion and many do not have clear functions associated with

their QS circuitry (Shrout and Nerenberg, 2012). Moreover,

many microorganisms possess QS genes but not all of these

genes are expressed. Different microorganisms also generate

different QSs. Therefore, for QQ-based strategies to hold more

promise in biofouling management, it is important to learn

more about the eco-physiology of biofouling microorganisms.

The practicality, specific design and operational re-

quirements as well as the economic feasibility of expanding

lab-scale experiments to real-scale systems still need deeper

assessment and more research on real-scale applications.

Also, most of the well-studied systems in biological control

studies have been studied for pure cultures while uncovering

the details for mixed microbial cultures and other species

such as Gram-positive bacteria, archaea and fungi is needed

(Choudhary and Schmidt-Dannert, 2010; Calderon et al., 2011).

Through proper application of high throughput analytical

tools, the specific microorganisms and genes responsible for

MBR biofouling can be identified. This information can be

used in developing targeted and cost-effective biological

control approaches in contrast to the present status of

disjointed and still rudimentary trials followed in biological

control approaches. Consequently, studies on the effective-

ness of biofilm dispersal and inhibition techniques still need

to be broadened to validate their applicability to specific

operational conditions in MBRs and prove their effectiveness

on the key microbial species responsible for MBR biofouling.

4.2.2. The importance of an integrated control approach

Effective biofilm control practices are expected to originate

from well-selected and directed strategy combinations. A

single approach must be 100% effective, whereas in combi-

nation each individual approach can be partially effective

while the combination is still efficient (Vrouwenvelder et al.,

2010). As evidenced in several laboratory studies, any one

process modification alone is unlikely to solve all accumula-

tion of undesired biofilm growth (Shrout andNerenberg, 2012).

For example, a furanone inhibitor of P. aeruginosaQS increased

the efficacy of detergents or antibiotics in biofilm killing

(Hentzer et al., 2003) and exogenous addition of autoinducer

peptide (AIP) increased the sensitivity of biofilms to antibiotics

(Lauderdale et al., 2010). Combinations of phage enzymes and

disinfectants by adding the phage and then the disinfectant

have also been found to be more effective than adding either

alone (Tait et al., 2002). Similarly, Hughes et al. (1998)

demonstrated that phage polysaccharide depolymerases

afforded better access for disinfection and better EPS removal.

Chlorine treatment was also found to be 20 times more

effective at removing multispecies biofilms from water sys-

tems after exposure to nitric oxide, which can disrupt the

intracellular secondary messenger cyclic di-GMP in microor-

ganisms (Barraud et al., 2009). Another agent that disrupts

intracellular signaling, not necessarily through QS pathways,

is cis-2-decanoic acid, which acts as a fatty acid messenger

that can induce biofilmdispersion (Davies andMarques, 2009).

Both nitric oxide and cis-2-decanoic acid seem to be appealing

due to their multispecies efficacy, natural synthesis, and

readily biodegradable characteristics (Shrout and Nerenberg,

2012); however, their incorporation to cleaning protocols in

real-scale systems and in properly designed and cost-effective

strategies remains a challenge.

Gino et al. (2010) proved the potential of combined

chemical-biological treatment in preventing groundwater

well clogging as a result of biotic iron oxidation. Glycolic acid

(2%) and isolated bacteriophages showed inhibition of biofilm

formation while earlier biofilm treatment with reduced gly-

colic acid concentration revealed efficient EPS digestion thus

allowing phages to be more efficient against biofilm matrix

bacteria. The above examples show that combined ap-

proachesmight offer a greater potential for effectively limiting

biofouling problems than a single control method. The

appropriate doses, designs and operational aspects to be

applied in real systems still need to be determined. Newly

developed approaches should also not interfere with the bio-

degrading activities and desired functions of microorganisms

in the mixed liquor. For instance, Meng et al. (2012) reported

that ciprofloxacin could inhibit filaments in MBRmixed liquor

but with an adverse effect on denitrification.

In addition to integrating effective biological-based stra-

tegies with optimized cleaning methods, a combined sys-

tematic approach that links microbiology with MBR design

and operational aspects is essential to understand how these

parameters are interrelated and to effectively manage

biofouling for the wide range of conditions governing MBR

operation.

