The Membrane Bioreactor, can no longer be considered a novel process, it ensures improved effluent quality for wastewater treatment, while requiring a smaller footprint. The most costly part is the membrane, which is susceptible to fouling. Fouling of membrane bioreactors, results in higher operational expenses, reduced stability and operational performance. This review paper focuses on the biofouling of membrane bioreactors for wastewater treatment. The biofouling factors and biological control strategies are discussed. Lastly, future studies on membrane biofouling research are addressed.
Keywords: Membrane Bioreactor; biofouling, Factors; biological Control
Over the past two decades, the membrane bioreactor (MBR) has emerged as one of the innovative technologies in leading wastewater treatment . MBRs are widely used for wastewater treatment that requires high effluent quality, e.g., water reuse or water recycling [2, 3]. The MBR system has many advantages over conventional wastewater treatment methods. These consist of small footprint, high effluent quality, good disinfection capability, higher volumetric loading, and less sludge production . However, membrane biofouling decreases MBR filtration performance with filtration time because of the bacterial cell's deposition, and its growth onto and into the membrane . Extracellular polymeric substances (EPS) and Soluble microbial products (SMP) secreted by bacteria also play vital role in biofouling . This major hindrance and limitation of process has been under analysis since the early MBRs, and it is one of the most demanding issues facing further MBR development .
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This review paper is mainly focused on biofouling of the membrane bioreactor, particularly on the fundamentals of such biofouling, the major biofouling factors, control strategies, and some future prospects. It is anticipated that this review may serve as a stepping stone for further development and application of biological methods towards effective control of membrane biofouling. In recent years, a number of papers were published on membrane biofouling (Google scholar) (Fig. 1).
Biofouling is the adhesion, growth, and metabolism of bacteria cells or flocs on the membranes, which is a significant concern in membrane filtration . The fig. 2 shows membrane biofouling. For a low pressure driven membrane, such as ultrafiltration and microfiltration for treating wastewater, biofouling is a major hitch because most foulants (microbial flocs) in MBRs are much larger than the membrane pore size. Biofouling may start with the deposition of cell cluster or a cell on the surface of membrane, after which the cells multiply and form a biocake. Many researchers propose that SMP and EPS also play roles in the synthesis of biological foulants and cake layer on membrane surfaces [9, 6]. The structure of biofilm is known to vary greatly, ranging from patchy, discontinuous colonies to thick, continuous films, and it affects characteristics such as resistance to antimicrobial agents depending on environmental conditions and mass transport phenomena .
Techniques used to study the biofouled membrane
The biofouled membrane can be visualized by different techniques presented in Table 1. Zhang et al.  used DOTM to examine the interactions between bioflocs and the membrane surface. They observed that the bioflocs could move across the surface of membrane via sliding and rolling. Nowadays, a powerful approach for characterization of membrane biofouling is CLSM; it can present the three 3D structure of fouling layer and can identify the attached cells. Ng et al.  applied CLSM to visualize the distribution of bacterial cells on the membrane surface, and found that bacteria were extensively present on the fouled membrane. The combination of image analysis and CLSM can quantify or visualize the structural design of the biocake layer . Yun et al.  characterized the biofilm structure and analyzed its effect on membrane flux in MBR for dye wastewater treatment. They found that membrane filterability was narrowly associated with the structural parameters of the biofilms (i.e., porosity, biovolume). The study of biofouling through the application of these techniques is useful for understanding of the floc/cell deposition process and the structure of the cake layer.
A handful researches have been carried out to study the microbial community structures and colonization microbes on membranes in MBRs [15, 16]. The microbial community structures can be studied using methods such as Fluorescence In Situ Hybridization (FISH) and polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) [15, 17]. Bereschenko et al.  used a combination of molecular ((fluorescence in situ hybridization [FISH], denaturing gradient gel electrophoresis (DGGE), and microscopic (epifluorescence, field emission scanning electron, and confocal laser scanning microscopy)) techniques to analyze the architecture, composition, abundance, and 3D structure of biofilm communities. Jinhua et al.  reported that Proteobacteria more selectively adhered and grew on membranes than other microorganisms, and that the deposited cells have higher surface hydrophobicity than the suspended sludge. The high shear stress provoked by aeration can choose the deposition of cells. Few cells can be removed easily by the shear stress, but others adhere tightly to the membrane surface. The discerning deposition of the cell depends on the affinity of cells to membranes. Moreover, due to the anoxic condition in the cake layer, the temporal change of microbial community structure would take place. We can view that few of the bacteria in the sludge should play pivotal role in membrane biofouling. Understanding the deposition behavior of bacterial cell/flocs and mechanisms of cell attachment in MBRs will be vital for the development of suitable biofouling mitigation methods in the future.
Factors of biofouling
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In this review paper, the major factors contributing to biofouling, including EPS and SMP are discussed.
Microbial EPS and SMP
Microbial biofilm on the membrane surface decreases the flux but can be controlled by backwash. In general, two kinds of substances are produced during biological activities, which cause fouling: extracellular polymeric substances (EPS) and soluble microbial products (SMP). EPS are insoluble macromolecules polymerized by microorganisms or substances like capsules, gels, and humic acid. SMP are produced by cell metabolism or self-digestion, which can be considered to be soluble, large molecules. They form colloidal substances and gradually accumulate in the membrane hole, decreasing the effective pore size of the membrane .
Effects of EPS on membrane fouling
Bound EPS have been found not only as main sludge floc components keeping the floc in a 3D matrix, but also as critical membrane foulants in MBR systems. Cho et al.  found a close association between the attached EPS and the specific cake resistance. Ahmed et al.  also observed that as attached EPS concentration increased, the specific cake resistance rose, and this inevitably resulted in the rise of TMP. A recent study by Ji and Zhou  indicated that both composition and quantity of attached EPS on the membrane surface influenced membrane fouling, and the total biopolymers in sludge suspension played a more important role than bound EPS in reflecting the extent of membrane fouling. Ramesh et al.  separated bound EPS into tightly bound EPS and loosely bound EPS, and confirmed that the resistance was primarily caused by the loosely bound EPS. The EPS that loosely bound correlated with the activity of flocculation and sedimentation processes . A number of researches, however, have declared that bound EPS had minute correlation with membrane fouling. Rosenberger and Kraume  observed that, contrary to some literature, no impact of bound EPS on filterability could be seen. Instead, the soluble EPS or SMP were found to have great impact on the filterability of sludge. This was confirmed by a more recent work reporting no clear relation between bound EPS and membrane fouling as the concentration of bound EPS was smaller than 10 mg/g SS . Several explorations have been carried out to gain a better knowledge of sludge features and their knocks on membrane fouling . The above mentioned inquiries showed that activated sludge has very complicated impressions on the membrane fouling process.
In spite of the verity that the research results on bound EPS are distinct from each other, it must be pointed out that bound EPS concentrations are closely connected to sludge characteristics, such as sludge flocculation ability, hydrophobicity, sludge viscosity volume index, and surface charge. Therefore, in view of the critical function of bound EPS in sludge features and membrane fouling, bound EPS should be restrained to mitigate membrane fouling.
Effect of SMP on membrane fouling
In fact, the complex nature of sludge suspension, the behavior of fouling cannot be assigned entirely to bound EPS. The effect of SMP on MBR fouling has attracted much attention [33, 34]. Due to the membrane rejection, SMP accumulate more easily in MBRs, which results in the low filterability of the sludge suspension. Geng and Hall  found that the distribution of floc size and the amount of soluble EPS in the mixed liquor were the most vital characteristics that influenced the fouling tendency of sludge. Moreover, some studies have shown that polysaccharide-like substances in SMP contribute to fouling more than protein-like substances [33, 36].
Since SMP have been identified as membrane foulants, research on SMP has become one of the main areas in the research on membrane fouling. Rosenberger et al.  found that the SMP of sludges had an impact on fouling and caused the difference in membrane performance between two similar MBRs. Iritani et al. (37: 2007) observed that the input of the supernatant to the membrane fouling of an anaerobic activated sludge is nearly 100%, indicating that SMP constitute the main factor in filtration of activated sludge. Lyko et al.  studied the SMP in supernatant and permeate as well as attached EPS from fouled membranes in the full-scale MBR, and found a vital effect of soluble humic substances and carbohydrates with metal cations on membrane fouling. They also found that dissolved organic carbon (DOC) was choice for complex and costly measurements of SMP components . These researches suggest that the presence of SMP in MBRs knocks on membrane fouling substantially, and SMP concentration and SMP composition would establish the fouling tendency.