4.3. Linking operation to microbiology

In MBRs, many factors, including reactor design and opera-

tion, cleaning strategies, membrane properties and biomass

conditions can have direct impact on MBR fouling potential

(Yamato et al., 2006; Meng et al., 2009). Kraume et al. (2009)

noted that differently sized modules, lack of appropriate hy-

drostatic head, presence of aerators, differing backflush or

relaxation hours might result in different fouling mecha-

nisms. These parameters are also expected to impact the

microbial community structure; yet, systematic correlations

that link the biofouling community structure and function to

important operational and environmental parameters are still

wat e r r e s e a r c h 4 7 ( 2 0 1 3 ) 5 4 4 7e5 4 6 3 5457

lacking. Miura et al. (2007) found that with higher aeration

rates, beta-proteobacteria became the most dominant

biofouling species. Lim et al. (2004) reported the presence of

Paracoccus sp. and Flavobacterium sp. for continually aerated

and Pseudomonas, Moraxella, Vibrio, Staphylococcus warneri,

Micrococcus sp. and Nocardia sp. for intermittently aerated

hollow fiber MF membranes in MBRs. Ahmed et al. (2007) re-

ported the growth of microorganisms from the delta- and

epsilon-subclass of Proteobacteria and members of the Cyto-

phagaeFlavobacterium cluster increased at longer sludge

retention times (SRT) whereas in the study by Duan et al.

(2009), beta-proteobacteria dominated at all SRTs. Huang et al.

(2008a) found that at lower fluxes, the MBR biofilm commu-

nity composition was similar independent of sludge age

whereas distinct biofilm communities developed on mem-

brane surfaces at high fluxes. Despite the significance of these

research findings, more studies on other parameters are still

needed particularly relating to reactor design, operation and

applied cleaning protocols and how these impact biofouling

communities.

Similarly the amount and temporal sequence of EPS for-

mation in response to different physical and biological con-

ditions are unknown and the potential to predict EPS

production is the weakest point in EPS research (Flemming

and Wingender, 2010). In general, EPS composition, amount

and properties are likely to be unique for each species but very

little is known about EPS production for most fouling species

(Shrout and Nerenberg, 2012). Using the emerging advanced

molecular and imaging tools, it may be possible to study the

particular genes responsible for biofouling at each stage of

biofilm development e.g. initial attachment, growth, EPS

secretion and detachment and how the expression of these

genes varies for different conditions.

When characterizing the microbial communities and their

associated products in MBRs, it is important to consider: a) the

use of real wastewater compared to synthetic wastewater. A

lower microbial diversity index is generally attributed to sys-

temswith synthetic influent regardless of operationmode and

reactor scale (Hiraishi et al., 1998); b) long-term monitoring of

the biofouling communities since deviations in operational

conditions and in membrane properties, as foulants attach to

the surface, can have a significant impact on the bacterial eco-

physiology. It has been reported that short-term membrane

adhesion tests are not a suitable predictor for biofouling in MF

and UF membranes, which showed significantly reduced

adhesion of biofoulants in short-term tests, but no reduction

during longer biofouling experiments (Miller et al., 2012); and

c) influence of recurrent cleaning and the relationship be-

tween the biofouling bacterial communities and the cleaning

option adopted. Fig. 1 depicts the interacting variables as well

as the possible integration of relevant analytical tools for

developing a better understanding of MBR biofouling.

5. Conclusion

Membrane biofouling is an inevitable problem that has

become an intensive research area, demanding a methodical

Fig. 1 e Membrane biofouling: a comprehensive overview for an integrated control approach.

wat e r r e s e a r c h 4 7 ( 2 0 1 3 ) 5 4 4 7e5 4 6 35458

and unified investigation approach. In practice, a more

effective integrated control approach should be based on

three considerations: system design and operation, biomass

growth conditions and cleaning strategies. The key argument

presented here to pursue microbiological routes as an essen-

tial ingredient in an integrated control of biofouling stems

from their high effectiveness potential, sustainability and

possibly lower costs. Biological methods offer a promising

control alternative but more investigations on the structure

and function of biofouling bacterial populations and their EPS

are needed. Also, future studies should specifically address

the relationship between the biofilm communities and envi-

ronmental, operational (including cleaning) and membrane

conditions, as well as the application of successful biofilm

control methods to MBRs.

Acknowledgments

The preparation of this article was supported by discretionary

investigator funds (Pascal Saikaly) at King Abdullah University

of Science and Technology (KAUST).

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