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Moreover, the presence of SMP in MBR effluent is a concern in the execution of post-treatment for water recycling, and the release of SMP rich water brings further problems to the native environment, e.g., the presence of dissolved organic nitrogen. Table 2 presents the relationship between various biofouling factors and membrane biofouling.
Biological mitigation of membrane biofouling
So far, comprehensive research has been undertaken to explore the possible methods to prevent or reduce membrane biofouling. The traditional methods for reducing membrane biofouling are mainly based on physico-chemical principles, such as modification of the solid surface, optimization of operation conditions, regular physical and chemical cleaning, etc. [44, 48], and may not be effective and energy efficient.
Biofouling is hard to control, even by reducing the number of microorganisms, they can multiply even if their number is strongly diminished, and they will again grow if nutrients are available . Recently, various novel biological methods for controlling microbial attachment and membrane biofouling have been developed, and there are some recent reviews on quorum sensing (QS)-associated biofilm control and biofouling . In this review paper, biological methods (Fig. 3) to control biofouling of membrane bioreactors such as enzymatic, nitric oxide, bacteriophage and Quorum sensing inhibition are discussed. A summary of biological methods to control membrane biofouling have been given in table 3.
Bacterial EPS facilitate the formation of bioflocs in activated sludge and have role in its structural, surface charge and settling properties [51, 52]. Bioflocs are produced through the interaction of filamentous bacterial strains, and organic and inorganic particles, which are held together by EPS [53, 54]. EPS cannot be removed by traditional physico-chemical chemical methods. However, enzymes could be used to hydrolyse the EPS, implying a novel means to control membrane biofouling .
Enzymes, such as the trypsin, subtilisin, proteinase K, etc., have been used to remove the biofilms. Proteinase K, has been commonly applied to disperse the established biofilm. Trypsin hydrolyzes lysine peptides, can also remove the mature S. aureus 383 biofilm . Leroy et al.  compared the antifouling potential of some commercial enzymes, such as Amano Protease A, papain, umamizyme, and subtilisin, and the other hydrolases in the adhesion of Pseudoalteromonas sp. D41. It was found that the most efficient protease was subtilisin, which had the ability to inhibit microbial adhesion as well as detach adhered bacteria. Poele and Van der Graaf  used protease to remove biofouling on ultrafiltration (UF) membrane for wastewater treatment. Compared with the traditional cleaning method by alkaline, enzymatic cleaning by protease exhibited a much higher efficiency in removing biofouling, leading to a high-efficiency recovery of the permeate flux.
In addition to the traditional prevention and cleaning technologies for membrane biofouling, enzymatic disruption of EPS appears to be a promising alternative for high-efficiency control of microbial attachment and membrane biofouling.
To date, extracellular DNA (eDNA) has been shown to function as a structural support to maintain biofilm architecture, and has also been known to serve as a vital factor to induce the development of biofilm [59, 60]. A study on S. epidermidis biofilm reveals that eDNA was an important factor for the initial development of S. Epidermidis biofilm on polystyrene and glass surfaces under static or hydrodynamic conditions . These suggest that disruption of eDNA would lead to detachment or dispersal of biofilms.
These indicate that DNase I would be more effective for controlling microbial attachment or biofilm development than disrupting mature biofilm. Therefore, DNase may provide a feasible and efficient means for controlling microbial attachment as well as membrane biofouling. Lastly, it should be realized that enzymatic disruption of EPS would have some inherent drawbacks that, to some extent, may limit its large-scale application. Enzyme is unstable in the environment, e.g., the activity of enzyme would be reduced or even totally lost when the optimal pH value is altered. Moreover, enzymes are highly temperature sensitive. High temperature may make enzymes denatured, while low temperature could decrease the enzymatic activity substantially. In addition, enzymes are also sensitive to elevated salt concentration. Compared with the chemical methods for EPS removal, the enzymatic method is nontoxic and environmentally friendly, while the problem associated with the bacterial resistance to the antibouling agents would be minimized.
The hydrolytic enzymes of cell walls, such as lysozyme, can damage bacterial cell walls. Consequently, they have been employed to prevent microbial attachment and biofilm formation onto solid surfaces. Lysozyme-immobilized polymeric packaging films exhibit bacterial growth-inhibiting properties . Caro et al.  coated the glycosidase hen egg white lysozyme to a stainless steel surface and found that microbial attachment on the substrate coated with lysozyme could be completely inhibited as compared with the control without immobilization of lysozyme. The antimycotic protein lysozyme is environmentally friendly and acts more specifically than the traditional biocides for biofilm control and has been used for the control of Candida biofilm development on denture acrylic .
Consequently, lysozyme or capsulated lysozyme could be a safe and highly effective agent for controlling microbial attachment and disrupting established biofilms. It should be pointed out that lysozyme, as a type of hydrolase, is not stable in the environment, and its efficiency would depend on pH, temperature, ionic strength, etc.
Control by nitric oxide
Nitric oxide (NO), as a biologically ubiquitous gas molecule, can antagonize useless cell proliferation . This specific molecule has been identified as an important messenger molecule that regulates biofilm dispersal. Similar to the QS inhibitors, NO attenuates the bacterial infection by signaling mechanisms rather than by the toxic effect. NO can inhibit the infection of K. pneumonia, a kind of lung bacteria, and efficiently protect the lung system . Since the gene encoding for regulators of the Cyclic di-GMP commonly exists in bacteria [66, 67, 68] and several factors associated with NO production and response system seem to be preserved across microorganisms [69, 70], the NO-mediated dispersal mechanism may exist in a wide spectrum of microbial species. It has been reported that NO at a flux of 30 pmol/(cm2s) could efficiently reduce adhesion of S. aureus, Staphylococcus epidermidis, and E. coli, with a reduction rate of 96%, 48%, and 88%, respectively . These in turn suggest that NO would have a comparably universal effect on dispersal of sessile bacteria, including both Gram-positive and Gram-negative bacteria. S. aureus and S. epidermidis biofilms were repressed in the presence of nitrite (NO2âˆ’) presumably due to the involvement of NO . Besides the dispersal of pure culture biofilms of some stains, the dispersal of multispecies biofilms from water distribution and treatment systems can also be induced by various NO donors at picomolar or nanomolar levels, e.g., addition of the NO donors would result in an average of 63% decrease of total biofilm .
More importantly, combined low-dose NO and antimicrobial agents can greatly enhance biofilm removal. Compared with sole action of chlorine, a 20-fold increase in the efficiency was observed when NO was applied together with chlorine in removing multispecies biofilms . In addition, pretreatment of biofilm grown on the RO membrane by 100 nM of SNP that served as the donor of nitric oxide led to a twofold increase in the biofilm removal efficiency in contrast with the control .
As discussed above, NO has great application potential in controlling microbial attachment and membrane biofouling. On the other hand, it should be realized that NO has low solubility in water and is not stable as it can be easily oxidized. As a result, the direct addition of NO into aquatic solution would have low efficiency of biofouling control . Nevertheless, many NO donors, including the enzymatic and nonenzymatic NO donors (e.g., SNP, sodium nitrite, SNAP, diazeniumdiolate, etc.) have been proven to be efficient. This implies that the use of the NO donor would have the same effect as the direct addition of NO on dispersal of biofouling.
Control of microbial attachment and membrane biofouling by bacteriophage
An overview of the current scientific literature on "bacteriophages treatment" reveals several decades of "renaissance" on phages application in combating a large variety of bacteria in different experimental areas, such as foam formation reduction , slime and biofilm control , plant diseases , medicine , foodborne pathogen control, and detection , and shows phages to be an alternative agent . The use of phages has been abandoned due to our modest knowledge on their molecular biology and physiology at the time and the discovery of antibiotics .
Another important application of phage is to inhibit or disrupt biofilm development on solid surfaces such as membranes. Doolittle et al.  successfully used bacteriophages to disrupt E. coli and P. aeruginosa biofilms, respectively. Corbin et al.  reported that bacteriophage T4 at a multiplicity of infection of 10 would lead to a one-log decrease in biofilm density after a 90-min treatment. One of the studies also disclosed that phage F116 could pose a two-log decrease of P. aeruginosa biofilm cells . In addition, Curtin and Donlan  demonstrated that the infection of catheter by S. Epidermidis biofilm could be reduced by phage. Compared with the natural phage, an engineered phage with multifunction can enhance biofilm dispersal.
Lu and Collins  designed an enzymatic phage that can express Dispersin B, a polysaccharide-hydrolyzing enzyme. This engineered phage was found to have higher efficiency in the removal of biofilms, e.g., the reduction of cell count of biofilm by the engineered phage was two orders of magnitude higher than the nonengineered phage, leading to a nearly complete biofilm removal. In addition, a cocktail of multiple phages has been shown to be efficient in removing multispecies biofouling . Goldman et al. , probably for the first time, employed bacteriophage to control UF membrane biofouling. Results showed that the addition of phages could reduce microbial attachment to membrane surface by 40% on average, and the performance of the membrane bioreactor treating the effluents from the sewage treatment plant was improved significantly in terms of membrane permeability. The phages detected in permeative solution were only a few (1 to 10 PFU/100 ml), which might have originated from the penetration of phage across the membrane and/or possible permeate contamination by phage during sampling. Goldman et al.  thought that the growth of phage found in permeative solution would not be an issue due to the absence of bacteria in UF filtration permeative solution. As noted by Goldman et al. , "bacteriophages have a major advantage of continuous infection/multiplication as long as the host is present and grows" and "when several bacterial species are present, a combination of different phage may be used to stop concomitant adhesion and biofilm formation by bacteria." Although the study by Goldman et al.  sheds lights on the potential application of bacteriophage in mitigating membrane biofouling without the use of antimicrobial agents, the specific-parasite characteristics of bacteriophage would eventually pose a challenge on its application in large-scale wastewater treatment, which needs to be taken into serious account in future scenario.
Control by quorum sensing
Quorum sensing (QS) is cell to cell communication systems that used by microorganisms to assess their local densities. This mechanism is based on secretion, production, and detection of small signalling molecules (Fig. 4) . Several bacteria have been reported to biofilm formation through cell to cell communication or QS mediated by small, diffusible signals . It is also suggested that research into QS inhibition will provide some means for controlling the growth of biofilm without the use of growth-inhibitory agents that unavoidably select for resistant organisms . Compounds like salicylic acid , urosolic acid , cinnamaldehyde , extract from garlic , and cranberries have all shown various degrees of antibiofilm properties against a number of microorganisms in various studies. Furanones isolated from the marine red alga Delisa pulchera are among the most extensively studied classes of natural compounds with respect to their role in inhibiting biofilm. Ponnusamy et al.  recently reported the role of 2(5-H) furanone in suppressing biofilm formation by environmental strains of bacteria isolated from fouled RO membrane.
In view of the essential role of QS in biofilm formation, disruption or inhibition of the QS systems of microorganisms appears to be a promising alternative for controlling microbial attachment as well as membrane biofouling (Fig. 4). Therefore, strategies to disrupt QS systems are discussed herein with emphasis on the control of microbial attachment and membrane biofouling. Some of the QS applications for biofouling control are given in fig. 5.
The evidence shows that inhibition or disruption of QS systems (e.g., QS signal molecules, QS regulator gene) would be an effective means for controlling microbial attachment and membrane biofouling. However, the recent study by Yeon et al.  showed that membrane biofouling due to a mixed culture of Gram-negative and Gram-positive bacteria could also be efficiently removed by the addition of AHL inhibitors, e.g., Acylase I as discussed earlier. Kim et al.  reported that biochemical control of quorum sensing could be an effective alternative to control biofilm formation and, thus, the reduction in biofouling of RO membranes. Obviously, further study is strongly needed to test the QS inhibitors for biofouling control in industrial-scale membrane systems.
Conclusions and future research outlooks
This review paper discussed research on membrane biofouling, fouling factors, and some biofouling control strategies. In long-run operation, membrane biofouling needs more attention. According to the recent literature, future studies on membrane biofouling should take the following directions:
At present, most operating concerns of MBRs have arisen from the pilot studies or previous operational experiences. A modest research has been done in modeling MBRs. The expansion of mathematical models for different MBR systems will help optimize operational performance, process control, and the ways of dealing with biofouling.
Research is also needed on the biofouling behaviour in full-scale MBR plants in order to reveal the real biofouling behaviour.
The biological toxicity effects of pollutants in MBRs have received less attention. Studying these effects will not only expand the application of MBRs, which means treating different kinds of wastewater, but will also enhance the biological activity and treatment efficiency.
Much more needs to be learned about the impact of antimicrobial products on microbial biofilms and their recovery responses to damage, as microorganisms can develop resistance and subsequently survive previously effective control strategies.